AU2015203203B2 - Improved nitrile hydratase - Google Patents

Abstract

Provided is an improved nitrile hydratase with improved catalytic activity. Also provided are DNA for coding the improved nitrile hydratase, a recombinant vector that contains the DNA, a transformant that contains the recombinant vector, nitrile hydratase acquired from a culture of the transformant, and a method for producing the nitrile hydratase. Also provided is a method for producing an amide compound that uses the culture or a processed product of the culture. The improved nitrile hydratase contains an amino acid sequence represented by SEQ ID NO: 50 (GXiX 2X3X4 DX 5 X6 R) in a beta subunit, and is characterized in that X4 is an amino acid selected from a group comprising cysteine, aspartic acid, glutamic acid, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, serine and threonine. - 61 -

Description

TITLE OF THE INVENTION: IMPROVED NITRILE HYDRATASE 2015203203 15 Jun2015

Related Applications [0000]

This is a divisional of Australian Patent Application No. 2012265680, which is the Australian National Phase of PCT/JP2012/003745 filed 7 June 2012, which claims priority from Japanese patent application no. 2011-127466, filed 7 June 2011, Japanese patent application no. 2011-144378, filed 29 June 2011 and Japanese patent application no. 2011-145061, filed 30 June 2011. All publications, patent applications, patents, and other references mentioned herein are incorporated by referenced in their entirety.

Technical Field [0001]

The present invention relates to improving a nitrile hydratase (mutation) and its production method. Moreover, the present invention relates to genomic DNA that encodes the enzyme, a recombinant vector containing the genomic DNA, a transformant containing the recombinant vector, and a method for producing an amide compound.

Description of Background Art [0002]

In recent years, a nitrile hydratase was found, which is an enzyme having nitrile hydrolysis activity that catalyses the hydration of a nitrile group to its corresponding amide group. Also, methods are disclosed to produce corresponding amide compounds from nitrile compounds using the enzyme or a microbial cell or the like containing the enzyme. Compared with conventional chemical synthetic methods, such methods are known by a high conversion or selectivity rate from a nitrile compound to a corresponding amide compound.

[0003]

Examples of microorganisms that produce a nitrile hydratase are the genus Corynebacterium, genus Pseudomonas, genus Rhodococcus, genus Rhizobium, genus Klebsiella, genus Pseudonoeardia and the like. Among those, Rhodococcus rhodocbrous strain J1 has been used for industrial production of acrylamides, and its usefulness has been verified. Furthermore, a gene encoding a nitrile hydratase produced by strain J1 has been identified (see patent publication 1).

[0004]

Meanwhile, introducing a mutation into a nitrile hydratase has been attempted not - 1 -only to use a nitrile hydratase isolated from a naturally existing microorganism or its gene, but also to change its activity, substrate specificity, Vmax, Km, heat stability, stability in a substrate, stability in a subsequent product and the like of a nitrile hydratase. Regarding the nitrile hydratase in Pseudonocardia thermophila JCM 3095, from its conformational data, sites relating to the substrate specificity or thermal stability are anticipated, and mutant enzymes with modified substrate specificity were obtained (see patent publications 2~4). Also, nitrile hydratase genes with improved heat resistance and amide-compound resistance have been produced by the inventors of the present invention (see patent publications 5-9). 2015203203 15 Jun2015 [0005]

To produce acrylamide for industrial applications using enzyme production methods, it is useful to develop a nitrile hydratase with improved catalytic activity when production costs such as catalyst costs are considered. Developing enzymes with improved activity is especially desired so as to achieve a reduction in the enzyme amount for reactions and in production costs or the like.

Prior Art Publication Patent Publication - 1A- 2015203203 01 May 2017 [0006]

Patent publication 1: Japanese patent publication 3162091 Patent publication 2: International publication pamphlet W02004/056990 Patent publication 3: Japanese laid-open patent publication 2004-194588 Patent publication 4: Japanese laid-open patent publication 2005-16403 Patent publication 5: International publication pamphlet W02005/116206 Patent publication 6: Japanese laid-open patent publication 2007-143409 Patent publication 7: Japanese laid-open patent publication 2007-43910 Patent publication 8: Japanese laid-open patent publication 2008-253182 Patent publication 9: Japanese laid-open patent publication 2010-172295

SUMMARY OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION

[0007]

The present invention provides an improved nitrile hydratase so as to provide an improved nitrile hydratase with enhanced catalytic activity. The present invention also provides a nitrile hydratatse collected from DNA encoding such an improved nitrile hydratase, a recombinant vector containing the DNA, a transformant containing the recombinant vector, and a culture of the transformant, as well as a method for producing such a nitrile hydratase. The present invention further provides a method for producing an amide compound using the culture or the processed product of the culture.

SOLUTIONS TO THE PROBLEMS

[0008]

The inventors of the present invention have conducted extensive studies to solve the above problems. As a result, in the amino acid sequence of a nitrile hydatase, the inventors have found that a protein in which a specific amino-acid residue is substituted with another amino-acid residue has nitrile hydratase activity and exhibits enhanced catalytic activity. Accordingly, the present invention is completed.

[0008A]

The present invention provides an improved nitrile hydratase, comprising improved nitrile hydratase, comprising at least one of the following (a)~(c): (a) in the a subunit, a nitrile hydratase contains an amino-acid sequence as shown in SEQ ID NO: 119 below AX1X2X3X4GX5X6GX7X8 (SEQ ID NO: 119) (A is alanine, G is glycine, and Χι~Χγ each independently indicate any amino-acid residue), wherein Xs is an amino acid selected from among alanine, leucine, methionine, asparagine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, lysine, proline, arginine, serine, threonine and tryptophan; (b) in the a subunit, a nitrile hydratase has the amino-acid sequence as shown in SEQ ID NO: 132 below - 2- 2015203203 01 May 2017 AX1X2X3X4GX5X6GX7Q (SEQ ID NO: 132) (A is alanine, G is glycine, Q is glutamine, and Χι~Χδ each independently indicate any amino-acid residue), wherein X7 is substituted with an amino acid different from that in a wild type; and (c) in the a subunit, a nitrile hydratase has the amino-acid sequence as shown in SEQ ID NO: 136 below AX1X2X3X4GX5X6GX7QX8X9 (SEQ ID NO: 136) (A is alanine, G is glycine, Q is glutamine, and Xi~Xs each independently indicate any amino-acid residue), wherein X9 is substituted with an amino acid different from that in a wild type.

[0009]

Namely, the present invention is described as follows. (1) An improved nitrile hydratase characterized by at least one of the following (aMe): (a) in the β subunit, a nitrile hydratase contains an amino-acid sequence as shown in SEQ ID NO: 50 below GX1X2X3X4DX5X6R (SEQ ID NO: 50) (G is glycine, D is aspartic acid, R is arginine, and Xi, X2, X3, X5 and Xe each independently indicate any amino-acid residue), in which X4 is an amino acid selected from among cysteine, aspartic acid, glutamic acid, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, serine and threonine; (b) in the β subunit, a nitrile hydratase contains an amino-acid sequence as shown in SEQ ID NO: 81 below WEX1X2X3X4X5X6 X7X8X9X10X11X12X13X14X15X16X17X18D (SEQ ID NO: 81) (W is tryptophan, E is glutamic acid, D is aspartic acid, and Xi~X6, and Xg-Xis each independently indicate any amino-acid residue), in which X7 is an amino acid selected from among alanine, valine, aspartic acid, threonine, phenylalanine, isoleucine and - 2A- 2015203203 15 Jun2015 methionine; (c) in the a subunit, a nitrile hydratase contains an amino-acid sequence as shown in SEQ ID NO: 119 below AXiX2X3X4GX5X6GX7X8 (SEQ ID NO: 119) (A is alanine, G is glycine, and Xi~X7 each independently indicate any amino-acid residue), in which Xg is an amino acid selected from among alanine, leucine, methionine, asparagine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, lysine, proline, arginine, serine, threonine and tryptophan; (d) in the a subunit, a nitrile hydratase has the amino-acid sequence as shown in SEQ ID NO: 132 below, AX1X2X3X4GX5X6GX7Q (SEQ ID NO: 132) (A is alanine, G is glycine, Q is glutamine, and Xi~X6 each independently indicate any amino-acid residue), in which X7 is substituted with an amino acid different from that in a wild type; (e) in the a subunit, a nitrile hydratase has the amino-acid sequence as shown in SEQ ID NO: 136 below AX,X2X3X4GXsX6GX7QX8X9 (SEQ ID NO: 136) (A is alanine, G is glycine, Q is glutamine, and Xt~Xs each independently indicate any amino-acid residue), in which X9 is substituted with an amino acid different from that in a wild type. (2) The improved nitrile hydratase described in (1), characterized in that X2 in SEQ ID NO: 50 is S (serine). (3) The improved nitrile hydratase described in (1), characterized in that X| is I (isoleucine), X2 is S (serine), X3 is W (tryptophan), X5 is K (lysine), and X6 is S (serine) in SEQ ID NO: 50. (4) The improved nitrile hydratase described in any of (1)--(3), having an amino-acid sequence as shown in SEQ ID NO: 51 that includes the amino-acid sequence as shown in SEQ ID NO: 50. (5) The improved nitrile hydratase described in (1), characterized in that X]4 in SEQ ID NO: 81 is G (glycine). (6) The improved nitrile hydratase described in (1), characterized in that X] is G (glycine), X2 is R (arginine), X3 is T (threonine), X4 is L (leucine), X5 is S (serine), X6 is I (isoleucine), X8 is T (threonine), X9 is W (tryptophan), X]0 is M (methionine), XM is H (histidine), X|2 is L (leucine), X[3 is K (lysine), and X|4 is G (glycine) in SEQ ID NO: 81. (7) The improved nitrile hydratase described in any of (1), (5) and (6), having an amino-acid sequence as shown in SEQ ID NO: 82 that includes the amino-acid sequence as shown in SEQ ID NO: 81. (8) The improved nitrile hydratase described in (1), characterized in that X| is M (methionine), X2 is A (alanine), X3 is S (serine), X4 is L (leucine), X5 is Y (tyrosine), X6 is A (alanine) and X7 is E (glutamic acid) in SEQ ID NO: 119. (9) The improved nitrile hydratase described in (1) or (8), having an amino-acid sequence as shown in SEQ ID NO: 120 that includes the amino-acid sequence as shown in SEQ ID NO: 119. (10) The improved nitrile hydratase described in (1), characterized by containing the amino-acid sequence of the a subunit as shown in SEQ ID NO: 132, in which X7 is an amino acid selected from among cysteine, phenylalanine, histidine, isoleucine, lysine, methionine, glutamine, arginine, threonine and tyrosine. 2015203203 15 Jun2015 (11) The improved nitrile hydratase described in (1) or (10), characterized in that Xi is M (methionine), X2 is A (alanine), X3 is S (serine), X4 is L (leucine), X5 is Y (tyrosine), and Xg is A (alanine) in SEQ ID NO: 132. (12) The improved nitrile hydratase described in (1), (10) or (11), having an amino-acid sequence as shown in SEQ ID NO: 131 that includes the amino-acid sequence as shown in SEQ ID NO: 132. (13) The improved nitrile hydratase described in (1), characterized by containing an amino-acid sequence of the a subunit as shown in SEQ ID NO: 136, in which X9 is an amino acid selected from among cysteine, glutamic acid, phenylalanine, isoleucine, asparagine, glutamine, serine and tyrosine. (14) The improved nitrile hydratase described in (1) or (13), characterized in that Xi is M (methionine), X2 is A (alanine), X3 is S (serine), X4 is L (leucine), X5 is Y (tyrosine), Xg is A (alanine), X7 is E (glutamic acid), and Xg is A (alanine) in SEQ ID NO: 136. (15) The improved nitrile hydratase described in (1), (13) or (14), having an amino-acid sequence as shown in SEQ ID NO: 135 that includes the amino-acid sequence as shown in SEQ ID NO: 136. (16) The improved nitrile hydratase described in any one of (1) to (15) is a nitrile hydratase derived from Rhodococcus bacterium or Nocardia bacterium. (17) DNA encoding the improved nitrile hydratase described in any one of (1) to (16). (18) DNA hybridized with the DNA described in (17) under stringent conditions. (19) A recombinant vector containing the DNA described in (17) or (18). (20) A transformant containing the recombinant vector described in (19). (21) A nitrile hydratase collected from a culture obtained by incubating the transformant described in (20). (22) A method for producing a nitrile hydratase, such a method characterized by incubating the transformant described in (20) and by collecting the nitrile hydratase from the obtained culture. (23) A method for producing an amide compound, such a method characterized by bringing a nitrile compound into contact with a culture, or a processed product of the culture, obtained by incubating the improved nitrile hydratase described in any of (1)-(16) or the transformant described in (20).

EFFECTS OF THE INVENTION

[0010]

According to the present invention, a novel improved (mutant) nitrile hydratase is obtained to have enhanced catalytic activity. The improved nitrile hydratase with enhanced catalytic activity is very useful to produce amide compounds at a high yield.

[0011]

According to the present invention, an improved nitrile hydratase and its production method are provided; such a nitrile hydratase is obtained from genomic DNA encoding the improved nitrile hydratase, a recombinant vector containing the genomic DNA, a transformant containing the recombinant vector and a culture of the transformant.

Also provided by the present invention is a method for producing an amide compound using the protein (improved nitrile hydratase) and the culture or a processed product of -4- the culture. 2015203203 01 May 2017 [0011 A]

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[001 IB]

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 is a view showing the structure of plasmid pSJ034; FIG. 2-1 is a list showing the alignment results in β subunits of known nitrile hydratases; FIG. 2-2 is a list showing the alignment results in β subunits of known nitrile hydratases; FIG. 3 shows the amino-acid sequence of the β subunit identified as SEQ ID NO: 51 related to the present invention; FIG. 4 is a photograph showing results of SDS-PAGE; FIG. 5 is a view showing the structure of plasmid pER855A; FIG. 6-1 is a list showing amino-acid sequences (part of N-terminal side) of β subunits in wild-type nitrile hydratases derived from various microorganisms; FIG. 6-2 is a list showing amino-acid sequences (part of C-terminal side) subsequent to the amino-acid sequences in FIG. 6-1; FIG. 7 shows the amino-acid sequence in the β subunit identified as SEQ ID NO: 82 related to the present invention; FIG. 8-1 is a list showing amino-acid sequences (part of N-terminal side) in a subunits of nitrile hydratases derived from various microorganisms; FIG. 8-2 is a list showing amino-acid sequences subsequent to the amino-acid sequences in FIG. 8-1; FIG. 9 shows the amino-acid sequence in the a subunit identified as SEQ ID NO: 121 related to the present invention; FIG. 10-1 is a list showing amino-acid sequences (part of N-terminal side) in a subunits of nitrile hydratases derived from various microorganisms; FIG. 10-2 is a list showing amino-acid sequences the same as in FIG. 2-1, and shows the sequences subsequent to the amino-acid sequences in FIG. 10-1; FIG. 11 shows the amino-acid sequence in the a subunit identified as SEQ ID NO: 131 related the present invention; -5 - FIG. 12-1 is a list showing amino-acid sequences (part of N-terminal side) in a subunits of nitrile hydratases derived from various microorganisms; 2015203203 01 May 2017 FIG. 12-2 is a list showing amino-acid sequences subsequent to the amino-acid sequences in FIG. 12-1; and FIG. 13 shows the amino-acid sequence in the a subunit identified as SEQ ID NO: 135 related to the present invention.

MODE TO CARRY OUT THE INVENTION

[0013]

In the following, the present invention is described in detail. 1. Nitrile Hvdratase (a) Known Nitrile Hydratase

The improved nitrile hydratase of the present invention is obtained by modifying a known nitrile hydratase and is not limited to being derived from any specific type. For example, those registered as nitrile hydratases in the GenBank database (http://www.ncbi.nlm.nih. gov/entrez/query.fcgi?CMD=search&DB=protein) provided by the U.S. National Center for Biotechnology Information (NCBI), or those described -5A-as nitrile hydratases in publications, may be referred to for a use. Examples of such nitrile hydratases are those described in patent publications 5~9 (which are incorporated by reference in the present application). Nitrile hydratases in patent publications 5~9 have heat resistance and acrylamide resistance, and by employing amino-acid substitutions according to the present invention, enhanced catalytic activity is further added to their properties. In particular, nitrile hydratases having amino-acid sequences shown in SEQ ID NOs: 53~57 are listed as reference. 2015203203 15 Jun2015 [0014]

Furthermore, by introducing a mutation from the gene encoding the amino-acid sequences described above using a well-known method, and by evaluating and screening mutant enzymes which have desired properties, improved enzymes with further enhanced activity are achieved. In particular, nitrile hydratases with amino-acid sequences shown in SEQ ID NOs: 58~61 are listed.

[0015] A “nitrile hydratase” has a conformation formed with a and β subunit domains, and contains a non-heme iron atom or a non-corrin cobalt atom as a prosthetic molecule. Such a nitrile hydratase is identified and referred to as an iron-containing nitrile hydratase or a cobalt-containing nitrile hydratase.

[0016]

An example of an iron-containing nitrile hydratase is such derived from Rhodococcus N-771 strain. The tertiary structure of such an iron-containing nitrile hydratase has been identified by X-ray crystal structural analysis. The enzyme is bonded with non-heme iron via four amino-acid residues in a cysteine cluster (Cys-Ser-Leu-Cys-Ser-Cys) (SEQ ID NO: 48) forming the active site of the a subunit.

[0017]

As for a cobalt-containing nitrile hydratase, examples are those derived from Rhodococcus rhodochrous J1 strain (hereinafter may be referred to as “J1 strain”) or derived from Pseudonocardia thermophila.

[0018] A cobalt-containing nitrile hydratase derived from the J1 strain is bound with a cobalt atom via a site identified as a cysteine cluster (Cys-Thr-Leu-Cys-Ser-Cys) (SEQ ID NO: 49) that forms the active site of the a subunit. In the cysteine cluster of a cobalt-containing nitrile hydratase derived from Pseudonocardia thermophila, cysteine (Cys) at position 4 from the upstream side (N-terminal side) of the cysteine cluster derived from the J1 strain is cysteine sulfinic acid (Csi), and cysteine (Cys) at position 6 from the furthermost downstream side (C-terminal side) of the cysteine cluster derived from the J1 strain is cysteine sulfenic acid (Cse).

[0019]

As described above, a prosthetic molecule is bonded with a site identified as cysteine clusters “C(S/T)LCSC” (SEQ ID NO: 48, 49) in the a subunit. Examples of a nitrile hydratase containing a binding site with such a prosthetic molecule are those that have amino-acid sequences and are encoded by gene sequences derived from the -6-following: Rhodococcus rhodochrous J1 (FERM BP-1478), Rhodococcus rhodochrous M8 (SU 1731814), Rhodococcus rhodochrous M3 3 (VKM Ac-1515D), Rhodococcus rhodochrous ATCC 39484 (JP 2001-292772), Bacillus smithii (JP H9-248188), Pseudonocardia thermophila (JP H9-275978), or Geobacillus thermoglucosidasius. 2015203203 15 Jun2015 [0020]

On the other hand, the β-subunit is thought to be attributed to structural stability.

