AU751921B2 - D-pantolactone hydrolase and gene encoding the same - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT

SEC

Applicant: 104 Lu Appliant: Co, c/ FrUJI YAKUIIIN KOGYO KADUSIIKI KAIS V Oe Invention Title: D-Pantolactone Hydrolase and Gene Encoding the Same The following statement is a full description of this invention, including the best method of performing it known to me/us: la- D-PANTOLACTONE HYDROLASE AND GENE ENCODING THE SAME TECHNICAL FIELD The present invention relates to a novel enzyme which is useful for an optical resolution of D,L-pantolactone through a D-selective asymmetric hydrolysis process and also to a gene encoding the same. More particularly, the present invention relates to proteins having a natural D-pantolactone hydrolase activity, produced by Fusarium oxysporum, or an activity substantially equivalent to the same and genes coding for the same. Specifically, the present invention relates to DNA 15 containing a nucleotide sequence coding for said protein; to host cells transformed or transfected with said DNA; to a process for the production of said D-pantolactone hydrolase protein via using said host cells; and to the use of such proteins and host cells.

BACKGROUND ART D-Pantolactone has been known as an intermediate in 25 the preparation of D-pantothenic acid and pantethine which are useful as vitamins of medical or physiplogical importance.

D-Pantolactone has heretofore been prepared through an optical resolution of a chemically-synthesized D,L-pantolactone.

Such a process, however, has disadvantages in that it requires the use of expensive optical resolving agents such as quinine or brucine and further that the recovery of D-pantolactone is not easy. In order to solve such problems, the present inventors already proposed an optical resolving method by an enzymatic asymmetric hydrolysis of D,L-pantolactone in Unexamined Japanese Patent Publication (KOKAI TOKKYO) Nos.

Hei 03-65,198 and Hei 04-144,681.

2 Thus, it is a process for the production of D-pantolactone, wherein the D-pantolactone in D,L-pantolactone mixtures is selectively subjected to an asymmetric hydrolysis using a microorganism possessing a lactone-hydrolyzing activity to form D-pantoic acid, which is then separated and converted into D-pantolactone, wherein said microorganism is a member selected from the group consisting of microorganisms belonging to the genera: Fusarium, Cylindrocarpon, Gibberella, Aspergillus, Penicillium, Rhizopus, Volutella, Gliocladium, Eurotium, Nectoria, Schizophyllum, Myrothecium, Neurospora, Acremonium, Tuberculina, Absidia, Sporothrix, Verticillium and Arthroderma. It is also a process for producing D-pantolactone hydrolase which comprises using a microorganism belonging to the above-mentioned genus.

However, it cannot be always said that many of those S. microorganisms disclosed as above possess a hydrolyzing activity to such an extent that they are immediately applicable in industry. Furthermore, in increasing the enzymatic activity 20 of said microorganisms to an industrially applicable level, troublesome and difficult investigations requiring long time are needed for establishing conditions for growth of cells, conditions for enzyme activity induction, etc. There is another problem that, since said microorganisms are true fungus, their 25 cell bodies are in variously shaped hyphae and, as compared with bacteria having a single shape, it is considerably difficult to prepare immobilized cells which are advantageous for industrial production. There is still another problem that, in purifying the enzyme from the cells, its recovery rate is considerably poor so far as D-pantolactone hydrolase is concerned.

3 DISCLOSURE OF THE INVENTION An object of the present invention is to solve those problems and also to provide means for making a significant increase of the enzymatic activity possible, for example, means for modifying and improving the D-pantolactone hydrolase per se.

Thus, one aspect of the present invention is to disclose and provide a novel gene which codes for a protein having either a naturally-occurring D-pantolactone hydrolase activity (such as a Fusarium oxysporum D-pantolactone hydrolase activity) or an activity substantially equivalent thereto; a host cell transformed with DNA containing a nucleotide sequence coding for said protein; a process for producing said 15 protein via using said host cell; and uses of said proteins and host cells.

The present invention directed to a gene coding for D-pantolactone hydrolase isolated from the above-mentioned 20 microorganisms possessing the ability to hydrolyze a lactone and a system, with a high efficiency and rich productivity, for producing D-pantolactone is successfully developed through utilizing the D-pantolactone hydrolase gene isolated as such, not only solves the above-mentioned various problems but also 25 greatly contributes to the development of enzymes possessing the ability to hydrolyze a lactone, together with new functions; and to the development of techniques using the novel enzyme. Particularly, the present inventors have succeeded in isolating a novel gene coding for a hydrolase with a D-pantolactone hydrolyzing ability, derived from microorganisms of the genus Fusarium (such as Fusarium oxysporum) which produces the D-pantolactone hydrolase, whereby the present invention has been achieved.

The present invention relates to: a recombinant protein having D-pantolactone hydrolase activity or an activity substantially equivalent thereto or a salt thereof; or (ii) a recombinant protein having a primary structural conformation substantially equivalent thereto or a salt thereof; (iii) a characteristic partial peptide of said recombinant protein or a salt thereof; (iv) genes, such as DNA and RNA, coding for said recombinant protein; vectors or plasmids, containing said gene operably in a gene recombination technique; (vi) host cells transformed with such a vector etc.; (vii) a process for producing said recombinant protein or a salt thereof which comprises culturing said host cell; (viii) a process for producing D-pantolactone which comprises an optical resolution of D,L-pantolactone with such a gene-manipulated host cell (transformant), such a 20 recombinant protein or a salt thereof etc.; and (ix) a system means, such as an immobilized enzyme, for producing D-pantolactone.