[0021]

For example, in the a subunit derived from Rhodococcus rhodochrous J1 strain (FERM BP-1478), its amino-acid sequence is shown as SEQ ID NO: 4, and its base sequence is shown as SEQ ID NO: 3. Also, in the β subunit, its amino-acid sequence is shown as SEQ ID NO: 2, its base sequence is shown as SEQ ID NO: 1 and its accession number is “P21220.” In addition, in Rhodococcus rhodochrous M8 (SU 1731814), the accession number of the a subunit is “ATT 79340” and the accession number of the β subunit is “AAT 79339.”

The accession number of the nitrile hydratase gene derived from Rhodococcus pyridinivorans MW3 is “AJ582605,” and the accession number of the nitrile hydratase gene derived from Rhodococcus pyridinivorans S85-2 is “AJ582605.” The nitrile hydratase gene of Rhodococcus ruber RH (CGMCC No. 2380) is described in CN 101463358. Moreover, the accession number of the nitrile hydratase gene derived from Nocardia YS-2002 is “X86737,” and the accession number of the nitrile hydratase gene derived from Nocardia sp. JBRs is “AY141130.” [0022] (b-1) Improved Nitrile Hydratase (β48) FIGs. 2-1 and 2-2 show the alignments of amino-acid sequences (in one-letter code) in β-subunits of known nitrile hydratases derived from various microorganisms. FIGs. 2-1 and 2-2 each show amino-acid sequences in sequence ID numbers 2, 5—12, and 42-47 of amino-acid sequences from the top.

[0023]

Furthermore, the improved nitrile hydratase of the present invention includes examples in which one or more (for example, 1—10, preferred to be approximately 1—5) amino-acid residues are deleted, substituted and/or added in the amino-acid sequences of known nitrile hydratases, excluding the amino-acid sequence identified as SEQ ID NO: 50.

[0024]

An example of the improved nitrile hydratase of the present invention has an amino-acid sequence identified as SEQ ID NO: 51 in the β subunit as shown in FIG. 3. Here, the amino-acid sequence shown as SEQ ID NO: 50 is located at positions 44-52 counted from the N-terminal.

[0025]

According to an embodiment of the example above, in the improved nitrile hydratase that has the amino-acid sequence as shown in SEQ ID NO: 51, X], X2, X3, X5, -7 -and Xg each independently indicate any amino-acid residue, and X4 is an amino acid selected from among cysteine, aspartic acid, glutamic acid, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, serine and threonine. 2015203203 15 Jun2015 [0026]

In addition, according to another embodiment, in the improved nitrile hydratase that has the amino-acid sequence as shown in SEQ ID NO: 51, Xi, X3, X5, and Xe each independently indicate any amino-acid residue, X2 is S (serine), and X4 is an amino acid selected from among cysteine, aspartic acid, glutamic acid, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, serine and threonine.

[0027]

Moreover, according to yet another embodiment, in the improved nitrile hydratase that has the amino-acid sequence as shown in SEQ ID NO: 51, Xi is I (isoleucine), X2 is S (serine), X3 is W (tryptophan), and X5 is K (lysine), Xe is S (serine), and X4 is an amino acid selected from among cysteine, aspartic acid, glutamic acid, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, serine and threonine.

[0028]

Another example of the improved nitrile hydratase of the present invention is as follows: in the amino-acid sequence of a known nitrile hydratase identified as SEQ ID NO: 2, the amino-acid residue (tryptophan) at position 48 of the β subunit is substituted with cysteine, aspartic acid, glutamic acid, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, serine or threonine.

[0029]

Modes of such amino-acid substitutions are denoted, for example, as Wp48C, Wp48D,'Wp48E, Ψβ48Η, \\φ48Ι, Ψβ48Κ, Ψβ48Μ, Wp48N, Ψβ48Ρ, 1'A^48Q, W348S or \νβ48Τ. Amino acids are identified by a single-letter alphabetic code. The letter to the left of the numeral showing the number of amino-acid residues counted from the terminal to the substituted position (for example, “48”) represents the amino acid in a one-letter code before substitution, and the letter to the right represents the amino acid in a one-letter code after substitution.

[0030]

In particular, when the amino-acid sequence of the β subunit as shown in SEQ ID NO: 2 is denoted as “Ψβ480” in the improved nitrile hydratase, the abbreviation means that, in the amino-acid sequence of the β subunit (SEQ ID NO: 2), tryptophan (W) at . position 48 counted from the N-terminal amino-acid residue (including the N-terminal amino-acid residue itself) is substituted with cysteine (C).

[0031]

Modes of amino acid substitutions in more preferred embodiments of the improved nitrile hydratase according to the present invention are shown as the following 1-12: 1. Ψβ480, 2. Ψβ48ϋ, 3. \νβ48Ε, 4. Ψβ48Η, -8- 5. Wp48I, 2015203203 15 Jun2015 6. Wp48K, 7. Wp48M, 8. Ψβ48Ν, 9. Wp48P, 10. Wp48Q, 11. Wp48S,and 12. Wp48T.

Preferred embodiments of base substitutions to cause the above amino-acid substitutions are shown below.

[0032] WP48C: a base sequence TGG (at positions 142—144 in SEQ ID NO: 1) is preferred to be substituted with TGC (TGG—»TGC). WP48D: a base sequence TGG (at positions 142-144 in SEQ ID NO: 1) is preferred to be substituted with GAC (TGG—>GAC).

Wp48E: a base sequence TGG (at positions 142-144 in SEQ ID NO: 1) is preferred to be substituted with GAG (TGG—»GAG).

Wp48F: a base sequence TGG (at positions 142-144 in SEQ ID NO: 1) is preferred to be substituted with TTC (TGG—>TTC).

Wp48H: a base sequence TGG (at positions 142-144 in SEQ ID NO: 1) is preferred to be substituted with CAC (TGG—>CAC). WP48I: a base sequence TGG (at positions 142—144 in SEQ ID NO: 1) is preferred to be substituted with ATC (TGG—>ATC),

Wp48K: a base sequence TGG (at positions 142-144 in SEQ ID NO: 1) is preferred to be substituted with AAG (TGG—>AAG). WP48M: a base sequence TGG (at positions 142-144 in SEQ ID NO: 1) is preferred to be substituted with ATG (TGG—>ATG). Ψβ48Ν: a base sequence TGG (at positions 142-144 in SEQ ID NO: 1) is preferred to be substituted with AAC (TGG—>AAC).

Wp48P: a base sequence TGG (at positions 142-144 in SEQ ID NO: 1) is preferred to be substituted with CCG (TGG—GCG).

Wp48Q: a base sequence TGG (at positions 142-144 in SEQ ID NO: 1) is preferred to be substituted with CAG (TGG—GAG).

Wp48S: a base sequence TGG (at positions 142-144 in SEQ ID NO: 1) is preferred to be substituted with TCC (TGG-^TCC). WP48T: a base sequence TGG (at positions 142-144 in SEQ ID NO: 1) is preferred to be substituted with ACC (TGG—>ACC).

[0033] (b-2) Improved Nitrile Hydratase (β37) FIGs. 6-1 and 6-2 show the alignments of amino-acid sequences (in the one-letter code) in β-subunits of known nitrile hydratases derived from various microorganisms. FIGs. 6-1 and 6-2 each show amino-acid sequences in sequence ID numbers 2,5-12, and 42-49 of amino-acid sequences from the top.

[0034]

Furthermore, the improved nitrile hydratase of the present invention includes -9-examples in. which one or more (for example, 1-10, preferred to be approximately 1-5) amino-acid residues are deleted, substituted and/or added in the amino-acid sequences of known nitrile hydratases, excluding the amino-acid sequence identified as SEQ ID NO: 81. 2015203203 15 Jun2015 [0035]

An example of the improved nitrile hydratase of the present invention has an amino-acid sequence identified as SEQ ID NO: 82 in the β subunit as shown in FIG. 7. Here, the amino-acid sequence shown in SEQ ID NO: 81 is located at positions 29-49 counted from the N-terminal.

[0036]

According to an embodiment, in the improved nitrile hydratase that has the amino-acid sequence shown in SEQ ID NO: 82, Xi-Xs and Xg~ Xis each independently indicate any amino-acid residue, and Xy is an amino acid selected from among alanine, aspartic acid, threonine, phenylalanine, isoleucine and methionine.

[0037]

According to another embodiment, in the improved nitrile hydratase that has the amino-acid sequence shown in SEQ ID NO: 82, Xi-Xg, Xs~Xi3 and Xis- X|g, each independently indicate any amino-acid residue, X4 is G (glycine), and X7 is an amino acid selected from among alanine, valine, aspartic acid, threonine, phenylalanine, isoleucine and methionine.

[0038]

According to yet another embodiment, in the improved nitrile hydratase that has the amino-acid sequence as shown in SEQ ID NO: 82, Xis-Xiseach independently indicate any amino-acid residue, X| is G (glycine), X2 is R (arginine), X3 is T (threonine), X4 is L (leucine), X5 is S (serine), X$ is I (isoleucine), X$ is T (threonine), X9 is is W (tryptophan), X10 is M (methionine), Xn is H (histidine), X12 is L (leucine), X13 is K (lysine), X|4 is G (glycine), X7 is an amino acid selected from among alanine, valine, aspartic acid, threonine, phenylalanine, isoleucine and methionine.

[0039]

Another example of the improved nitrile hydratase of the present invention is as follows: in the amino-acid sequence of a known nitrile hydratase identified as SEQ ID NO: 2, the amino-acid residue (leucine) at position 37 of the β subunit is substituted with alanine, valine, aspartic acid, threonine, phenylalanine, isoleucine or methionine.

[0040]

Modes of such amino-acid substitutions are denoted, for example, as ίβ37Α, ίβ370, Εβ37Ρ, Εβ37Ι, Εβ37Μ, LP37T or Εβ37ν. Amino acids are identified by a single-letter alphabetic code. The letter to the left of the numeral showing the number of amino-acid residues counted from the terminal to the substituted position (for example, “37”) is the amino acid in the one-letter code before substitution, and the letter to the right represents the amino acid in the one-letter code after substitution.

[0041] -10-

In particular, when the amino-acid sequence of the β subunit (SEQ ID NO: 2) identified as SEQ ID NO: 2 is denoted as “Εβ37Α” in the improved nitrile hydratase, the abbreviation means that, in the amino-acid sequence of the β subunit (SEQ ID NO: 2), leucine (L) at position 37 counted from the N-terminal amino-acid residue (including the N-terminal amino-acid residue itself) is substituted with alanine (A). 2015203203 15 Jun2015 [0042]

Modes of amino acid substitutions in more preferred embodiments of the improved nitrile hydratase according to the present invention are shown as the following 1-7: 1. Ι,β37Α, 2. I^37D, 3. L^37F, 4. ίβ37Ι, 5. ίβ37Μ, 6. ίβ37Τ and 7. Τβ37Υ

Preferred embodiments of base substitutions to cause the above amino-acid substitutions are shown in Table 1 below.

[0043]

Table 1 amino-acid substitution base substitution LP37A Base sequence CTG (positions at 109-111 in SEQ ID NO: 1) is preferred to be substituted with GCA, GCC, GCG or GCT. Especially preferred to be substituted is C at·position 109 with G, T at position 110 with C, and G at position 111 with C (CTG-t-GCC). ίβ37ϋ Base sequence CTG (positions at 109-111 in SEQ ID NO: 1) is preferred to be substituted with GAC or GAT. Especially preferred to be substituted is C at position 109 with G, T at position 110 with A, and G at position 111 with C (CTG->GAC1. LP37F Base sequence CTG (positions at 109-111 in SEQ ID NO: 1) is preferred to be substituted with TTC orTTT. Especially preferred to be substituted is C at position 109 with T and G at position 111 with C (CTG-»TTC). Lp37l Base sequence CTG (positions at 109-111 in SEQ ID NO: 1) is preferred to be substituted with ATT, ATC orATA. Especially preferred to be substituted is C at position 109 with A and G at position 111 with C (CTG-iATC). LP37M Base sequence CTG (positions at 109-111 in SEQ ID NO: 1) is preferred to be substituted with ATG. Especially preferred to be substituted is C at position 109 with A (CTG—>ATG). Lp37T Base sequence CTG (positions at 109-111 in SEQ ID NO: 1) is preferred to be substituted with ACA, ACC, ACG or ACT. Especially preferred to be substituted is C at position 109 with A, T at position 110 with C and G at position 111 with C (CTG^ACC). Lp37V Base sequence CTG (positions at 109-111 in SEQ ID NO: 1) is preferred to be substituted with GTA, GTC, GTG or GTT. Especially preferred to be substituted is C at position 109 with G and G at position 111 with C (CTG-*GTC).

[0044] (b-3) Improved Nitrile Hydratase (a83) FIGs. 8-1 and 8-2 show amino-acid sequence alignments (in one-letter code) in α-subunits of known nitrile hydratases derived from various microorganisms: FIGs. 8-1 and 8-2 each show amino-acid sequences in sequence ID numbers 4,105-108,121, 109,110, 112, 111, 122-124,113, 114,125 from the top.

[0045]

Furthermore, the improved nitrile hydratase of the present invention includes examples in which one or more (for example, 1~10, preferred to be approximately 1-5) - 11 -amino-acid residues are deleted, substituted and/or added in amino-acid sequences of known nitrile hydratases, excluding the amino-acid sequence identified as SEQ ID NO: 2015203203 15 Jun2015 119. Examples of such a nitrile hydratase are described in patent publications 5-9 (the contents are incorporated by reference into the present application). Nitrile hydratases in patent publication 5~9 each exhibit heat resistance and acrylamide resistance. Moreover, as a result of amino-acid substitutions of the present invention, enhanced catalytic activity is further added to their properties.

[0046]

An example of the improved nitrile hydratase of the present invention has an amino-acid sequence as shown in SEQ ID NO: 120 in the a subunit as shown in FIG. 9. Here, an amino-acid sequence shown in SEQ ID NO: 119 is located at positions 73-83 counted from the N-terminal.

[0047]

According to an embodiment, in the improved nitrile hydratase that has the amino-acid sequence shown in SEQ ID NO: 120, Xi~X7 each independently indicate any amino-acid residue, and Xg is an amino acid selected from among alanine, leucine, methionine, asparagine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, lysine, pro line, arginine, serine, threonine, tyrosine and tryptophan.

[0048]

According to another embodiment, in the improved nitrile hydratase that has the amino-acid sequence shown in SEQ ID NO: 120, Xi is M (methionine), X2 is A (alanine), X3 is S (serine), X4 is L (leucine), X5 is Y (tyrosine), Xg is A (alanine), X7 is E (glutamic acid), and Xg is an amino acid selected from among alanine, leucine, methionine, asparagine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, lysine, proline, arginine, serine, threonine, tyrosine and tryptophan.

[0049]

Another example of the improved nitrile hydratase of the present invention is as follows: in the amino-acid sequence of a known nitrile hydratase identified as SEQ ID NO: 4, the amino-acid residue at position 83 (glutamine) of the a subunit is substituted with alanine, leucine, methionine, asparagine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, lysine, proline, arginine, serine, threonine, tyrosine or tryptophan.

[0050]

Modes of such amino-acid substitutions are denoted, for example, as Qa83A, Qa83C, Qa83D, Qa83E, Qa83F, Qa83G, Qa83H, Qa83K, Qa83L, Qa83M, Qa83N, Qa83P, Qa83R, Qa83S, Qa83T, Qa83Y and Qa83W. Amino acids are identified by a single-letter alphabetic code. The letter to the left of the numeral showing the number of amino-acid residues counted from tire terminal to the substituted position (for example, “83”) represents the amino acid in a one-letter code before substitution, and the letter to the right represents the amino acid in a one-letter code after substitution.

[0051]

In particular, when the amino-acid sequence of the a subunit in SEQ ID NO: 4 is -12- denoted as “Qa83A” in the improved nitrile hydratase, the abbreviated notation means that, in the amino-acid sequence of the a subunit (SEQ ID NO: 4), glutamine (Q) at position 83 counted from the N-terminal amino-acid residue (including the N-terminal amino-acid residue itself) is substituted with alanine (A). 2015203203 15 Jun2015 [0052]

Modes of amino-acid substitutions in more preferred embodiments of the improved nitrile hydratase according to the present invention are shown as the following 1~17: 1. Qa83A, 2. Qa83C, 3. Qa83D, 4. Qa83E, 5. Qa83F, 6. Qa83G, 7. Qa83H, 8. Qa83K, 9. Qa83L, 10. Qct83M, 11. Qa83N, 12. Qa83P, 13. Qa83R, 14. Q<x83S, 15. Qa83T, 16. Qa83Y and 17. Qa83W.

Preferred embodiments of base substitutions to cause the above amino-acid substitutions are shown below.

[0053]

Table 2 amino-acid substitution base substitution Qa83A Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with GCA, GCC, GCG, or GCT. Especially preferred to be substituted is C at position 247 with G, A at position 248 with C. and G at position 249 with C (CAG-*GCC). Qa83C Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with TGC or TGT. Especially preferred to be substituted is C at position 247 with T, A at position 248 with G, and G at position 249 with C (CAG->TGC). Qa83D Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with GAC or GAT. Especially preferred to be substituted is C at position 247 with G, and G at position 249 with C (CAG—> GAC). Qa83E Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with GAG or GAA. Especially preferred to be substituted is C at position 247 with G (CAG—* GAG) Qa83F Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with TTC or TTT, Especially preferred to be substituted is C at position 247 with T, A at position 248 with T, and G at position 249 with C (CAG^TTC). Qa83G Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with GGA, GGC, GGG or GGT. Especially preferred to be substituted is C at position 247 with G, A at position 248 with G, and G at position 249 with C (CAG—>GGC). Qa83H Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with CAC or CAT. Especially preferred to be substituted is G at position 249 with C (CAG-»CAC). Qa83K Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with AAA or AAG. Especially preferred to be substituted is C at position 247 with A (CAG-+AAG) Qa83L Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with CTA, CTC, CTG, CTT, TTA or TTG. Especially preferred to be substituted is A at position 248 with T, and -13 - 2015203203 15 Jun2015 G at position 249 with C (CAG—»CTC), Qa83M Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with ATG. Especially preferred to be substituted is C at position 247 with A, and A at position 248 with T (CAG-tATG). Qa83N Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with AAC or AAT. Especially preferred to be substituted is C at position 247 with A, and G at position 249 with C (CAG—>AAC). Qa83P Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with CCA, CCC, CCG or CCT. Especially preferred to be substituted is A at position 248 with C (CAG->CCG). Qa83R Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with CGA, CGC, CGG, CGT, AGA or AGG. Especially preferred to be substituted Is A at position 248 with G (CAG-»CGG). Qa83S Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with TCA, TCC, TCG, TCT, AGC or AGT. Especially preferred to be substituted is C at position 247 with T, A at position 248 with C, and G at position 249 with C (CAG->TCC). Qa83T Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with AGA, ACC, ACG or ACT. Especially preferred to be substituted is C at position 247 with A, A at position 248 with C, and G at position 249 with C (CAG—* ACC). Qa83Y Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with TAC or TAT. Especially preferred to be substituted is C at position 247 with T, and G at position 249 with C (CAG—*TAC). Qa83W Base sequence CAG (positions at 247-249 in SEQ ID NO: 3) is preferred to be substituted with TGG. Especially preferred to be substituted is C at position 247 with T, and A at position 248 with G (CAG-»TGG).

[0054] (b-4) Improved Nitrile Hydratase (a82) FIGs. 10-1 and 10-2 show amino-acid sequence alignments (in the one-letter code) in α-subunits of known nitrile hydratases derived from various microorganisms. FIGs. 10-1 and 10-2 each show amino-acid sequences in sequence ID numbers 4, 105-108, 121,109, 110, 112, 111, 122-124, 113, 114, 125 from the top.

[0055]

Furthermore, the improved nitrile hydratase of the present invention includes examples in which one or more (for example, 1-10, preferred to be approximately 1-5) amino-acid residues are deleted, substituted and/or added in the amino-acid sequences of known nitrile hydratases, excluding the amino-acid sequence identified as SEQ ID NO: 131. Examples of the improved nitrile hydratase are described in patent publications 5-9 (the contents are incorporated by reference into the present application). Nitrile hydratases in patent publication 5-9 each exhibit heat resistance and acrylamide resistance. Moreover, as a result of amino-acid substitutions of the present invention, enhanced catalytic activity is further added to their properties.

[0056]

An example of the improved nitrile hydratase of the present invention has an amino-acid sequence as shown in SEQ ID NO: 131 in the a subunit as shown in FIG. 11. Here, an amino-acid sequence shown in SEQ ID NO: 132 is located at positions 73-83 counted from the N-terminal.

[0057]

According to an embodiment of the present invention, in the improved nitrile hydratase that has the amino-acid sequence shown in SEQ ID NO: 131, Xi~Xg each independently indicate any amino-acid residue, and X7 is an amino acid selected from among cysteine, phenylalanine, histidine, isoleucine, lysine, methionine, glutamine, arginine, threonine and tyrosine. - 14-[0058] 2015203203 15Jun2015

According to another embodiment, in the improved nitrile hydratase that has the amino-acid sequence shown in SEQ ID NO: 131, Xi is M (methionine), X2 is A (alanine), X3 is S (serine), X4 is L (leucine), X5 is Y (tyrosine), X6 is A (alanine), and X7 is an amino acid selected from among cysteine, phenylalanine, histidine, isoleucine, lysine, methionine, glutamine, arginine, threonine and tyrosine.

[0059]

Another example of the improved nitrile hydratase of the present invention is as follows: in the amino-acid sequence of a known nitrile hydratase shown in SEQ ID NO: 4, the amino-acid residue at position 82 (glutamic acid) of the a subunit is substituted with cysteine, phenylalanine, histidine, isoleucine, lysine, methionine, glutamine, arginine, threonine or tyrosine.

[0060]

Modes of such amino-acid substitutions are denoted, for example, as Ea82C, Ea82F, Ea82H, Ea82I, Ea82K, Ea82M, Ea82Q, Ea82R, Ea82T and Ea82Y. Amino acids are identified by a single-letter alphabetic code. The letter to the left of the numeral showing the number of amino-acid residues counted from the terminal to the substituted position (for example, “82”) is the amino acid in a one-letter code before substitution, and the letter to the right represents the amino acid in a one-letter code after substitution.

[0061]

In particular, when the amino-acid sequence of the a subunit in SEQ ID NO: 4 is denoted as “Ea82C” in the improved nitrile hydratase, the abbreviated notation means among the amino-acid sequence of the a subunit, glutamic acid (E) at position 82 counted from the N-terminal amino-acid residue (including the N-terminal amino-acid residue itself) is substituted with cysteine (C).

[0062]

Modes of amino acid substitutions in more preferred embodiments of the improved nitrile hydratase according to the present invention are shown as the following 1~10: 1. Ea82C, 2. Ea82F, 3. Ea82H, 4. Ea82I, 5. Ea82K, 6. Ea82M, 7. Ea82Q, 8. Ea82R, 9. Ea82T and 10. Ea82Y.

Preferred embodiments of base substitutions to cause above amino-acid substitutions are shown below.

[0063]

Table 3 - 15 - 2015203203 15 Jun2015 amino-acid substitution base substitution Ea82C Base sequence GAG (positions at 244-246 in SEQ ID NO: 3) is preferred to be substituted with TGC or TGT. Especially preferred to be substituted is G at position 244 with T, A at position 245 with G, and G at position 246 with C (GAG—>TGC). Ea82F Base sequence GAG (positions at 244-246 in SEQ ID NO: 3) is preferred to be substituted with TTC or TTT. Especially preferred to be substituted is G at position 244 with T, A at position 245 with T, and G at position 246 with C (GAG—>TTC). Ea82H Base sequence GAG (positions at 244-246 in SEQ ID NO: 3) is preferred to be substituted with CAT or CAC. Especially preferred to be substituted is G at position 244 with C, and G at position 246 with C (GAG-CAC). Ea82l Base sequence GAG (positions at 244-246 in SEQ ID NO: 3) is preferred to be substituted with ATT, ATC or ATA. Especially preferred to be substituted is G at position 244 with A, A at position 245 with T, and G at position 246 with C (GAG-»ATC). Ea82K Base sequence GAG (positions at 244-246 in SEQ ID NO: 3) is preferred to be substituted with AAA or AAG. Especially preferred to be substituted is G at position 244 with A (GAG-*AAG). Ea82M Base sequence GAG (positions at 244-246 in SEQ ID NO: 3) is preferred to be substituted with ATG. Especially preferred to be substituted is G at position 244 with A, and A at position 245 with T (GAG-ATG). Ea82Q Base sequence GAG (positions at 244-246 in SEQ ID NO: 3) is preferred to be substituted with CAA or CAG. Especially preferred to be substituted is G at position 244 with C (GAG->CAG). Ea82R Base sequence GAG (positions at 244-246 in SEQ ID NO: 3) is preferred to be substituted with CGA, CGC, CGG, CGT, AGA or AGG. Especially preferred to be substituted is G at position 244 with C, and A at position 245 with G (GAG-*CGG). Ea82T Base sequence GAG (positions at 244-246 in SEQ ID NO: 3) is preferred to be substituted with ACA, ACC, ACG or ACT. Especially preferred to be substituted is G at position 244 with A, A at position 245 with C, and G at position 246 with C (GAG—>ACC). Ea82Y Base sequence GAG (positions at 244-246 in SEQ ID NO: 3) is preferred to be substituted with TAT or TAC. Especially preferred to be substituted is G at position 244 with T, and G at position 246 with G (GAG->TAC).

[0064] (b-5) Improved Nitrile Hydratase (a85) FIGs. 12-1 and 12-2 show the alignments of amino-acid sequences (in the one-letter code) in a-subunits of known nitrile hydratases derived from various microorganisms. FIGs. 12-1 and 12-2 each show amino-acid sequences in sequence ID numbers 4, 105-108, 121, 109, 110, 112, 111, 122-124, 113, 114, 125 from the top.

[0065]

Furthermore, the improved nitrile hydratase of the present invention includes examples in which one or more (for example, 1-10, preferred to be approximately 1-5) amino-acid residues are deleted, substituted and/or added in the amino-acid sequences of biown nitrile hydratases, excluding the amino-acid sequence identified as SEQ ID NO: 135. Examples of such a nitrile hydratase are described in patent publications 5-9 (the contents are incorporated by reference into the present application). Nitrile hydratases in patent publication 5-9 each exhibit heat resistance and acrylamide resistance. Moreover, as a result of amino-acid substitutions of the present invention, enhanced catalytic activity is further added to their properties.

[0066]

An example of the improved nitrile hydratase of the present invention has an amino-acid sequence as shown in SEQ ID NO: 135 in the a subunit as shown in FIG. 13. Here, an amino-acid sequence shown in SEQ ID NO: 136 is located at positions 73-85 counted from the N-terminal.

[0067]

According to an embodiment of the present invention, in the improved nitrile -16-hydratase that has the amino-acid sequence shown in SEQ ID NO: 135, Xi~Xg each independently indicate any amino-acid residue, and X9 is an amino acid selected from among cysteine, glutamic acid, phenylalanine, isoleucine, asparagine, glutamine, serine and tyrosine. 2015203203 15Jun2015 [0068]

According to another embodiment, in the improved nitrile hydratase that has the amino-acid sequence shown in SEQ ID NO: 135, Xi is M (methionine), X2 is A (alanine), X3 is S (serine), X4 is L (leucine), X5 is Y (tyrosine), X6 is A (alanine), X7 is E (glutamic acid), Xs is A (alanine), and X9 is an amino acid selected from among cysteine, glutamic acid, phenylalanine, isoleucine, asparagine, glutamine, serine and tyrosine.

[0069]

Another example of the improved nitrile hydratase of the present invention is as follows: in the amino-acid sequence of a known nitrile hydratase shown in SEQ ID NO: 4, the amino-acid residue at position 85 (histidine) of the a subunit is substituted with cysteine, glutamic acid, phenylalanine, isoleucine, asparagine, glutamine, serine or tyrosine.

[0070]

Modes of such amino-acid substitutions are shown, for example, as Ha85C, Ha85E, Ha85F, Ha85I, Ha85N, Ha85Q, Ha85S and Ha85Y. Amino acids are identified by a single-letter alphabetic code. The letter to the left of the numeral showing the number of amino-acid residues counted from the terminal to the substituted position (for example, “85”) is the amino acid in a one-letter code before substitution, and the letter to the right represents the amino acid in a one-letter code after substitution.

[0071]

In particular, when the amino-acid sequence of the a subunit in SEQ ID NO: 4 is denoted as “Ha85C” in the improved nitrile hydratase, the abbreviated notation means that, in the amino-acid sequence of the a subunit (SEQ ID NO: 4), histidine (H) at position 85 counted from the N-terminal amino-acid residue (including the N-terminal amino-acid residue itself) is substituted with cysteine (C).

[0072]

Modes of amino acid substitutions in more preferred embodiments of the improved nitrile hydratase according to the present invention are shown as the following 1~8: 1. Ha85C, 2. Ha85E, 3. Ha85F, 4. Ha85I, 5. Ha85N, 6. Ha85Q, 7. Ha85S and 8. Ha85Y.

Preferred embodiments of base substitutions to cause the above amino-acid substitutions are shown below. - 17- 2015203203 15 Jun2015 [0073] Table 4 amino-acid substitution base substitution HaS5C Base sequence CAC (positions at 253-255 in SEQ ID NO: 3) is preferred to be substituted with TGC or TGT. Especially preferred to be substituted Is C at position 253 with T, and A at position 254 with G (CAC—»TGC), Ha85E Base sequence CAC (positions at 253-255 in SEQ ID NO: 3) is preferred to be substituted with GAG or GAA. Especially preferred to be substituted is C at position 253 with G, and C at position 255 with G (CAC-+GAG). Ha85F Base sequence CAC (positions at 253-255 in SEQ ID NO: 3) is preferred to be substituted with TTC or TTT. Especially preferred to be substituted is C at position 253 with T. and A at position 254 with T (CAC—►TTC). Ha85l Base sequence CAC (positions at 253-255 in SEQ ID NO: 3) Is preferred to be substituted with ATT, ATC or ATA. Especially preferred to be substituted is C at position 253 with A, and A at position 254 with T (CAC—»ATC). Ha85N Base sequence CAC (positions at 253-255 in SEQ ID NO: 3) Is preferred to be substituted with AAC or AAT. Especially preferred to be substituted is C at position 253 with A (CAC—* AAC). Ha85Q Base sequence CAC (positions at 253-255 in SEQ ID NO: 3) is preferred to be substituted with CAA or CAG. Especially preferred to be substituted is C at position 255 with G (CAC—>CAG). Ha85S Base sequence CAC (positions at 253-255 in SEQ ID NO: 3) is preferred to be substituted with TCA, TCC, TCG, TCT, AGC or AGT. Especially preferred to be substituted is C at position 253 with T, and A at position 254 with C (CAC—>TCC). Ha85Y Base sequence CAC (positions at 253-255 in SEQ ID NO: 3) is preferred to be substituted with TAT or TAC. Especially preferred to be substituted is C at position 253 with T (CAC->TAC), [0074] (b-6) Nitrile Hydratase Activity

Among the activity properties of the improved nitrile hydratase according to the present invention, catalytic activity is improved relative to that in a nitrile hydratase before a mutation is introduced.

[0075]

Here, “nitrile hydratase activity” means an enzyme to catalyze the hydration for converting a nitrile compound to a corresponding amide compound (RCN+H2O—>-RCONH2). Determining the activity is conducted by bringing a nitrile compound as a substrate into contact with a nitrile hydratase for conversion to a corresponding amide compound and by determining the resultant amide compound.

Any nitrile compound may be used as a substrate as long as nitrile hydratase reacts with such a compound, but acrylonitrile is preferred.

[0076]

Reaction conditions are a substrate concentration of 2.5%, reaction temperature of 10°C to 30°C and duration of 10-30 minutes. The enzymatic reactions are terminated by adding phosphoric acid. Then, using HPLC (high-performance liquid chromatography) or gas chromatography, the produced acrylamide is analyzed to measure the amount of the amide compound.

[0077] “Improved catalytic activity” means that when activity is measured in the culture of a transformant containing the improved nitrile hydratase or the improved nitrile hydratase isolated from the transformant, the catalytic activity of the improved nitrile hydratase is at least 10% higher than that of the parent strain measured under the same conditions. The parent strain in the present application means a transformant into - 18 -which a template plasmid for mutation was introduced. 2015203203 15 Jun2015

As for an amide compound, an amide compound represented by the general formula (1) below, for example, is preferred. R-CONH2......(1) (Here, R is an optionally substituted linear or branched alkyl or alkenyl group having 1~10 carbon atoms, an optionally substituted cycloalkyl or allyl group having 3—18 carbon atoms, or an optionally substituted saturated or unsaturated heterocyclic group.) Especially preferred is an acrylamide in which “R” in the formula is “CH2=CH-.” [0078]

The above improved nitrile hydratase is obtained by performing amino-acid substitution on a nitrile hydratase. For example, such an improved nitrile hydratase is obtained by modifying the amino-acid sequence (SEQ ID NO: 2) of a nitrile hydratase derived from Rhodococcus rhodocrous J1 strain, and by screening a nitrile hydratase with an improved catalytic activity.

[0079]

Rhodococcus rhodochrous J1 strain is internationally registered under accession number “PERM BP-1478” at the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6,1-1-1 Higashi, Tsukuba, Ibaraki), deposited September 18,1987.

[0080]

Using a nitrile hydratase derived from bacteria other than the J1 strain, catalytic activity is thought to be improved as well when a mutation is introduced by modifying a position, type of amino acid or DNA sequence described above. Preferred strains are: Rhodococcus rhodocrous M8 (SU 1731814) (SEQ ID NO: 5), Rhodococcus ruber TH (SEQ ID NO: 6), Rhodococcus rhodocrous M33 (VKM Ac-1515D), Rhodococcus pyridinivorans MW3 (SEQ ID NO: 7), Rhodococcus pyridinivorans S85-2 (SEQ ID NO: 8), Rhodococcus pyridinivorans MS-38 (SEQ ID NO: 9), Rhodococcus ruber RH (CN 101463358) (SEQ ID NO: 52), Nocardia sp. JBRs (SEQ ID NO: 10), Nocardia sp. YS-2002 (SEQ ID NO: 11), Rhodococcus rhodocrous ATCC 39384 (SEQ ID NO: 12), uncultured bacterium SP1 (SEQ ID NO: 42), uncultured bacterium BD2 (SEQ ID NO: 43), Comamonas testosterone (SEQ ID NO: 44), Geobacillus thermoglucosidasius Q6 (SEQ ID NO: 45), Pseudonocardia thermophila JCM 3095 (SEQ ID NO: 46), Rhodococcus rhodocrous Cr 4 (SEQ ID NO: 47), or the like. Obtained through natural mutation from the M8 strain above (SU 1731814), Rhodococcus rhodocrous M33 (VKM Ac-1515D) was selected because it is capable of constitutive expression of a nitrile hydratase. The amino-acid or gene sequence of the nitrile hydratase itself is not mutated (US Patent 5,827,699). In the β subunit in a bacterium listed above, the amino-acid residue at position 48 from the N-terminal of the improved nitrile hydratase is substituted with cysteine, aspartic acid, glutamic acid, histidine, isoleucine, lysine, methionine, asparagine, praline, glutamine, serine or threonine.

[0081]

Methods for conducting amino-acid substitution on a wild-type nitrile hydratase are as follows: a bacterium having nitrile hydratase activity is brought into contact for reactions with chemicals such as hydroxyl amine or nitrous acid as a mutation source; - 19- UV rays are irradiated to induce mutation; error-prone PCR or site-directed mutagenesis is employed to introduce a mutation at random into the gene that encodes a nitrile hydratase; and the like. 2015203203 15 Jun 2015 [0082]

(b-7) Error-Prone PCR

To study functions and characteristics of proteins using a mutant, random mutagenesis is known. Random mutagenesis is a method to introduce a random mutation to the gene encoding a specific protein so that a mutant is produced. In random mutagenesis by PCR, stringency conditions are set low for the DNA amplification period so that a mutant base is introduced (error-prone PCR).

[0083]

In such an error-prone PCR method, a mutation is introduced randomly into any position of the entire DNA site to be amplified. Then, by examining the function of the obtained mutant, which occurred through the mutation introduced at a random site, information of the amino acid or domain important for a specific function of a protein is obtained.

[0084]

As a nitrile hydratase used for the template of error-prone PCR, the nitrile hydratase gene derived from a wild-type strain or DNA obtained as an amplified product by error-prone PCR is used.

[0085]

As reaction conditions for error-prone PCR, for example, a composition ratio of any one, two or three among dNTP (dGTP, dCTP, dATP or dTTP) in the reaction mix is reduced relative to another dNTP. In so setting, during the DNA synthesis, at a position that requires a dNTP whose ratio is reduced, another dNTP is more likely to be used by error and that may lead to mutation. In addition, other preferred reaction conditions are a composition in which the amount of MgCh and/or MnCL in the reaction mix is increased.

[0086] (b-8) Improved Nitrile Hydratase Mutagenesis

Based on a known nitrile hydratase gene, DNA that encodes such an improved nitrile hydtratase is produced by site-directed mutagenesis methods described in Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley and Sons (1987-1997) and the like. To introduce a mutation into DNA by well-known methods such as the Kunkel method or Gapped Duplex method, mutagenesis kits applying site-directed mutagenesis methods such as follows are used: QuickChange™ XL Site-Directed Mutagenesis Kit (made by Stratagene), GeneTailor™ Site-Directed Mutagenesis System (made by Invitrogen Corporation), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km and the like, made by Takara Bio Inc.) and the like.

[0087] -20-

Furthermore, the DNA related to the present invention includes DNA which is hybridized under stringent conditions with a DNA made up of a base sequence complementary to the base sequence of the DNA of the present invention, and which encodes a protein having nitrile hydratase activity. 2015203203 15 Jun2015 [0088]

Such an improved nitrile hydratase DNA is obtained by introducing a mutation into a wild-type gene as described above. Alternatively, using the DNA sequence or its complementary sequence or a DNA fragment as a probe, improved nitrile hydratase DNA may also be obtained from cDNA libraries and genomic libraries by employing well-known hybridization methods such as colony hybridization, plaque hybridization, Southern blot or the like. Libraries constructed by a well-known method may be used, or commercially available cDNA libraries and genomic libraries may also be used.

[0089] “Stringent conditions” are those for washing after hybridization; a salt concentration of 300-2000 mM and a temperature of 40~75°C, preferably a salt concentration of 600-900 mM and a temperature of 65°C. For example, conditions 2xSSC at 50°C may be employed. In addition to such a salt concentration of the buffer, temperature and the like, a person skilled in the art may set conditions for obtaining DNA that encodes a nitrile hydratase of the present invention by adding various conditions such as probe concentration, probe length and reaction time.

[0090]

For detailed procedures for hybridization, Molecular Cloning, A Laboratory Manual, 2nd edition (Cold Spring Harbor Laboratory Press (1989)) or the like may be referred to. DNA to be hybridized includes DNA or its fragment, containing a base sequence which is at least 40%, preferably 60%, more preferably 90% or greater, homologous to the genomic DNA of the present invention.

[0091] (c) Recombinant Vector, Transformant

It is necessary for a nitrile hydratase gene to be put into a vector so that nitrile hydratase is expressed in the host organism to be transformed. Examples of such vectors are plasmid DNA, bacteriophage DNA, retrotransposon DNA, artificial chromosome DNA and the like.

[0092]

In addition, a host to be used in the present invention is not limited to any specific type as long as it can express the target nitrile hydratase after the recombinant vector is introduced into the host. Examples are bacteria such as E. coli and Bacillus subtilis, yeasts, animal cells, insect cells, plant cells and the like. When E. coli is used as a host, an expression vector with high expression efficiency, such as expression vector pkk 233-2 with a trc promoter (made by Amersham Biosciences Corp.), pTrc 99A (made by Amersham Biosciences Corp.) or the like, is preferred.

[0093]

In addition to a nitrile hydratase gene, a vector may be coupled with a promoter, -21 -terminator, enhancer, splicing signal, poly A addition signal, selection marker, ribosome binding sequence (SD sequence) or the like. Examples of selection markers are kanamycin resistance gene, dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like. 2015203203 15 Jun2015 [0094]

When a bacterium is used as a host, Escherichia coli may be used, for example, and a Rhodococcus strain such as Rhodococcus rhodochrous ATCC 12674, Rhodo coccus rhodochrous ATCC 17895 and Rhodococcus rhodochrous ATCC 19140 may also be used. Those ATCC strains are obtained from the American type culture collection.

[0095]

When E. coli is used as a host for producing a transformant to express a nitrile hydratase, since most of the expressed nitrile hydratase is formed as an inclusion body and is insoluble, a transformant with low catalytic activity is obtained. On the other hand, if a Rhodococcus strain is used as a host, nitrile hydratase is present in the soluble fraction, and a transformant with high activity is obtained. Those transformants may be selected based on purposes. However, when an improved enzyme is selected under stringent conditions, a transformant with high activity derived from a Rhodococcus strain is preferred.

[0096]

Introducing a recombinant vector into a bacterium is not limited to any specific method as long as DNA is introduced into the bacterium. For example, a method using calcium ions, electroporation or the like may be employed.

[0097]

When yeast is used as a host, examples are Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and the like. As a method for introducing a recombinant vector into yeast, it is not limited specifically as long as DNA is introduced into the yeast. For example, an electroporation method, spheroplast method, lithium acetate method or the like may be employed.

[0098]

When animal cells are used as a host, monkey cells COS-7, Vero, CHO cells, mouse L cells, rat GH3 cells, human FL cells or the like may be employed. As a method for introducing a recombinant vector into animal cells, for example, an electroporation method, calcium phosphate method, lipofection method or the like may be used.

[0099]

When insect cells are used as a host, Sf9 cells, S£21 cells or the like may be used. A method for introducing a recombinant vector into insect cells, for example, a calcium phosphate method, lipofection method, electroporation method or the like may be used.

[0100]

When plant cells are used as a host, tobacco BY-2 cells or the like may be used. However, that is not the only option. A method for introducing a recombinant vector -22-into plant cells, for example, an Agrobacterium method, particle gun method, PEG method, electroporation method or the like may be used. 2015203203 15 Jun2015 [0101] (d) Method for Producing Culture and Improved Nitrile Hydratase

An improved nitrile hydratase of the present invention is obtained by incubating the above transformant and by collecting from the obtained culture.

[0102]

The present invention also relates to a method for producing an improved nitrile hydratase, and the method is characterized by collecting an improved nitrile hydratase from the culture above.

[0103]

In the present invention, “culture” means any of culture supernatant, cell cultured cell, bacterial-cell culture, and cell homogenates or bacterial-cell homogenates. To incubate a transformant of the present invention, a generally used method for incubating a host is used. The target nitrile hydratase is accumulated in the culture.

[0104]

As for a culture to incubate a transformant of the present invention, a natural or synthetic culture medium is used as long as it contains a carbon source, a nitrogen source, inorganic salts or the like for the host bacteria to assimilate, and incubation of a transformant is performed efficiently. Examples of a carbon source are carbohydrates such as glucose, galactose, fructose, sucrose, raffmose and starch; organic acids such as acetic acid and propionic acid; alcohols such as ethanol and propanol; and the like. Examples of a nitrogen source are inorganic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate; ammonium salts of organic acids; and other nitrogen-containing compounds.

[0105]

In addition, peptone, yeast extract, meat extract, corn steep liquor, various amino acids or the like may also be used. Examples of minerals are monopotassium phosphate, potassium dihydrogenphosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, zinc sulfate, copper sulfate, calcium carbonate and the like. Also, if necessary, a defoaming agent may be used to prevent foaming during the incubation process. Moreover, cobalt ions or iron ions as prosthetic molecules of a nitrile hydratase, or nitriles and amides as an inducer of the enzyme, may also be added to the culture.

[0106]

Incubation may be conducted by adding selective pressure to prevent the vector and the target gene from being eliminated. Namely, if a selection marker is a drug-resistant gene, a corresponding chemical agent may be added; or if a selection marker is an auxotrophic complementary gene, corresponding nutrition factors may be removed.

Also, if a selection marker has a genetic assimilation trait, an equivalent assimilation factor may be added as a sole factor if necessary. For example, when E. coli transformed by a vector containing an ampicillin-resistant gene is incubated, -23 -ampicillin may be added as needed during the incubation process. 2015203203 15 Jun2015 [0107]

When incubating a transformant transformed by an expression vector containing an inducible promoter, such an inducer may be added to the culture if necessary. For example, when incubating a transformant transformed by an expression vector with a promoter inducible with i sopropyl-P-D-thiogalactopyrano side (IPTG), IPTG or the like may be added to the culture. Likewise, when incubating a transformant transformed by an expression vector with a trp promoter inducible with indoleacetic acid (IAA), IAA or the like may be added to the culture.

[0108]

Incubation conditions of a transformant are not limited specifically as long as the productivity of the target nitrile hydratase and growth of the host are not prohibited. Generally, conditions are preferred to be 10°C~40°C, more preferably 20°C~37°C, for 5—100 hours. The pH value is adjusted using inorganic or organic acid, alkaline solution or the like. If it is an E. coli, the pH is adjusted to be 6~9.

[0109]

As for incubation methods, solid-state culture, static culture, shaking culture, aeration-agitation culture and the like may be used. When an E. coli transformant is incubated, it is especially preferred to use shaking culture or aeration-agitation culture (jar fermentation) under aerobic conditions.

[0110]

When incubated in culture conditions above, the improved nitrile hydratase of the present invention is accumulated at a high yield in the above culture medium, namely, at least in any of culture supernatant, cell culture, bacterial-cell culture, cell homogenates or bacterial-cell homogenates.

[0111]

When an improved nitrile hydratase is incubated and produced in a cell or bacterial cell, the target nitrile hydratase is collected by homogenizing the cells or bacterial cells. Cells or bacterial cells are homogenized by high-pressure treatment using a French press or homogenizer, supersonic treatment, grinding treatment using glass beads or the like, enzyme treatment using lysozyme, cellulose, pectinase and the like, freezing and thawing treatment, hypotonic solution treatment, bacteriolysis induction treatment by phage, and so on.

[0112]

After the homogenization process, residues of cell homogenates or bacterial-cell homogenates (including insoluble fractions of the cell extract) are removed if necessary. To remove residues, centrifugal or filtration methods are employed. To increase the efficiency of removing residues, a coagulant or filter aid may be used. The supernatant obtained after the removal of residues is soluble fractions of the cell extract, which are used as a crudely purified improved nitrile hydratase solution.

[0113] -24-

Also, when an improved nitrile hydratase is produced in a bacterial cell or in cells, it is an option to collect the bacterial cell or the cells themselves by a centrifuge or membrane filtration and to use without homogenizing them. 2015203203 15 Jun2015 [0114]

When an improved nitrile hydratase is produced outside cells or bacterial cells, the culture may be used as is, or the cells or bacterial cells are removed using a centrifugal or filtration method. Then, the improved nitrile hydratase is collected from the culture by being extracted through ammonium sulfate precipitation, if necessary. Furthermore, dialysis or various chromatography techniques (gel filtration, ion exchange chromatography, affinity chromatography, etc.) may be used to isolate and purify the nitrile hydratase.

[0115]

To check the production yield of a nitrile hydratase obtained by incubating a transformant is not limited to using any specific method, but SDS-PAGE (polyacrylamide gel electrophoresis), nitrile hydratase activity measurements or the like may be used to determine the yield per culture, per wet or dry weight in a bacterial cell, or per crude enzymatic protein. SDS-PAGE may be conducted by a method well known by a person skilled in the art. Also, the activity described above may be applied to nitrile hydratase activity.

[0116]

Without using any living cells, an improved nitrile hydratase of the present invention may be produced using a cell-free protein synthesis system.

[0117]

In a cell-free protein synthesis system, a protein is produced in an artificial vessel such as a test tube using a cell extract. A cell-free protein synthesis system used in the present application includes a cell-free transcription system that synthesizes RNA using DNA as a template.

[0118]

In such a case, an organism corresponding to the above host is the organism from which the cell extract is derived. Here, for the cell extract, extracts of eukaryotic or prokaryotic origin, such as the extract from wheat germ, E. coli and the like, may be used. Such cell extracts may be concentrated or not.

[0119]

The cell extract is obtained by ultrafiltration, dialysis, polyethylene glycol (PEG) precipitation or the like. In the present invention, a commercially available kit may also be used for cell-free protein synthesis. Examples of such a kit are a reagent kit PROTEIOS™ (Toyobo), TNT™ system (Promega KK), a synthesizer PG-Mate™ (Toyobo), RTS (Roche Diagnostics) and the like.

[0120]

An improved nitrile hydratase obtained by cell-free protein synthesis as described above is also purified by properly selecting a chromatography type. -25-[0121] 2015203203 15 Jun2015 2. Method for Producing Amide Compound

The improved nitrile hydratase obtained above is used as an enzymatic catalyst for material production. For example, an amide compound is produced by bringing a nitrile compound into contact with the improved nitrile hydratase. Then, the amide compound produced upon contact is collected. Accordingly, an amide compound is produced.

[0122]

The isolated and purified nitrile hydratase as described above is used as an enzymatic catalyst. In addition, a gene is introduced so as to express an improved nitrile hydratase in a proper host as described above and the culture after the host is incubated or the processed products of the culture may also be used. Processed products are, for example, incubated cells immobilized with acrylamide gel or the like, those processed by glutaraldehyde, those supported by inorganic carriers such as alumina, silica, zeolite, diatomaceous earth and the like.

[0123]

Here, “contact” means that an improved nitrile hydratase and a nitrile compound are present in the same reaction system or incubation system: for example, an isolated and purified improved nitrile hydratase and a nitrile compound are mixed; a nitrile compound is added into a incubation vessel of a cell to express an improved nitrile hydratase gene; cells are incubated in the presence of a nitrile compound; a cell extract is mixed with a nitrile compound; and so on.

[0124] A nitrile compound as a substrate is selected by considering the substrate specificity of the enzyme, stability of the enzyme in the substrate and the like. As for a nitrile compound, acrylonitrile is preferred. The reaction method and the method for collecting an amide compound after the completion of reactions are properly selected depending on the characteristics of the substrate and the enzymatic catalyst.

[0125]

The enzymatic catalyst is preferred to be recycled as long as its activity is not deactivated. From the viewpoint of preventing deactivation and of recycling ease, the enzymatic catalyst is preferred to be used as a processed product.

EXAMPLES

[0126]

In the following, examples of the present invention are described in detail.

However, the present invention is not limited to those. Rhodococcus rhodocrous J1 strain is registered under accession number “FERM BP-1478” at the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6,1-1-1 Higashi, Tsukuba, Ibaraki), deposited September 18,1987.

[0127] [Preparation Example 1] -26-

Preparation of Plasmid pSJ034 2015203203 15 Jun2015

As a template to perform the amino-acid substitution of the present invention, plasmid pSJ034 (FIG. 1) having the nitrile hydratase gene of the J1 strain was produced by the following method.

[0128]

Plasmid pSJ034 is capable of expressing nitrile hydratase in a Rhodococcus strain. Plasmid pSJ034 was produced from pSJ023 by the method disclosed in JP publication H10-337185. Namely, partially cleaved at the Xbal site and ligated with the Sse8387I linker, plasmid pSJ033 was prepared so that one Xbal site of plasmid pSJ023 was substituted with Sse8387I. Next, plasmid pSJ033 was partially cleaved at the Sse8387I site, and a Klenow fragment was used to blunt the ends so as to cause self ligation. Accordingly, plasmid pSJ034 was obtained. Here, pSJ023 is a transformant “R- rhodochrous ATCC 12674/pSJ023,” and is internationally registered under accession number “FERM BP-6232” at the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6,1-1-1 Higashi, Tsukuba, Ibaraki), deposited March 4,1997.

[0129] [Preparation Example 2]

Preparation of Plasmid pFR005 (1) C onstruction of Mutant Gene Library

As for a template plasmid, pER855A (FIG. 5) was used, prepared by modifying plasmid pER855 (see JP publication 2010-172295) as follows: counted downstream from the N-terminal amino-acid residue of the amino-acid sequence (SEQ ID NO: 2) in the β subunit, an amino-acid residue at position 167 was mutated from asparagine (N) to serine (S); an amino-acid residue at position 219 was mutated from valine (V) to alanine (A); an amino-acid residue at position 57 was mutated from serine (S) to methionine (M); an amino-acid residue at position 114 was mutated from lysine (K) to tyrosine (Y); and an amino-acid residue at position 107 was mutated from threonine (T) to lysine (K).

[0130]

First, introduction of a mutation into the nitrile hydratase gene was conducted as follows: '<composition of PCR reaction mixture>

sterile water 20 p.T, pER855A(l ng/mL) 1 pL Forward primer (10 mM) 2 pL Reverse primer (10 mM) 2 pL PrimeSTAR MAX (2x) 25 uL total 50 pL <PCR reaction conditions> (98°C for 10 sec, 55°C for 5 sec, 72°C for 90 sec) x 30 cycles <primers> primers for saturation mutagenesis at β17 β17ΙΙΜ-Ρ: ggatacggaccggtcNNStatcagaaggacgag (SEQ ID NO: 63) βΠΙΙΜ-R: ctcgtccttctgataSNNgaccggtccgtatcc (SEQ ID NO: 64) -27-<reaction conditions> 2015203203 15 Jun2015 (94°C for 30 sec, 65°C for 30 sec, 72°C for 3 min) x 30 cycles [0131]

After the completion of PCR, 5 pL of the reaction mixture was provided for 0.7% agarose gel electrophoresis, an amplified fragment of 11 kb was confirmed, and 1 pL Dpnl (provided with the kit) was added to the PCR reaction mixture, which was then reacted at 37°C for an hour. Accordingly, the template plasmid was removed. After that, the reaction mixture was purified using Wizard SV Gel and PCR Clean-Up System (Promega KK), and transformation was introduced into JM109 using the purified PCR reaction product. A few thousand obtained colonies were collected from the plate, and plasmid DNA was extracted using QIAprep Spin Miniprep Kit (Qiagen) to construct a mutant gene library.

[0132] (2) Producing Rhodococcus Transformant

The cells of Rhodococcus rhodochrous strain ATCC 12674 at a logarithmic growth phase were collected by a centrifugal separator, washed with ice-cooled sterile water three times and suspended in the sterile water. Then, 1 pL of plasmid prepared in (2) above and 10 pL of the bacterial-cell suspension were mixed and ice-cooled. The plasmid DNA and the bacterial-cell suspension were supplied into a cuvette, and electric pulse treatment was conducted at 2.0 KV and 200 Ω using an electroporation device, Gene Pulser II (Bio-Rad Laboratories, Inc.).

[0133]

The cuvette with the mixture processed by electric pulse was let stand for 10 minutes under ice-cold conditions, and a heat-shock treatment was conducted at 37°C for 10 minutes. Then, 500 pL of an MYK culture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bacto malt extract, 0.2% K2HPO4, 0.2% KH2PO4) was added and let stand at 30°C for 5 hours, and the strain was then applied on an MYK agar medium containing 50 pg/mL kanamycin. The colony obtained after being incubated at 30°C for 3 days was used as a transformant. In the same manner, transformant pER S55A was prepared as a comparative strain.

[0134] (3) Amide Treatment on Rhodococcus Strain Transformant

The Rhodococcus transformant containing nitrile hydratase gene, obtained in (2) above and ATCC 12674/pER855A as a comparative strain were used for screening. In a 96-hole deep-well plate, 1 mL each of a GGPK culture medium (1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05% K2HPO4, 0.05% KH2P O4, 0.05% MgS04-7H20, 1% C0CI2, 0.1% urea, 50 pg/mL kanamycin, pH 7.2) was supplied. In each culture medium, the above strain was inoculated, and subjected to liquid culture at 30°C for 3 days.

[0135]

Next, 30 pL of the liquid culture obtained above was dispensed in a 96-hole plate and the culture medium was removed by centrifugation. Lastly, 40 pL of a 50% -28- acrylamide solution was added to suspend the bacteria. The transformant suspended in a high-concentration acrylamide solution was put in an incubator to completely deactivate the comparative strain through heat treatment conducted at 50°C for 30 minutes. The remaining nitrile hydratase activity was measured as follows. 2015203203 15 Jun2015 [0136]

First, after the acrylamide treatment, a transformant was washed with a 50 mM phosphate buffer (pH 7.0) and the activity was measured by the following method.

The washed transformant and 50 mM phosphate buffer (pH 7.0) were supplied to a test tube and preincubated at 30°C for 10 minutes, and an equivalent volume of a 5% acrylonitrile solution (pH 7.0) was added and reacted for 10 minutes. Then, one tenth volume of 1 M phosphoric acid was added to terminate the reaction. Next, the transformant was removed from the terminated reaction mixture by centrifugation, and the mixture was diluted to a proper concentration for analysis by HPLC (WAKOSIL 5C8 (Wako Pure Chemical Industries) 250 mm long, 10% acetonitrile containing 5 mM phosphoric acid, flow rate of mobile phase at 1 mL/min, wavelength of a UV absorption detector 260 nm). Using untreated cells for which acrylamide treatment was not conducted, activity was measured for comparison. Then, based on the obtained activity values, the remaining activity after acrylamide treatment was determined.

[0137]

Among hundreds of transformants containing a mutant nitrile hydratase gene obtained above, mutant enzyme pFR005 showing resistance to a high-concentration acrylamide was selected.

[0138] (4) Confirming Base Sequence

To confirm the base sequence of the nitrile hydratase gene, plasmid was recovered from the selected strains. Rhodococcus transformants were inoculated in 10 mL of an MYK culture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% malt extract, 1% glucose, 50 pg/mL kanamycin) and incubated for 24 hours, and a 20% sterile glycine solution was added to make the final concentration of 2%, and further incubated for another 24 hours. Then, the bacterial cells were recovered by centrifugation, washed with a TES buffer (10 mM Tris-HCl (pH 8)-10 mM NaCl-1 mM EDTA), suspended in 2 mL of 50 mM Tris-HCl (pH8)-12.5% sucrose-100 mM NaCl-1 mg/mL lysozyme, and subjected to shaking culture at 37°C for 3 hours. Then, 0.4 mL of 10% SDS was added and the mixture was shaken gently for an hour at room temperature, to which 2.1 mL of 5 M sodium acetate buffer (pH 5.2) was added and let stand in ice for an hour. Next, the mixture was centrifuged for an hour at 10,000 xg at 4°C to obtain a supernatant, to which a 5-times volume ethanol was added and let stand at -20°C for 30 minutes. Then, the mixture was centrifuged at 10,000 xg for 20 minutes. The precipitate was washed with 10 mL of 70% ethanol and dissolved in 100 pL of a TE buffer. Accordingly, a DNA solution was obtained, [0139]

Next, the sequence including nitrile hydratase was amplified by a PCR method. <composition of PCR reaction mixture> template plasmid 1 pL -29-

10 x PCR buffer (made by NEB) 10 pL 2015203203 15 Jun2015

primer NH-19 (50 μΜ) 1 μι primer NH-20 (50 μΜ) 1 pL 2.5 mM dNTPmix 8 pL sterile water 79 pL

Taq DNA polymerase (made by NEB) 1 pL <primers> NH-19: GCCTCTAGATATCGCCATTCCGTTGCCGG (SEQ ID NO: 65) NH-20: ACCCTGCAGGCTCGGCGCACCGGATGCCCAC (SEQ ID NO: 66) <reaction conditions> (94°C for 30 sec, 65 °C for 30 sec, 72°C for 3 min) χ 30 cycles [0140]

After completion of PCR, 5 pL of the reaction mixture was subjected to 0.7% agarose gel electrophoresis to detect a 2.5 kb PCR amplified product. After Exo-SAP treatment (Amersham Pharmacia Biotech) on the PCR reaction mixture, samples for alignment analysis were prepared by a cycle sequencing method, and were analyzed using CEQ-2000XL (Beckman Coulter). As a result, the mutation positions of pFR005 were confirmed to be Npl67S, νβ219Α, Sp57M, Κβ114Υ, Τβ107Κ and Ppl7G. Namely, in plasmid pFR005, proline at position 17 in the β subunit was mutated to glycine, serine at position 57 in the β subunit was mutated to lysine, tyrosine at position 107 in the β subunit was mutated to lysine, lysine at position 114 in the β subunit was mutated to tyrosine, asparagine at position 167 in the β subunit was mutated to serine, and valine at position 219 in the β subunit was mutated to alanine. EXAMPLE 1 [0141]

Preparation of Improved Nitrile Hydratase

Using pSJ034 formed in preparation example 1, amino-acid substitution was conducted. The following composition of a reaction mixture, reaction conditions and primers were used for the PCR.

Composition of PCR reaction mixture>

sterile water 20 pL pSJ034 (1 ng/mL) 1 pL Forward primer (10 mM) 2 pL Reverse primer (10 mM) 2 pL PrimeSTAR MAX (2^ 25 uL total 50 pL <PCR reaction conditions> (98°C for 10 sec, 55°C for 5 sec, 72°C for 90 sec) χ 30 cycles <primers> [0142]

Table 5 -30- 2015203203 15 Jun2015 substituted amino acid name of primer sequence SEQ ID NO C 348C-F TCGTGGTGCGACMGTCGCGGTTCTTC 13 348C-R CTT GTCG CACCAC G ATAT G CC CTT GAG 14 D 348D-F TCGTGGGACGACAAGTCGCGGTTCTTC 15 348D-R CTTGTCGTCCCACGATATGCCCTTGAG 16 E 348E-F TCGTGGGAGGACAAGTCGCGGTTCTTC 17 348E-R CTTGTCCTCCCACGATATGCCCTTGAG 18 H 348H-F TCGTGGCACGACAAGTCGCGGTTCTTC 19 343H-R CTTGTCGTGCCACGATATGCCCTTGAG 20 1 3481-F TCGTGGATCGACAAGTCGCGGTTCTTC 21 3481-R CTTGTCGATCCACGATATGCCCTTGAG 22 K B48K-F TCGTGGAAGGACAAGTCGCGGTTCTTC 23 B48K-R CTTGTCCTTCCACGATATGCCCTTGAG 24 M 348M-F TCGTGGATGGACAAGTCGCGGTTCTTC 25 B48M-R CTTGTCCATCCACGATATGCCCTTGAG 26 N 348N-F TCGTGGAAGGACAAGTCGCGGTTCTTC 27 348N-R CTTGTCGTTCCACGATATGCCCTTGAG 28 P 348P-F TCGTGGCCGGACAAGTCGCGGTTCTTC 29 348P-R CTTGTCCGGCCACGATATGCCCTTGAG 30 Q 348Q-F TCGTGGCAGGACAAGTCGCGGTTCTTC 31 348Q-R CTTGTCCTGCCACGATATGCCCTTGAG 32 S 348S-F rTCGTGGTCCGACAAGTCGCGGTTCTTC 33 348S-R CTTGTCGGACCACGATATGCCCTTGAG 34 T 348T-F TCGTGGACCGACAAGTCGCGGTTCTTC 35 348T-R CTTGTCGGTCCACGATATGCCCTTGAG 36 [0143]

After the completion of PCR, 5 pL of the reaction mixture was subjected to 0.7% agarose gel electrophoresis and an 11 -kb PCR amplified product was detected. Then, 1 pL of Dpnl (provided in the kit) was added to the PCR reaction mixture and reacted at 37“C for an hour to remove the template plasmid. After the reaction was completed, the reaction mixture was purified using Wizard SV Gel and PCR Clean-Up System (made by Promega KK), and the purified PCR product was used to transform JM109. From the obtained culture, plasmid DNA was extracted using QIAprep Spin Miniprep Kit (made by Qiagen), and the base sequence of the nitrile hydratase was confirmed using automated sequencer CEQ 8000 (made by Beckman Coulter, Inc.). Obtained plasmids were named as follows.

[0144] Table 6

name of plasmid amino-acid substitution PSJ102 WB48C PSJ103 WB48D PSJ104 W348E PSJ107 W348H PSJ108 W348! pSJ109 W348K PSJ111 W348M PSJ112 W348N PSJ113 W348P PSJ114 WB48Q PSJ116 WB48S pSJ117 W348T -31 - EXAMPLE 2 [0145] 2015203203 15 Jun2015

Preparation of Rhodococcus Transformant

Cells of Rhodococcus rhodocrous strain ATCC 12674 in a logarithmic growth phase were collected using a centrifuge, washed three times with ice-cold sterile water, and suspended in the sterile water. Then, 1 pL of plasmid prepared in example 1 and 10 pL of the bacterial-cell suspension were mixed and ice-cooled. The DNA and the bacterial-cell suspension were supplied in a cuvette, and electric pulse treatment was conducted using an electroporation device, Gene Pulser (Bio-Rad Laboratories), under conditions of 2.0 kV and 200 Ω. The electric-pulse processed mixture was let stand in an ice-cold condition for 10 minutes, and subjected to heat shock at 37°C for 10 minutes. After 500 pL of an MYK culture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bacto malt extract, 0.2% K2HPO4, 0.2% KH2PO4) was added and let stand at 30°C for 5 hours, the strain was applied onto an MYK agar culture medium containing 50 pg/mL kanamycin and incubated at 30°C for 3 days. The obtained colony after incubating at 30°C for 3 days was used as a transformant.

[0146]

Each transformant obtained above was inoculated into an MYK culture medium (50 pg/mL kanamycin), and subjected to shaking culture at 30°C for 2days. Then, 1% culture was inoculated into a GGPK culture medium (1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05%o K2HPO4, 0.05% KH2P O4, 0.05% Mg204-7H20, 1% CoCl2, 0.1% urea, 50 pg/mL kanamycin, pH 7.2), and subjected to shaking culture at 30°C for 3 days. Bacterial cells were collected by using a centrifuge, and were washed with a 100 mM phosphate buffer (pH 7.0) to prepare a bacterial-cell suspension. EXAMPLE 3 [0147]

Improved Nitrile Hydratase Activity

The nitrile hydratase activity in the obtained bacterial-cell suspension was measured by the following method: 0.2 mL of the bacterial-cell mixture and 4.8 mL of a 50 mM phosphate buffer (pH 7.0) were mixed, to which 5 mL of a 50 mM phosphate buffer (pH 7.0) containing 5.0% (w/v) acrylonitrile was further added. Next, the mixture was reacted while being shaken at 10°C for 10 minutes. Then, bacterial cells were filtered and the amount of produced acrylamide was determined using gas chromatography. <analysis conditions> analysis instrument: detector: column: column temperature: gas chromatograph GC-14B (Shimadzu Corporation) FID (detection at 200°C) lm glass column filled with PoraPak PS (column filler made by Waters Corp.) 190°C [0148]

Nitrile hydratase activity was determined by conversion from the amount of acrylamide. Here, regarding nitrile hydratase activity, the amount of enzyme to produce 1 pmol of acrylamide per 1 minute is set as 1 U. Table 7 shows relative -32-activities when the parent strain activity without amino-acid substitution was set at 1.0.

[0149] Table 7

Vleasurement result ts of catalytic activity amino-acid substitution name of plasmid catalytic activity (relative value) none (parent strain) PSJ034 1.0 (comp, example) W348D PSJ103 1.2 W348E PSJ104 1.6 W648K PSJ109 1.1 W848M PSJ111 3.1 WI348N pSJ112 1.8 W848P pSJ113 2.0 W|348S PSJ116 1.1 W348T PSJ117 1.3 2015203203 15 Jun2015 [0150]

From the results above, enhanced enzymatic activity was confirmed in the improved nitrile hydratase in which an amino acid at position 48 in the β subunit was substituted with aspartic acid, lysine, asparagine, proline, serine or threonine. EXAMPLE 4 [0151]

Preparation and Evaluation of Improved Nitrile Hydratase

Plasmid pFR005 formed in preparation example 2 as a template plasmid was used to substitute an amino acid at position 48 of the β subunit.

[0152]

Namely, using the method in example 1, each of the improved nitrile hydratases with a substituted amino acid were prepared, and a transformant was obtained by the method in example 2. Further, the enzymatic activity was measured by the same method in example 3. The results are shown in Table 8.

[0153]

Table 8

Vleasurement results of catalytic activity name of plasmid amino-acid substitution catalytic activity (relative value) pFR005 Ρβ176, Sp57K, Τβ107Κ, Κβ114Υ, N8167S. V3219A 1.0 (comp, example) pER11Q2 Pp17G, 5β57Κ, Τβ107Κ, Κβ114Υ, N3167S, V3219A. WB48C 1.6 pER1103 PP17G, 5β57Κ, Τβ107Κ, Κβ114Υ, N3167S, V3219A. W348D 1.7 pER1104 Pp17G, S357K, Τβ107Κ, Κβ114Υ, N3167S. V3219A, WB48E 1.3 PER1107 PP17G, SP57K, Τβ107Κ, Κβ114Υ, N3167S, V3219A. W348H 1.2 pER1108 P317G, SP57K, Τβ107Κ, Κβ114Υ, N3167S, VB219A. W348I 1.6 PER1109 P317G, Sp57K, Τβ107Κ, Κβ114Υ, N3167S, V3219A. W348K 1.4 PER1112 P317G, 5β57Κ, Τβ107Κ, Κβ114Υ, N3167S, V3219A. WB48M 3.7 -33- pER1113 Pp17G, Sp57K, Τβ107Κ, Κβ114Υ, N3167S, V3219A. WB48N 1.7 pER1114 Ρβ17Θ, Sp57K, Τβ107Κ, Κβ114Υ, NP167S, V3219A. WB48P 1.7 pER1116 PJ317G, 3β57Κ, Τβ107Κ, Κβ114Υ, N3167S. νβ219Α. WB48Q 1.9 pER1117 Pp17Gt 3β57Κ,Τβ107Κ, Κβ114Υ, Νβ1673, VB219A. WB48S 1.8 PER1119 PB17G. SP57K, Τβ107Κ, Κβ114Υ, N3167S. VB219A. W348T 1.1 [0154] 2015203203 15 Jun2015

From the results above, the same enzymatic activity was confirmed in the mutant nitrile hydratase when the amino acid at X4 (corresponding to an amino acid at position 48 in the β subunit) in the amino-acid sequence shown in SEQ ID NO: 50 was substituted with an amino acid selected from among cysteine, glutamic acid, aspartic acid, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, serine and threonine, EXAMPLE 5 [0155] SDS-Polyacrylamide Gel Electrophoresis

Using a sonicator VP-300 (TAITEC Corporation), the bacterial-cell suspension prepared in example 2 was homogenized for 10 minutes while being ice-cooled. Next, the bacterial-cell homogenate was centrifuged at 13500 rpm for 30 minutes and a cell-free extract was obtained from the supernatant. After the protein content of the cell extract was measured using a Bio-Rad protein assay kit, the cell extract was mixed with a polyacrylamide gel electrophoresis sample buffer (0.1 M Tris-HCl (pH 6.8), 4% w/v SDS, 12% v/v β mercaptoethanol, 20% v/v glycerol, and a trace ofbromophenol blue), and boiled for 5 minutes for denaturation. A 10% acrylamide gel was prepared and denatured samples were applied to have an equivalent protein mass per one lane to conduct electrophoresis analysis (FIG. 4).

[0156]

As a result, since hardly any difference was observed in the band strength of nitrile hydratase in all the samples, the expressed amount of nitrile hydratase was found to be the same. Accordingly, the enzymatic specific activity was found to be attributed to the improved enzymatic activity. EXAMPLE 6 [0157]

Preparation of Transformant Containing Nitrile Hydratase Derived from Rhodococcus Rhodocrous M8 Strain (hereinafter referred to as M8 strain) (1) Preparation of Chromosomal DNA from M8 Strain

The M8 strain (SU 1731814) is obtained from the Russian Institute of Microorganism Biochemistry and Physiology (VKPM S-926). In 100 mL of an MYK culture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3%» Bacto malt extract, 0.2% K2HPO4, 0.2% KH2PO4, pH 7.0), the M8 strain was subjected to shaking culture at 30°C for 72 hours. The culture mixture was centrifuged, and the collected bacterial cells were suspended in 4 mL of a Saline-EDTA solution (0.1 M EDTA, 0.15 M NaCl, -34-pH 8.0). Then, 8 mg of lysozyme was added to the suspension, which was shaken at 37°C for 1~2 hours and was frozen at -20°C. 2015203203 15 Jun2015 [0158]

Next, 10 mL of Tris-SDS solution (1% SDS, 0.1M NaCl, 0.1 M Tris-HCl (pH 9.0)) was added to the suspension while the suspension was gently shaken. Proteinase K (Merck KGaA) was further added (final concentration of 0.1 mg) and shaken at 37°C for 1 hour. Next, an equivalent volume of TE saturated phenol was added, agitated (TE: 10 mM Tris-HCl, 1 mM EDTA (pH 8.0)) and then centrifuged. The supernatant was collected and a double volume of ethanol was added and DNA strands were wrapped around a glass rod. Then, the phenol was removed through centrifugation by successively adding 90%, 80%, and 70% ethanol.

[0159]

Next, the DNA was dissolved in a 3 mL TE buffer, to which a Ribonuclease A solution (processed at 100°C for 15 minutes) was added to have a 10 pg/rnL concentration and shaken at 37°C for 30 minutes. Proteinase K (Merck KGaA) was further added and shaken at 37°C for 30 minutes. After an equivalent volume of TE saturated phenol was added and centrifuged, the mixture was separated into upper and lower layers.

[0160]

An equivalent volume of TE saturated phenol was further added to the upper layer and centrifuged to separate into upper and lower layers. Such a process was repeated. Then, an equivalent volume of chloroform (containing 4% isoamyl alcohol) was added, centrifuged and the upper layer was collected. Then, a double volume of ethanol was added to the upper layer and the DNA strands were collected by wrapping them around a glass rod. Accordingly, chromosomal DNA was obtained.

[0161] (2) Using PCR, Preparation of Improved Nitrile Hydratase from Chromosomal DNA Derived from M8 Strain

The nitrile hydratase derived from the M8 strain is described in a non-patent publication (Veiko, V.P. et al., “Cloning, Nucleotide Sequence of Nitrile Hydratase Gene from Rhodococcus rhodochrous M8,” Russian Biotechnology (Mosc.) 5, 3-5 (1995)). The sequences of β subunit, a subunit and activator are respectively identified in SEQ ID NOs: 37, 38 and 39. Based on the sequence information, primers of SEQ ID numbers 40 and 41 in the sequence listing were synthesized and PCR was performed using the chromosomal DNA prepared in step (1) above as a template.

[0162] <composition of PCR reaction mixture>

sterile water 20 pL template DNA (chromosomal DNA) 1 pL primer M8-1 (10 mM) , 2pL primer M8-2 (10 mM) 2 pL PrimeSTAR MAX (2*)_25 uL total 50 pL -35 - [0163] 2015203203 15 Jun2015 <primers> M8-1: GGTCTAGAATGGATGGTATCCACGACACAGGC (SEQ ID NO: 40) M8-2: cccctgcaggtcagtcgatgatggccatcgattc (SEQ ID NO: 41) [0164] creaction conditions> (98°C for 10 sec, 55°C for 5 sec, 72°C for 30 sec) χ 30 cycles [0165]

After the completion of PCR, 5 pL of the reaction mixture was subjected to 0.7% agarose gel electrophoresis (0.7 wt.% Agarose I, made by Dojin Chemical Co., Ltd.) and an amplified fragment of 1.6 kb was detected. The reacted mixture was purified using Wizard SV gel and PCR Clean-Up System (Promega KK).

[0166]

Next, the collected PCR product was coupled with a vector (pUC118/Hinc II site) using a ligation kit (made by Takara Shuzo Co., Ltd.) so that competent cells of E. coli JM109 were transformed using the reaction mixture. A few clones from the obtained transformant colony were inoculated into 1.5 mL of an LB-Amp culture medium, and incubated at 37°C for 12 hours while being shaken. After incubation was finished, the bacterial cells were collected from the culture through centrifugation. Plasmid DNA was extracted from the collected bacterial cells using QIAprep Spin Miniprep Kit (Qiagen). The base sequence of nitrile hydratase in the obtained plasmid DNA was confirmed using a sequencing kit and automated sequencer CEQ 8000 (Beckman Coulter, Inc.) (SEQ ID NO: 62).

[0167]

Next, the obtained plasmid DNA was cleaved with restriction enzymes Xbal and Sse8387I, and subjected to 0.7% agarose gel electrophoresis so as to collect a nitrile hydratase gene fragment (1.6 kb), which was then inserted into XbaI-Sse8387I site of plasmid pSJ042. The obtained plasmid was named pSJ-ΝΟΙΑ. Here, pSJ042 as a plasmid capable of expressing nitrile hydratase in Rhodococcus J1 strain was prepared by a method described in JP publication 2008-154552 (the content is incorporated in this application by reference). Plasmid pSJ023 used for preparation of pSJ042 is registered as transformant ATCC 12674/pSJ023 (FERM BP-6232) at the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6,1-1-1 Higashi, Tsukuba, Ibaraki), deposited March 4,1997. EXAMPLE 7 [0168]

Preparation and Evaluation of Improved Nitrile Hydratase

Using plasmid pSJ-NOl A obtained in example 6, the amino acid at position 48 of the β subunit was substituted. The same method in example 1 was employed for amino-acid substitution to prepare an improved nitrile hydratase. Next, using the same method in example 3, a transformant of Rhodococcus rhodocrous ATCC 12674 strain and its bacterial-cell suspension were prepared. Then, the enzymatic activity was -36-measured by the same method as in example 4. The results are shown in Table 9. 2015203203 15 Jun2015 [0169]

Table 9

Measurement results of catalytic activity name of plasmid amino-acid substitution catalytic activity (relative value) PSJ-N01A none (parent strain) 1.0 (comp, example) PSJR13 WB48M 2.4 PSJR21 W348N 2.3 [0170]

From the results in table 9, when the amino acid at position 48 of the β subunit was substituted, the enzymatic activity of the improved nitrile hydratase was confirmed to be enhanced the same as in example 3. EXAMPLE 8 [0171]

Preparation of Improved Nitrile Hydratase [0172]

Using plasmid pSJ034 formed in preparation example 1, amino-acid substitution was conducted. The following composition of reaction mixture, reaction conditions and primers shown in table 2 were used for the PCR.

Composition of PCR reaction mixture> sterile water 20 μι

pSJ034 (1 ng/mL) 1 pL

Forward primer (10 mM) 2 pL

Reverse primer (10 mM) 2 pL

PrimeSTAR MAX (2*1 25 uL total . 50 pL <PCR reaction conditions> (98°C for 10 sec, 55°C for 5 sec, 72°C for 90 sec) x 30 cycles <primers> [0173] Table 10 substituted amino acid name of primer sequence SEQ ID NO A P37A-F GT C AATT GC G ACTT G G ATGC AT CTCAAG 67 P37A-R CCAAGTCGCAATTGACAGGGTCCGACC 68 D I337D-F GT C AATT GACACTTGGATGCATCT CAAG 69 (337D-R CCAAGT GT CAATT GACAGG GTCC GACC 70 F P37F-F GTCAATTTTCACTTGGATGCATCTCAAG 71 P37F-R CCAAGTGAAAATTGACAGGGTCCGACC 72 i P37I-F GTCAATTATCACTTGGATG CATCTCAAG 73 -37 3371-R CCAAGTGATAATTGACAGGGTCCGACC 74 M (337M-F GTCAATTATGACTTGGATGCATCTCAAG 75 337M-R CCAAGTCATAATTGACAGGGTCCGACC 76 T 337T-F GTCAATTACCACTTGGATGCATCTCAAG 77 337T-R CCAAGTGGTAATTGACAGGGTCCGACC 78 V 337V-F GTCAATTGTCACTTGGATGCATCTCAAG 79 337V-R CCAAGTGACAATTGACAGGGTCCGACC 80 [0174] 2015203203 15 Jun2015

After the completion of PCR, 5 pL of the reaction mixture was subjected to 0.7% agarose gel electrophoresis and an amplified fragment of 1 kb was confirmed. Then, 1 pL of Dpnl (provided with a kit) was added to the PCR reaction mixture and reacted at 37°C for an hour to remove the template plasmid. The reacted mixture was purified using Wizard SV gel and PCR Clean-Up System (Promega), and JM109 was transformed using the purified PCR reaction product. Then, a plasmid DNA was extracted from the obtained culture using QIAprep Spin Miniprep Kit (Qiagen), and the base sequence of the nitrile hydratase was confirmed using an automated sequencer CEQ 8000 (Beckman Coulter, Inc.) Obtained plasmids were named as shown in Table 11.

[0175]

Table 11

name of plasmid amino-acid substitution PSJ120 LP37A PSJ122 L337D pSJ124 L337F PSJ127 L337I pSJ129 L337L PSJ130 L337M PSJ136 L337T PSJ137 L337V EXAMPLE 9 [0176]

Preparation of Rhodococcus Transformant [0177]

Cells of Rhodococcus rhodocrous ATCC 12674 strain in a logarithmic growth phase were collected using a centrifuge, washed three times with ice-cold sterile water, and suspended in the sterile water. Then, 1 pL of plasmid prepared in example 1 and 10 pL of the bacterial-cell suspension were mixed and ice-cooled. The DNA and the bacterial-cell suspension were supplied in a cuvette, and electric pulse treatment was conducted using an electroporation device, Gene Pulser (Bio-Rad Laboratories), under conditions of 2.0 kV and 200 Ω. The electric-pulse processed mixture was let stand in an ice-cold condition for 10 minutes, and subjected to heat shock at 37°C for 10 minutes. After 500 pL of an MYK culture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bacto malt extract, 0.2% K2HPO4, 0.2% KH2PO4) was added and let stand at 30°C for 5 hours, and applied onto an MYK agar culture medium containing 50 pg/mL kanamycin and incubated at 30°C for 3 days. The obtained colony after incubating at 30°C for 3 days was used as a transformant. -38-[0178] 2015203203 15 Jun2015

Each transformant obtained above was inoculated into an MYK culture medium (50 pg/mL kanamycin), and subjected to shaking culture at 30‘C for 2 days. Then, 1% culture was each inoculated into a GGPK culture medium (1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05% K2HP04, 0.05% KH2P04, 0.05% Mg204-7H20, 1% CoCl2, 0.1% urea, 50 μg/mL kanamycin, pH 7.2), and shaking culture was performed at 30°C for 3 days. Bacterial cells were collected by using a centrifuge and were washed with a 100 mM phosphate buffer (pH 7.0) to prepare a bacterial-cell suspension. EXAMPLE 10 [0179]

Improved Nitrile Hydratase Activity [0180]

The nitrile hydratase activity in the obtained bacterial-cell suspension was measured by the following method: 0.2 mL of the bacterial-cell mixture and 4.8 mL of a 50 mM phosphate buffer (pH 7.0) were mixed, to which 5 mL of a 50 mM phosphate buffer (pH 7.0) containing 5.0% (w/v) acrylonitrile was further added. Next, the mixture was reacted while being shaken at 10°C for 10 minutes. Then, bacterial cells were filtered and the amount of produced acrylamide was determined using gas chromatography. <analysis conditions> analysis instrument: gas chromatograph GC-14D (Shimadzu Corporation) detector: FID (detection at 200°C) column: 1 m glass column filled with PoraPak PS (column filler made by Waters Corp.)

column temperature: 190°C

[0181]

Nitrile hydratase activity was determined by conversion from the amount of acrylamide. Here, regarding nitrile hydratase activity, the amount of enzyme to produce 1 umol of acrylamide per 1 minute is set as 1 U. Table 12 shows relative activities when the parent strain activity without amino-acid substitution was set at 1.0.

[0182] Table 12 amino-acid substitution name of plasmid catalytic activity (relative value! none (parent strain! pSJ034 1.0 (comp, example! L337A PSJ120 1.3 L337D PSJ122 1.5 L337F PSJ124 1.2 L337J PSJ127 1,2 L337M pSJ130 1.2 L337T PSJ136 1.2 LB37V PSJ137 1.3 [0183] -39-

From the results above, enhanced enzymatic activity was confirmed in the enzyme in which an amino acid at position 37 in the β subunit was substituted with an amino acid selected from among alanine, valine, asparagine, threonine, phenylalanine, isoleucine and methionine. 2015203203 15 Jun2015 EXAMPLE 11 [0184] SDS-Polyacrylamide Gel Electrophoresis [0185]

Using a sonicator VP-300 (TAITEC Corporation), the bacterial-cell suspension prepared in example 2 was homogenized for 10 minutes while it was ice-cooled. Next, the bacterial-cell homogenate was centrifuged at 13500 rpm for 30 minutes and a cell-free extract was obtained from the supernatant. After the protein content of the cell extract was measured using a Bio-Rad protein assay kit, the cell extract was mixed with a polyacrylamide gel electrophoresis sample buffer (0.1 M Tris-HCl (pH 6.8), 4% w/v of SDS, 12% v/v of β mercaptoethanol, 20% v/v of glycerol, and a trace of bromophenol blue), and boiled for 5 minutes for denaturation. A10% acrylamide gel was prepared, and denatured samples were applied to have an equivalent protein mass per one lane to conduct electrophoresis analysis.

[0186]

As a result, since hardly any difference was observed in the band strength of nitrile hydratase in all the samples, the expressed amount of nitrile hydratase was found the same. Accordingly, enzymatic specific activity was found to be attributed to be the improved enzymatic activity. EXAMPLE 12 [0187]

Preparation and Evaluation of Improved Nitrile Hydratase

Plasmid pFROOS below was used as a template plasmid substitute an amino acid at position 37 of the β subunit.

[0188]

Namely, using the method in example 1, an improved nitrile hydratase with a substituted amino acid was prepared, and a transformant of Rhodococcus rhodocrous ATCC 12674 strain and its bacterial-cell suspension were obtained by the method in example 2. Further, the enzymatic activity was measured by the same method in example 3. The results are shown in Table 13.

[0189] Table 13 name of plasmid amino-acid substitution catalytic activity (relative value) pFROOS PP17G, SP57K, Np167S, Τβ107Κ, Κβ114Υ, VP219A 1.0 (comp, example) PER1121 PP17G, SJ357K, Νβ1675, Τβ107Κ, KP114Y, VP219A, LP37A 1.6 pER1140 Pp17G, Sp57K, Νβ1675, Τβ107Κ, KP114Y. VP219A. LB37D 1.3 -40-[0190] 2015203203 15 Jun2015

From the results above, the amino-acid substitution according to the present invention applies not only to a wild-type nitrile hydratase but to a mutant nitrile hydratase to exhibit the same effects. EXAMPLE 13 [0191]

Preparation of Improved Nitrile Hydratase [0192]

Using pSJ034 formed in preparation example 1, amino-acid substitution was conducted. The following composition of a reaction mixture, reaction conditions and primers shown in Table 14 were used for the PCR. <composition of PCR reaction mixture>

sterile water 20 pL pSJ034 (1 ng/mL) 1 pL Forward primer (10 mM) 2 pL Reverse primer (10 mM) 2 pL PrimeSTAR MAX (2x) 25 uL total 50 pL <PCR reaction conditions> (98°C for 10 sec, 55°C for 5 sec, 72°C for 90 sec) x 30 cycles <primers> [0193] Table 14 substituted amino acid name of primer Sequence SEQ ID NO A a83A-F GGTGAGGCGGCACACCAAATTTCGGCG 83 a83A-R GTGTGCCGCCTCACCGGCATAGCCC 84 C a83C-F GGTGAGTGCGCACACCAAATTTCGGCG 85 a83C-R GTGTGCGCACTCACCGGCATAGCCC 86 D a83D-F GGTGAGGACGCACACCAAATTTCGGCG 87 a83D-R GTGTGCGTCCTCACCGGCATAGCCC 88 E a83E-F GGTGAGGAGGCACACCAAATTTCGGCG 89 a83E-R GTGTGCCTCCTCACCGGCATAGCCC 90 F a83F-F GGTGAGTTCGCACACCAAATTTCGGCG 91 a83F-R GTGTGCGAACTCACCGGCATAGCCC 92 G a83G-F GGTGAGGGCGCACACCAAATTTCGGCG 93 a83G-R GTGTGCGCCCTCACCGGCATAGCCC 94 H a83H-F GGTGAGCACGCACACCAAATTTCGGCG 95 083H-R GTGTGCGTGCTCACCGGCATAGCCC 96 M a83M-F GGTGAGATGGCACACCAAATTTCGGCG 97 a83M-R GTGTGCCATCTCACCGGCATAGCCC 98 P a83P-F GGTGAGCCGGCACACCAAATTTCGGCG 99 a83P-R GTGTGCCGGCTCACCGGCATAGCCC 100 S a83S-F G GTGAGTC C GCACAC CAAATTT CG G C G 101 ct83S-R GTGTGCGGACTCACCGGCATAGCCC 102 T a83T-F G GT G AG ACCGC AC ACCAAATTTCGGCG 103 a83T-R GTGTGCGGTCTCACCGGCATAGCCC 104 -41 - [0194] 2015203203 15 Jun2015

After the completion of PCR, 5 pL of the reaction mixture was subjected to 0.7% agarose gel electrophoresis and an amplified fragment of 11 kb was confirmed. Then, 1 pL of Dpnl (provided with a kit) was added to the PCR reaction mixture and reacted at 37°C for an hour to remove the template plasmid. The reacted mixture was purified using Wizard SV gel and PCR Clean-Up System (Promega), and JM109 was transformed using the purified PCR reaction product. Then, a plasmid DNA was extracted from the obtained culture using QIAprep Spin Miniprep Kit (Qiagen), and the base sequence of the nitrile hydratase was confirmed using an automated sequencer CEQ 8000 (Beckman Coulter, Inc.) Obtained plasmids were named as shown in Table 15.

[0195]

Table 15

name of plasmid amino-acid substitution PSJ127 Q<x83A PSJ152 Qa83C pSJ153 Qa83D PSJ154 Qa83E PSJ155 Qa83F PSJ156 Qa83G PSJ157 Qa83H PSJ130 Qa83M PSJ132 Qa83N PSJ159 Qa83P PSJ161 Qa83S PSJ162 Q«83T EXAMPLE 14 [0196]

Preparation of Rhodococcus Transformant [0197]

Cells of Rhodococcus rhodocrous strain ATCC 12674 in a logarithmic growth phase were collected using a centrifuge, washed three times with ice-cold sterile water, and suspended in the sterile water. Then, 1 pL of plasmid prepared in example 1 and 10 pL of the bacterial-cell suspension were mixed and ice-cooled. The DNA and the bacterial-cell suspension were supplied in a cuvette, and electric pulse treatment was conducted using an electroporation device, Gene Pulser (Bio-Rad Laboratories), under conditions of 2.0 kV and 200 Ω. The electric-pulse processed mixture was let stand in an ice-cold condition for 10 minutes, and subjected to heat shock at 37°C for 10 minutes. After 500 pL of an MYK culture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bacto malt extract, 0.2% K2HPO4, 0.2% KH2PO4) was added and let stand at 30°C for 5 hours, and applied onto an MYK agar culture medium containing 50 pg/mL kanamycin and incubated at 30°C for 3 days. The obtained colony after incubating at 30°C for 3 days was used as a transformant.

[0198]

Each transformant obtained above were inoculated into an MYK culture medium -42-(50 pg/mL kanamycin), and subjected to shaking culture at 30°C for 2 days. Then, 1% culture was inoculated into a GGPK culture medium (1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05% K2HP04, 0.05% KH2P 04, 0.05% Mg204-7H20, 1% CoCl2,0.1% urea, 50 pg/mL kanamycin, pH 7.2), and shaking culture was performed at 30°C for 3 days. Then, bacterial cells were collected by using a centrifuge and were washed with a 100 mM phosphate buffer (pH 7.0) to prepare a bacterial-cell suspension. 2015203203 15Jun2015 EXAMPLE 15 [0199]

Improved Nitrile Hydratase Activity

The nitrile hydratase activity in the obtained bacterial-cell suspension was measured by the following method: 0.2 mL of the bacterial-cell mixture and 4.8 mL of a 50 mM phosphate buffer (pH 7.0) were mixed, to which 5 mL of a 50 mM phosphate buffer (pH 7.0) containing 5.0% (w/v) acrylonitrile was further added. Next, the mixture was reacted while being shaken at 10°C for 10 minutes. Then, bacterial cells were filtered and the amount of produced acrylamide was determined using gas chromatography. <analysis conditions> analysis instrument: detector: column: column temperature: gas chromatograph GC-14B (Shimadzu Corporation) FID (detection at 200°C) lm glass column filled with PoraPak PS (column filler made by Waters Corp.)

190°C

[0201]

Nitrile hydratase activity was determined by conversion from the amount of acrylamide. Here, regarding nitrile hydratase activity, the amount of enzyme to produce 1 pmol of acrylamide per 1 minute is set as 1 U. Table 16 shows relative activities when the parent strain activity without amino-acid substitution was set at 1.0.

[0202]

Table 16 name of plasmid amino-acid substitution catalytic activity (relative value) PSJ034 none (parent strain) 1.0 (comp, example) PSJ127 Qa83A 5.3 PSJ152 Qa83C 3.7 PSJ153 Qa83D 1.9 PSJ154 Qa83E 1.2 PSJ155 Qa83F 1.8 PSJ156 Qa83G 4.4 PSJ157 Qa83H 1.9 PSJ130 Qa83M 2.3 PSJ132 Qa83N 5.7 PSJ159 Qa83P 1.5 PSJ161 Qa83S 5.8 PSJ162 Q«83T 3.8 -43-[0203] 2015203203 15 Jun2015

From the results above, enhanced enzymatic activity was confirmed in the enzyme in which an amino acid at position 83 in the a subunit was substituted with an amino acid selected from among alanine, aspartic acid, phenylalanine, histidine, methionine and asparagine. EXAMPLE 16 [0204]

Preparation and Evaluation of Improved Nitrile Hydratase

Plasmid pFR005 formed below was used as a template plasmid to substitute an amino acid at position 83 of the a subunit.

[0205]

Namely, using the method in example 1, an improved nitrile hydratase with a substituted amino acid was prepared, and a transformant of Rhodococcus rhodocrous strain ATCC 12674 and its bacterial-cell suspension were obtained by the method in example 2. Further, the enzymatic activity was measured by the same method in example 3. The results are shown in Table 17.

[0206]

Table 17 name of plasmid amino-acid substitution catalytic activity (relative value) pFR005 P(317G, S057K, Τβ107Κ, Κβ114Υ, NB167S, V3219A 1.0 (comp, example) pER1127 PB17G, SB57K, Τβ107Κ, Κβ114Υ, NB167S, νβ219Α. Qo83A 5.1 PER1129 PB17G, 3β57Κ, Τβ107Κ, Κβ114Υ, Νβ167S, \/β219Α. Qo37L 1.9 pER1130 Ρβ17G, SB57K, Τβ107Κ, Κβ114Υ, NB167S, VB219A. Qo83M 2.7 pER1132 Ρβ17G, SB57K, Τβ107Κ, Κβ114Υ. NB167S. VB219A. Qo37N 4.8 [0207]

From the results above, the amino-acid substitution according to the present invention applies not only to a wild-type nitrie hydratase but to a mutant nitrile hydratase to exhibit the same effects. EXAMPLE 17 [0208]

Preparation of Transformant Containing Nitrile Hydratase Derived from Rhodococcus Rhodocrous M8 Strain (hereinafter referred to as M8 strain) [0209] (1) Preparation of Chromosomal DNA from M8 strain

The M8 strain (SU 1731814) is obtained from Russian Institute of Microorganism Biochemistry and Physiology (VKPM S-926). In a 100 mL MYK culture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bacto malt extract, 0.2% K2HPO4, 0.2% KH2PO4, pH 7.0), the M8 strain was subjected to shaking culture at 30°C for 72 -44- hours. The culture mixture was centrifuged, and the collected bacterial cells were suspended in 4 mL of Saline-EDTA solution (0.1 M EDTA, 0.15 M NaCl, pH 8.0). 2015203203 15 Jun2015

Then, 8 mg of lysozyme was added to the suspension, which was shaken at 37°C for 1~2 hours and was frozen at -20°C.

[0210]

Next, 10 mL of Tris-SDS solution (1% SDS, 0.1M NaCl, 0.1 M Tris-HCl (pH 9.0)) was added to the suspension while the suspension was gently shaken. Proteinase K (Merck KGaA) was further added (final concentration of 0.1 mg) and shaken at 37°C for 1 hour. Next, an equivalent volume of TE saturated phenol was added, agitated (TE: 10 mM Tris-HCl, 1 mM EDTA (pH 8.0)) and then centrifuged. The supernatant was collected, a double volume of ethanol was added and DNA strands were wrapped around a glass rod. Then, the phenol was removed through centrifugation by successively adding 90%, 80%, and 70% ethanol.

[0211]

Next, the DNA was dissolved in a 3 mL TE buffer, to which a Ribonuclease A solution (processed at 100°C for 15 minutes) was added to have a 10 pg/mL concentration and shaken at 37°C for 30 minutes. Proteinase K (Merck KGaA) was further added and shaken at 37°C for 30 minutes. After an equivalent volume of TE saturated phenol was added and centrifuged, the mixture was separated into upper and lower layers.

[0212]

An equivalent volume of TE saturated phenol was further added to the upper layer and centrifuged to separate into upper and lower layers. Such a process was repeated. Then, an equivalent volume of chloroform (containing 4% isoamyl alcohol) was added, centrifuged and the upper layer was collected. Then, a double volume of ethanol was added and the DNA strands were collected by wrapping them around a glass rod. Accordingly, chromosomal DNA was obtained.

[0213] (2) Using PCR, Preparation of Improved Nitrile Hydratase from Chromosomal DNA Derived from the M8 Strain

The nitrile hydratase derived from the M8 strain is described in a non-patent publication (Veiko, V.P. et al., “Cloning, Nucleotide Sequence of Nitrile Hydratase Gene from Rhodococcus rhodochrous M8,” Russian Biotechnology (Mosc.) 5,3-5 (1995)). The sequences of β subunit and a subunit are respectively identified as SEQ ID NOs: 17 and 18. Based on the sequence information, primers of SEQ ID NOs: 115 and 116 in the sequence listing were synthesized and PCR was performed using the chromosomal DNA prepared in step (1) above as a template.

Composition of PCR reaction mixture>

sterile water 20 pL template DNA (chromosomal DNA) 1 pL primer M8-1 (10 mM) 2 pL primer M8-2 (10 mM) 2 pL PrimeSTAR MAX (2 A_25 llL -45- 50 μι 2015203203 15 Jun2015 total <primers> M8-1: GGTCTAGAATGGATGGTATCCACGACACAGGC (SEQ ID NO: 115) M8-2: CCCCTGCAGGTCAGTCGATGATGGCCATCGATTC (SEQ ID NO: 116) <PCR reaction conditions> (98°C for 10 sec, 55°C for 5 sec, 72°C for 30 sec) x 30 cycles [0214]

After completion of PCR, 5 pL of the reaction mixture was subjected to 0.7% agarose gel electrophoresis (0.7 wt.% Agarose I, made by Dojin Chemical Co., Ltd.) and an amplified fragment of 1.6 kb was detected. The reacted mixture was purified using Wizard SV gel and PCR Clean-Up System (Promega KK).

[0215]

Next, the collected PCR product was coupled with a vector (pUC118/Hinc II site) using a ligation kit (made by Takara Shuzo Co., Ltd.) so that competent cells of E. coli JM109 were transformed using the reaction mixture. A few clones from the obtained transformant colonies were inoculated into 1.5 mL of an LB-Amp culture medium, and subjected to shaking culture at 37°C for 12 hours. After incubation was finished, the bacterial cells were collected from the culture through centrifugation. A plasmid DNA was extracted from the collected bacterial cells using QIAprep Spin Miniprep Kit (Qiagen). The base sequence of nitrile hydratase in the obtained plasmid DNA was confirmed using a sequencing kit and automated sequencer CEQ 8000 (Beckman Coulter, Inc.).

[0216]

Next, the obtained plasmid DNA was cleaved at restriction enzyme Xbal and Sse8387I, and subjected to 0.7% agarose gel electrophoresis so as to collect nitrile hydratase gene fragments (1.6 kb), which were then introduced into XbaI-Sse8387I site of plasmid pSJ042. The obtained plasmid was named pSJ-NOl A. Here, pSJ042 as a plasmid capable of expressing nitrile hydratase in Rhodococcus J1 strain was prepared by a method described in JP publication 2008-154552. Plasmid pS J023 used for preparation of pSJ042 is registered as transformant ATCC 12674/pSJ023 (FERM BP-6232) at the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6,1-1-1 Higashi, Tsukuba, Ibaraki), deposited March 4,1997. EXAMPLE 18 [0217]

Preparation and Evaluation of Improved Nitrile Hydratase [0218]

Using plasmid pSJ-NOl A obtained in example 5, the amino acid at position 83 of the a subunit was substituted. The same method as in example 1 was employed for amino-acid substitution to prepare an improved nitrile hydratase. Next, using the same method as in example 2, a transformant of Rhodococcus rhodocrous ATCC 12674 strain -46-and its bacterial-cell suspension were prepared. Then, the enzymatic activity was measured by the same method as in example 4. The results are shown in Table 18. 2015203203 15 Jun2015 [0219] Table 18 name of plasmid amino-acid substitution catalytic activity (relative value) PSJ-N01A none (parent strain) 1.0 (comp, example) PSJR17 Qa83M 6.9 [0220]

From the results above, pSJR17 in which the amino acid at position 83 of the a subunit was substituted with methionine was found to have an enhanced enzymatic activity the same as in example 4. EXAMPLE 19 [0221]

Preparation of Improved Nitrile Hydratase

Using plasmid pSJ034 formed in preparation example 1, amino-acid substitution was conducted. The following composition of a reaction mixture, reaction conditions and primers were used for the PCR.

Composition of PCR reaction mixture>

sterile water 20 pL pSJ034 (1 ng/mL) 1 pL Forward primer (10 mM) 2 pL Reverse primer (10 mM) 2 pL PrimeSTAR MAX (2 *1 25 uL total 50 pL <PCR reaction conditions> (98°C for 10 sec, 55 °C for 5 sec, 72°C for 90 sec) x 30 cycles <primers> saturation mutagenesis for a82 a82RM-F: ATGCCGGTNNSCAGGCACACCAAATTT (SEQ ID NO: 129) a82RM-R: TGTGCCTGSNNACCGGCATAGCCCAAT (SEQ ID NO: 130) [0222]

After the completion of PCR, 5 pL of the reaction mixture was subjected to 0.7% agarose gel electrophoresis and an amplified fragment of 1 kb was confirmed. Then, 1 pL of Dpnl (provided with a kit) was added to the PCR reaction mixture and reacted at 37°C for an hour to remove the template plasmid. The reacted mixture was purified using Wizard SV gel and PCR Clean-Up System (Promega), and JM109 was transformed using the purified PCR reaction product. Then, a plasmid DNA was extracted from the obtained culture using QIAprep Spin Miniprep Kit (Qiagen), and the base sequence of the nitrile hydratase was confirmed using an automated sequencer CEQ 8000 (Beckman Coulter, Inc.) Obtained plasmids were named as shown in Table 19. -47-[0223] Table 19

name of plasmid amino-acid substitution PSJ173 Ea82C PSJ174 Ea82F PSJ175 Ea82H PSJ176 Ea82l PSJ177 Ea82K PSJ178 Ea82M PSJ179 Ea82Q PSJ180 Ea82R PSJ181 Ea82T PSJ182 Ea82Y 2015203203 15 Jun2015 EXAMPLE 20 [0224]

Preparation of Rhodococcus Transformant

Cells of Rhodococcus rhodocrous ATCC 12674 strain in a logarithmic growth phase were collected using a centrifuge, washed three times with ice-cold sterile water, and suspended in the sterile water. Then, 1 pL of plasmid prepared in example 2 and 10 pL of the bacterial-cell suspension were mixed and ice-cooled. The DNA and the bacterial-cell suspension were supplied in a cuvette, and electric pulse treatment was conducted using an electroporation device, Gene Pulser (Bio-Rad Laboratories), under conditions of 2,0 kV and 200 Ω. The electric-pulse processed mixture was let stand in an ice-cold condition for 10 minutes, and subjected to heat shock at 37°C for 10 minutes. After 500 pL of an MYK culture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bacto malt extract, 0.2% K2HPO4, 0.2% KH2PO4) was added and let stand at 30°C for 5 hours, and applied onto an MYK agar culture medium containing 50 pg/mL kanamycin and incubated at 30°C for 3 days. The obtained colony after incubating at 3Q°C for 3 days was used as a transformant.

[0225]

Each transformant obtained above was inoculated into an MYK culture medium (50 pg/mL kanamycin), subjected to shaking culture at 30°C for 2 days. Then, 1% culture was inoculated into a GGPK culture medium (1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05% K2HPO4, 0.05% KH2PO4, 0.05% Mg2C>4-7H20,1% C0CI2, 0.1% urea, 50 pg/mL kanamycin, pH 7.2), and subjected to shaking culture at 30°C for 3 days. Then, bacterial cells were collected by using a centrifuge and were washed with a 100 mM phosphate buffer (pH 7.0) to prepare a bacterial-cell suspension. EXAMPLE 21 [0226]

Improved Nitrile Hydratase Activity

The nitrile hydratase activity in the obtained bacterial-cell suspension was measured by the following method.

[227]

After 0.2 mL of the bacterial-cell mixture and 4.8 mL of a 50 mM phosphate buffer (pH 7.0) were mixed, 5 mL of a 50 mM phosphate buffer (pEI 7.0) containing 5.0% -48-(w/v) acrylonitrile was further added, and the mixture was reacted while being shaken at 10°C for 10 minutes. Then, bacterial cells were filtered and the amount of produced acrylamide was determined by gas chromatography. 2015203203 15 Jun2015

gas chromatograph GC2014 (Shimadzu Corporation) FID (detection at 200'C) lm glass column filled with PoraPak PS (column filler made by Waters Corp.) 190°C <analysis conditions> analysis instrument: detector: column: column temperature: [0228]

Nitrile hydratase activity was determined by conversion from the amount of acrylamide. Here, regarding nitrile hydratase activity, the amount of enzyme to produce 1 pmol of acrylamide per 1 minute is set as 1 U. Table 20 shows relative activities when the parent strain activity without amino-acid substitution was set at 1.0.

[0229]

Table 20 name of plasmid amino-acid substitution catalytic activity (relative value) PSJ042 none (parent strain) 1.0 (comp, example) PSJ173 Ea82C 2.6 pSJ174 Ea82F 4.3 PSJ175 Ea82H 1.3 PSJ176 Ea82l 3.6 pSJ177 Ea82K 4.2 pSJ178 Ea82M 3.6 PSJ179 Ea82Q 2.3 pSJ180 Ea82R 4.2 PSJ181 Ea82T 1.2 PSJ182 Ea82Y 2.1

From the results above, enhanced enzymatic activity was confirmed in the enzyme in which an amino acid at position 82 in the a subunit was substituted with an amino acid selected from among cysteine, phenylalanine, histidine, isoleucine, lysine, methionine, glutamine, arginine, threonine and tyrosine. EXAMPLE 22 [0230] SDS-Polyacrylamide Gel Electrophoresis

Using a sonicator VP-300 (TAITEC Corporation), the bacterial-cell suspension prepared in example 3 was homogenized for 10 minutes while being ice-cooled. Next, the bacterial-cell homogenate was centrifuged at 13500 rpm for 30 minutes and a cell-free extract was obtained from the supernatant. After the protein content of the cell extract was measured using a Bio-Rad protein assay kit, the cell extract was mixed with a polyacrylamide gel electrophoresis sample buffer (0.1 M Tris-HCl (pH 6.8), 4% w/v SDS, 12% v/v β mercaptoethanol, 20% v/v glycerol, and a trace of bromophenol blue), and boiled for 5 minutes for denaturation. A 10% acrylamide gel was prepared, and denatured samples were applied to have an equivalent protein mass per one lane to -49-conduct electrophoresis analysis. 2015203203 15 Jun2015 [0231]

As a result, since hardly any difference was observed in the band strength of nitrile hydratase in all the samples, the expressed amount of nitrile hydratase was found the same. Accordingly, enzymatic specific activity was found to be attributed to the improved enzymatic activity. EXAMPLE 23 [0232]

Preparation of Improved Nitrile Hydratase

Using plasmid pSJ034 formed in preparation example 1, amino-acid substitution was conducted. The following composition of a reaction mixture, reaction conditions and primers were used for the PCR.

Composition of PCR reaction mixture>

sterile water 20 pL pSJ034 (1 ng/mL) 1 pL Forward primer (10 mM) 2 pL Reverse primer (10 mM) 2 pL PrimeSTAR MAX 12*1 25 uL total 50 pL <PCR reaction conditions> (98°C for 10 sec, 55°C for 5 sec, 72°C for 90 sec) x 30 cycles <primers> saturation mutagenesis primer for a85 a85RM-F: CAGGCANNSCAAATTTCGGCGGTCTTC (SEQ ID NO: 133) a85RM-R: AATTTGSNNTGCCTGCTCACCGGCATA (SEQ ID NO: 134) [0233]

After the completion of PCR, 5 pL of the reaction mixture was subjected to 0.7% agarose gel electrophoresis and an amplified fragment of 1 kb was confirmed. Then, 1 pL of Dpnl (provided with a kit) was added to the PCR reaction mixture and reacted at 37“C for an hour to remove the template plasmid. The reacted mixture was purified using Wizard SV gel and PCR Clean-Up System (Promega), and JM109 was transformed using the purified PCR reaction product. Then, a plasmid DNA was extracted from the obtained culture using QIAprep Spin Miniprep Kit (Qiagen), and the base sequence of the nitrile hydratase was confirmed using an automated sequencer CEQ 8000 (Beckman Coulter, Inc.) Obtained plasmids were named as shown in Table 21.

[0234] Table 21

name of plasmid amino-acid substitution PSJ165 Ha85C PSJ166 . Ha85E PSJ167 Ha85F -50-

PSJ168 Ha85l PSJ169 Ha85N PSJ170 Ha85Q PSJ171 Ha85S PSJ172 Ha85Y EXAMPLE 24 2015203203 15 Jun2015 [0235]

Preparation of Rhodococcus Transformant

Cells of Rhodococcus rhodocrous ATCC 12674 strain in a logarithmic growth phase were collected using a centrifuge, washed three times with ice-cold sterile water, and suspended in the sterile water. Then, 1 liL of plasmid prepared in example 2 and 10 μι of the bacterial-cell suspension were mixed and ice-cooled. The DNA and the bacterial-cell suspension were supplied in a cuvette, and electric pulse treatment was conducted using an electroporation device, Gene Pulser (Bio-Rad Laboratories), under conditions of 2.0 kV and 200 Ω. The electric-pulse processed mixture was let stand in an ice-cold condition for 10 minutes, and subjected to heat shock at 37°C for 10 minutes. After 500 pL of an MYK culture medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bacto malt extract, 0.2% K2HP04i 0.2% KH2P04) was added and let stand at 30°C for 5 hours, and applied onto an MYK agar culture medium containing 50 μg/mL kanamycin and incubated at 30°C for 3 days. The obtained colony after incubating at 30°C for 3 days was used as a transformant.

[0236]

Each transformant obtained above was inoculated into an MYK culture medium (50 μg/mL kanamycin), and subjected to shaking culture at 30°C for 2 days. Then, 1% culture was each inoculated into a GGPK culture medium (1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05% K2HP04, 0.05% KH2P04, 0.05% Mg204-7H20, 1% CoCl2, 0.1% urea, 50 pg/mL kanamycin, pH 7.2), and shaking culture was performed at 30°C for 3 days. Bacterial cells were collected by using a centrifuge and were washed with a 100 mM phosphate buffer (pH 7.0) to prepare a bacterial-cell suspension. EXAMPLE 25 [0237]

Improved Nitrile Hydratase Activity

The nitrile hydratase activity of the bacterial-cell suspension was measure as follows.

[0238]

After 0.2 mL of the bacterial-cell mixture and 4.8 mL of a 50 mM phosphate buffer (pH 7.0) were mixed, 5 mL of a 50 mM phosphate buffer (pH 7.0) containing 5.0% (w/v) acrylonitrile was further added, and the mixture was reacted while being shaken at 10°C for 10 minutes. Then, bacterial cells were filtered and the amount of produced acrylamide was determined by gas chromatography. <analysis conditions> analysis instrument: gas chromatograph GC2014 (Shimadzu Corporation) detector: FID (detection at 200°C) -51 - column: lm glass column filled with PoraPak PS (column filler made 2015203203 15 Jun2015

by Waters Corp.) column temperature: 190°C

[0239]

Nitrile hydratase activity was determined by conversion from the amount of acrylamide. Here, regarding nitrile hydratase activity, the amount of enzyme to produce 1 μηιοί of acrylamide per 1 minute is set as 1 U. Table 22 shows relative activities when the parent strain activity without amino-acid substitution was set at 1.0.

[0240] Table 22 name of plasmid amino-acid substitution catalytic activity (relative value) pSJ042 none (parent strain) 1.0 (comp, example) PSJ165 Ha85C 1.5 PSJ166 Ha85E 1.9 PSJ167 Ha85F 1.8 PSJ168 Ha85l 2.1 pSJ169 Ha85N 2.3 PSJ170 Ha85Q 2.1 PSJ171 Ha85S 2.5 PSJ172 Ha85Y 1.5 [0241]

From the results above, enhanced enzymatic activity was confirmed in the enzyme in which an amino acid at position 85 in the a subunit was substituted with an amino acid selected from among cysteine, glutamic acid, phenylalanine, isoleucine, asparagine, glutamine, serine and tyrosine. EXAMPLE 26 [0242] SDS-Polyacrylamide Gel Electrophoresis

Using a sonicator VP-300 (TAITEC Corporation), the bacterial-cell suspension prepared in example 3 was homogenized for 10 minutes while being ice-cooled. Next, the bacterial-cell homogenate was centrifuged at 13500 rpm for 30 minutes and a cell-free extract was obtained from the supernatant. After the protein content of the cell extract was measured using a Bio-Rad protein assay kit, the cell extract was mixed with a polyacrylamide gel electrophoresis sample buffer (0.1 M Tris-HCl (pH 6.8), 4% w/v SDS, 12% v/v β mercaptoethanol, 20% v/v glycerol, and a trace of bromophenol blue), and boiled for 5 minutes for denaturation. A 10% acrylamide gel was prepared, and denatured samples were applied to have an equivalent protein mass per one lane to conduct electrophoresis analysis.

[0243]

As a result, since hardly any difference was observed in the band strength of nitrile hydratase in all the samples, the expressed amount of nitrile hydratase was found to be the same. Accordingly, the enzymatic specific activity was found to be attributed to the improved enzymatic activity. -52-

POTENTIAL INDUSTRIAL APPLICABILITY 2015203203 15 Jun2015 [0244]

According to the present invention, a novel improved (mutant) nitrile hydratase is provided with enhanced catalytic activity. Such an improved nitrile hydratase with enhanced catalytic activity is very useful to produce amide compounds.

[0245]

According to the present invention, a nitrile hydratase is obtained from DNA encoding the improved nitrile hydratase above, a recombinant vector containing the DNA, a transformant containing the recombinant vector, and a culture of the transformant, and a method for producing such a nitrile hydratase is also provided. Moreover, a method for producing an amide compound using the protein (improved nitrile hydratase), the culture or the processed product of the culture is provided according to the present invention.

[0246]

According to the present invention, a novel improved (mutant) nitrile hydratase is provided with enhanced catalytic activity. Such an improved nitrile hydratase with enhanced catalytic activity is very useful to produce amide compounds.

[0247]

According to the present invention, a nitrile hydratase is obtained from genomic DNA encoding the improved nitrile hydratase above, a recombinant vector containing the genomic DNA, a transformant containing the recombinant vector, and a culture of the transformant, and a method for producing such a nitrile hydratase is also provided. Moreover, a method for producing an amide compound using the protein (improved nitrile hydratase), the culture or the processed product of the culture is provided according to the present invention.

ACCESSION NUMBERS

[0248]

Rhodococcus rhodochrous J1 strain is internationally registered under accession number “PERM BP-1478” at the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, (Central 6,1-1-1 Higashi, Tsukuba, Ibaraki), deposited September 18,1987.

[0249]

In addition, pSJ023 is a transformant “R. rhodochrous ATCC 12674/pSJ023,” and is internationally registered under accession number FERM BP-6232 at the International Patent Organism Depositary, National Institute of Advanced Industrial Science (Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki), deposited March 4,1997.

[0250] [Description of Sequence Listing] SEQ ID NO: 1 base sequence of β subunit derived from Rhodococcus rhodocrous J1 strain (FERM BP-1478) SEQ ID NO: 2 amino-acid sequence of β subunit derived from Rhodococcus rhodocrous J1 strain (FERM BP-1478) -53- ιη Ο SEQ ID NO: 3 base sequence of a subunit derived from Rhodococcus <N rhodocrous J1 strain (FERM BP-1478) α SEQ ID NO: 4 amino-acid sequence of a subunit derived from Rhodococcus £ rhodocrous J1 strain (FERM BP-1478) in SEQ ID NO: 5 amino-acid sequence of β subunit in Rhodococcus rhodocrous M8 SEQ ID NO: 6 (SU 1731814) amino-acid sequence of β subunit in Rhodococcus ruber TH cn SEQ ID NO: 7 amino-acid sequence of β subunit in Rhodococcus pyridinivorans o MW33 (VKM Ac-1515D) <N m o SEQ ID NO: 8 amino-acid sequence of β subunit in Rhodococcus pyridinivorans S85-2 <N SEQ ID NO: 9 amino-acid sequence of β subunit in Rhodococcus pyridinivorans in MS-38 O SEQ ID NO: 10 amino-acid sequence of β subunit in Nocardia sp.JBRs <N SEQ ID NO: 11 amino-acid sequence of β subunit in Nocardia sp.YS-2002 SEQ ID NO: 12 amino-acid sequence of β subunit in Rhodococcus rhodocrous ATCC 39384 SEQ ID NO: 13 β48(2-Ρ primer SEQ ID NO: 14 β48<2-Ε primer SEQ ID NO: 15 β48ϋ-Ρ primer SEQ ID NO: 16 p48D-R primer SEQ ID NO: 17 p48E-F primer SEQ ID NO: 18 p48E-R primer SEQ ID NO: 19 p48H-F primer SEQ ID NO: 20 p48H-R primer SEQ ID NO: 21 β48Ι-Ρ primer SEQ ID NO: 22 p48I-R primer SEQ ID NO: 23 β48Κ-Ρ primer SEQ ID NO: 24 [348K-R primer SEQ ID NO: 25 p48M-F primer SEQ ID NO: 26 P48M-R primer SEQ ID NO: 27 β48Ν-Ρ primer SEQ ID NO: 28 β48Ν-ΙΙ primer SEQ ID NO: 29 p48P-F primer SEQ ID NO: 30 β48Ρ^ primer SEQ ID NO: 31 β48Q-F primer SEQ ID NO:32 P48Q-R primer SEQ ID NO: 33 P48S-F primer SEQ ID NO: 34 P48S-R primer SEQ ID NO: 35 p48T-F primer SEQ ID NO: 36 P48T-R primer SEQ ID NO: 37 amino-acid sequence of β subunit in nitrile hydratase derived from M8 strain SEQ ID NO: 38 amino-acid sequence of a subunit in nitrile hydratase derived from M8 strain SEQ ID NO: 39 amino-acid sequence of activator in nitrile hydratase derived from M8 strain SEQ ID NO :40 M8-1 primer -54-

O <N in

m o (N m o<N in o <N SEQ ID NO: 41 M8-2 primer SEQ ID NO: 42 amino-acid sequence of β subunit in uncultured bacterium SP1 SEQ ID NO: 43 amino-acid sequence of β subunit in uncultured bacterium BD2 SEQ ID NO: 44 amino-acid sequence of β subunit in Comamonas testosterone SEQ ID NO: 45 amino-acid sequence of β subunit in Geobacillus thermoglucosidasius Q6 SEQ ID NO: 46 amino-acid sequence of β subunit in Pseudonocardia thermophila JCM 3095 SEQ ID NO: 47 amino-acid sequence of β subunit in Rhodococcus rhodocrous Cr4 SEQ ID NO: 48 amino-acid sequence of cysteine cluster of a subunit in iron-containing nitrile hydratase SEQ ID NO: 49 amino-acid sequence in cysteine cluster of a subunit in cobalt-containing nitrile hydratase SEQ ID NO: 50 predetermined amino-acid sequence to be used in the present invention SEQ ID NO: 51 amino-acid sequence of β subunit related to the present invention SEQ ID NO: 52 amino-acid sequence of β subunit in Rhodococcus ruber RH (CN 101463358) SEQ ID NO: 53 base sequence of nitrile hydratase J1D SEQ ID NO: 54 base sequence of nitrile hydratase 203 SEQ ID NO: 55 base sequence of nitrile hydratase 414 SEQ ID NO: 56 base sequence of nitrile hydratase 855 SEQ ID NO: 57 base sequence of the a subunit in nitrile hydratase D2 SEQ ID NO: 58 base sequence of nitrile hydratase 005 SEQ ID NO: 59 base sequence of nitrile hydratase 108A SEQ ID NO: 60 base sequence of nitrile hydratase 211 SEQ ID NO: 61 base sequence of nitrile hydratase 306A SEQ ID NO: 62 base sequence of a PCR fragment containing a primer sequence at both terminal of Rhodococcus rhodocrous M8 SEQ ID NO: 63 β17ϋΜ-Ε primer SEQ ID NO: 64 β17ΙΙΜ-Ε primer SEQ ID NO: 65 NH-19 primer SEQ ID NO: 66 NH-20 primer SEQ ID NO: 67 [337A-F primer SEQ ID NO: 68 β37Α4β. primer SEQ ID NO: 69 β3704Ρ primer SEQ ID NO: 70 β37ΰ-Ε primer SEQ ID NO: 71 β37Ε-Ε primer SEQ ID NO: 72 β37Ε^ primer SEQ ID NO: 73 P37I-F primer SEQ ID NO: 74 β37Ι-Ε primer SEQ ID NO: 75 P37M-F primer SEQ ID NO: 76 β37Μ^ primer SEQ ID NO:77 β37Τ-Ε primer SEQ ID NO: 78 β37Τ^ primer SEQ ID NO: 79 β37ν-Ρ primer SEQ ID NO :80 β37ν41 primer SEQ ID NO: 81 predetermined amino-acid sequence to be used in the present -55- ιη Ο<N α m ο <Ν m ο (Ν 1/3 Ο (Ν invention SEQ ID NO: 82 amino-acid sequence of β subunit related to the present invention SEQ ID NO: 83 a83A-F primer SEQ ID NO: 84 a83A-R primer SEQ ID NO: 85 a83C-F primer SEQ ID NO: 86 a83C-R primer SEQ ID NO: 87 a83D-F primer SEQ ID NO: 88 a83D-R primer SEQ ID NO: 89 a83E-F primer SEQ ID NO: 90 a83E-R primer SEQ ID NO: 91 a83F-F primer SEQ ID NO :92 a83F-R primer SEQ ID NO :93 a83G-F primer SEQ ID NO :94 a83G-R primer SEQ ID NO: 95 a83H-F primer SEQ ID NO :96 a83H-R primer SEQ ID NO: 97 a83M-F primer SEQ ID NO: 98 a83M-R primer SEQ ID NO: 99 a83P-F primer SEQ ID NO: 100 a83P-R primer SEQ ID NO: 101 a83S-F primer SEQ ID NO: 102 a83S-R primer SEQ ID NO: 103 a83T-F primer SEQ ID NO: 104 a83T-R primer SEQ ID NO: 105 amino-acid sequence of a subunit in Rhodococcus rhodocrous M8 (SU 1731814) SEQ ID NO: 106 amino-acid sequence of a subunit in Rhodococcus ruber TH SEQ ID NO: 107 amino-acid sequence of a subunit in Rhodococcus pyridinivorans MW33 (VKM Ac-1515D) SEQ ID NO: 108 amino-acid sequence of a subunit in Rhodococcus pyridinivorans S85-2 SEQ ID NO: 109 amino-acid sequence of a subunit in Nocardia sp.JBRs SEQ ID NO: 110 amino-acid sequence of a subunit in Nocardia sp.YS-2002 SEQ ID NO: 111 amino-acid sequence of a subunit in uncultured bacterium BD2 SEQ ID NO: 112 amino-acid sequence of a subunit in uncultured bacterium SP1 SEQ ID NO: 113 amino-acid sequence of a subunit in Pseudonocardia thermophila JCM 3095 SEQ ID NO: 114 amino-acid sequence of a subunit in Rhodococcus rhodocrous Cr4 SEQ ID NO: 115 M8-1 primer SEQ ID NO: 116 M8-2 primer SEQ ID NO: 117 amino-acid sequence in a cysteine cluster of a subunit in iron-containing nitrile hydratase SEQ ID NO: 118 amino-acid sequence in cysteine cluster of a subunit in cobalt-containing nitrile hydratase SEQ ID NO: 119 predetermined amino-acid sequence to be used in the present invention SEQ ID NO: 120 amino-acid sequence of a subunit related to the present -56- 2015203203 15 Jun2015 SEQ ID NO: 121 SEQ ID NO: 122 SEQ ID NO: 123 SEQ ID NO: 124 SEQ ID NO: 125 SEQ ID NO: 126 SEQ ID NO: 127 SEQ ID NO: 128 SEQ ID NO: 129 SEQ ID NO: 130 SEQ ID NO: 131 SEQ ID NO: 132 SEQ ID NO: 133 SEQ ID NO: 134 SEQ ID NO: 135 SEQ ID NO: 136 invention amino-acid sequence of a subunit in Rhodococcus pyridinivorans MS-3 8 amino-acid sequence of a subunit in Rhodococcus rhodocrous ATCC 39384 amino-acid sequence of a subunit in Sinorhizobium medicae WSM419 amino-acid sequence of a subunit in Geobacillus thermoglucosidasius Q6 amino-acid sequence of a subunit in Comamonas testosterone

amino-acid sequence of a subunit in Rhodococcus ruber RH (CN 101463358) a83N-F primer a83N-R primer a82RM-F primer a82RM-R primer amino-acid sequence of a subunit related to the present invention predetermined amino-acid sequence to be used in the present invention a85RM-F primer a85RM-R primer amino-acid sequence of a subunit related to the present invention predetermined amino-acid sequence to be used in the present invention -57-

Claims (16)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

   1. An improved nitrile hydratase, comprising at least one of the following (a)~(c): (a) in the a subunit, a nitrile hydratase contains an amino-acid sequence as shown in SEQ ID NO: 119 below AX1X2X3X4GX5X6GX7X8 (SEQ ID NO: 119) (A is alanine, G is glycine, and Χι-Χγ each independently indicate any amino-acid residue), wherein Xs is an amino acid selected from among alanine, leucine, methionine, asparagine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, lysine, proline, arginine, serine, threonine and tryptophan; (b) in the a subunit, a nitrile hydratase has the amino-acid sequence as shown in SEQ ID NO: 132 below AX1X2X3X4GX5X6GX7Q (SEQ ID NO: 132) (A is alanine, G is glycine, Q is glutamine, and Xi~Xe each independently indicate any amino-acid residue), wherein X7 is substituted with an amino acid different from that in a wild type; and (c) in the a subunit, a nitrile hydratase has the amino-acid sequence as shown in SEQ ID NO: 136 below AX1X2X3X4GX5X6GX7QX8X9 (SEQ ID NO: 136) (A is alanine, G is glycine, Q is glutamine, and X|~Xs each independently indicate any amino-acid residue), wherein X9 is substituted with an amino acid different from that in a wild type.
2. 2. The improved nitrile hydratase according to Claim 1, wherein Xi is M (methionine), X2 is A (alanine), X3 is S (serine), X4 is L (leucine), X5 is Y (tyrosine), Xe is A (alanine) and X7 is E (glutamic acid) in SEQ ID NO: 119.
3. 3. The improved nitrile hydratase according to Claim 1 or 2, further comprising an amino-acid sequence as shown in SEQ ID NO: 120 that includes the amino-acid sequence as shown in SEQ ID NO: 119.
4. 4. The improved nitrile hydratase according to Claim 1, further comprising an amino-acid sequence of the a subunit as shown in SEQ ID NO: 132, wherein X7 is an amino acid selected from among cysteine, phenylalanine, histidine, isoleucine, lysine, methionine, glutamine, arginine, threonine and tyrosine.
5. 5. The improved nitrile hydratase according to Claim 1 or 4, wherein Xi is M (methionine), X2 is A (alanine), X3 is S (serine), X4 is L (leucine), X5 is Y (tyrosine), and Xe is A (alanine) in SEQ ID NO: 132.
6. 6. The improved nitrile hydratase according to Claim 1, 4 or 5, further comprising an amino-acid sequence as shown in SEQ ID NO: 131 that includes the amino-acid sequence as shown in SEQ ID NO: 132.
7. 7. The improved nitrile hydratase according to Claim 1, further comprising an amino-acid sequence of the a subunit as shown in SEQ ID NO: 136, wherein X9 is an amino acid selected from among cysteine, glutamic acid, phenylalanine, isoleucine, asparagine, glutamine, serine and tyrosine.
8. 8. The improved nitrile hydratase according to Claim 1 or 7, wherein Xi is M (methionine), X2 is A (alanine), X3 is S (serine), X4 is L (leucine), X5 is Y (tyrosine), Xe is A (alanine), X7 is E (glutamic acid), and Xs is A (alanine) in SEQ ID NO: 136.
9. 9. The improved nitrile hydratase according to Claim 1, 7 or 8, further comprising an amino-acid sequence as shown in SEQ ID NO: 135 that includes the amino-acid sequence as shown in SEQ ID NO: 136.
10. 10. The improved nitrile hydratase according to any one of Claims 1 to 9, wherein the nitrile hydratase is derived from Rhodococcus bacterium or Nocardia bacterium.
11. 11. DNA encoding the improved nitrile hydratase according to any one of Claims 1 to 10.
12. 12. A recombinant vector containing the DNA according to Claim 11.
13. 13. A transformant containing the recombinant vector according to Claim 12.
14. 14. A nitrile hydratase collected from a culture obtained by incubating the transformant according to Claim 13.
15. 15. A method for producing a nitrile hydratase, comprising: incubating the transformant according to Claim 13; and collecting the nitrile hydratase from the obtained culture.
16. 16. A method for producing an amide compound, comprising: bringing a nitrile compound into contact with a culture, or a processed product of the culture, obtained by incubating the nitrile hydratase according to any one of Claims 1 to 10 or the transformant according to Claim 13.