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0*0* o 0 0000 0 0 .0.0 0 0 According to a first aspect of the present invention there is provided a recombinant peptide having Dpantolactone hydrolase activity and an amino acid sequence represented by SEQ ID NO:1 or an amino acid sequence represented by SEQ ID NO:1 but in which one or more amino acids is/are substituted, deleted, inserted, translocated or added, or a salt thereof.

According to a second aspect of the present invention there is provided an isolated nucleic acid having a nucleotide sequence encoding a peptide as described above.

According to a third aspect of the present invention there is provided a vector incorporating the nucleic acid described above.

According to a fourth aspect of the present invention there is provided a transformant in which a vector as described above is harbored.

According to a fifth aspect of the present invention there is provided a process for producing a 20 peptide as described above, which comprises: culturing the transformant as described above in a nutrient medium suitable for growing said transformant to produce the peptide.

According to a sixth aspect of the present invention there is provided a process for producing Dpantolactone, which comprises: carrying out an optical resolution of D,Lpantolactone in the presence of a peptide as described above or (ii) the transformant as described above.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the amino acid sequences obtained by sequencing of digestive peptides of D-pantolactone hydrolase.

Figure 2 shows sites each corresponding to a digestive peptide of D-pantolactone hydrolase on the amino acid sequence for which the isolated cDNA codes.

Figure 3 shows the structures of primers applied in PCR wherein a genomic DNA for D-pantolactone hydrolase is used as a template.

Figure 4 shows the structures of primers applied 20 in PCR for the construction of a vector used for expressing o*o recombinant D-pantolactone hydrolase.

Figure 5 shows the amino acid sequence and nucleotide sequence of D-pantolactone hydrolase.

7- DETAILED DESCRIPTION OF THE INVENTION The present invention provides techniques such as cloning of a gene coding for naturally-occurring D-pantolactone hydrolase (such as natural D-pantolactone hydrolase derived from (or originating in) Fusarium oxysporum) or a protein having an activity substantially equivalent thereto, identification of said gene and determination of the characteristic sequence (sequencing) of said gene as well as recombination of said gene to an expression vector; production and culture/growth of host cells transformed with DNA containing a nucleotide sequence coding for said protein (transformants); production of said protein via using said host cell; and use of such proteins and host cells.

Described herein below are detailed techniques and operations according to the present invention.

The present invention also provides various means for utilizing genes coding for the above-mentioned 20 D-pantolactone hydrolase and further provides a D-pantolactone hydrolase production system with a good efficiency and a more excellent productivity wherein said isolated D-pantolactone hydrolase gene is utilized.

The present invention relates to a protein having a 25 naturally-occurring D-pantolactone hydrolase activity or an activity substantially equivalent thereto or a salt thereof, or a protein having a primary structural conformation substantially equivalent thereto or a salt thereof; a characteristic partial peptide of said protein or a salt thereof; a gene, such as DNA and RNA, coding for said protein or peptide; a vector or plasmid (or vehicle) containing said gene operably in a gene recombination technique; a host cell transformed with such a vector, etc.; a process for producing said protein or a salt thereof which comprises culturing said host cell; a process for synthesizing D-pantolactone which comprises an optical resolution of D,L-pantolactone with such a gene-manipulated host cell, or said recombinant protein or 8 a salt thereof; and systems and means, such as immobilized enzymes, for producing D-pantolactone.

In the present invention, D-pantolactone hydrolase or a salt thereof which comprises, preferably, an amino acid sequence of SEQ ID NO:1 or a amino acid sequence substantially equivalent thereto is specifically illustrated but the D-pantolactone hydrolase of the present invention includes any enzyme which has a D-pantolactone hydrolyzing ability as long as it has a novel amino acid sequence. The D-pantolactone hydrolyzing ability refers to any ability which is in the same quality in view of hydrolyzing D-pantolactone.

More preferably, the D-pantolactone hydrolase of the present invention includes all substances having an amino acid sequence S 15 of SEQ ID NO:1; or having a substantially equivalent amino acid sequence thereto and/or the substantially same amino acid sequence.

The D-pantolactone hydrolase gene according to the 20 present invention may be cloned, for example, by the following processes: It should be noted that gene recombination techniques •may be conducted, for example, by the methods disclosed in T. Maniatis et al., "Molecular Cloning", 2nd Ed., Cold Spring 25 Harbor Laboratory, Cold Spring Harbor, N. T. (1989); Nippon Seikagaku Kai (Biochemical Society of Japan) ed., "Zoku-Seikagaku Jikken Kouza 1, Idensi Kenkyuho II Lectures on Biochemical Experiments (Second Series; Methods for Gene Study Tokyo Kagaku Dojin, Japan (1986); Nippon Seikagaku Kai (Biochemical Society of Japan) ed., "Shin-Seikagaku Jikken Kouza 2, Kakusan III (Kumikae DNA Gijutsu) (New Lectures on Biochemical Experiments 2, Nucleic Acids III (Recombinat DNA Technique))", Tokyo Kagaku Dojin, Japan (1992); R. Wu "Methods in Enzymology", Vol. 68, Academic Press, New York (1980); R. Wu et al. "Methods in Enzymology", Vols. 100 and 101, Academic Press, New York (1983); R. Wu et al. "Methods in Enzymology", Vols. 153, 9 154 and 155, Academic Press, New York (1987), etc. as well as by the techniques disclosed in the references cited therein, the disclosures of which are hereby incorporated by reference, or by the substantially same techniques as they disclose or modified techniques thereof. Such techniques and means may also be those which are individually modified/improved from conventional techniques depending upon the object of the present invention.

1) Cloning of Partial Genomic DNA of D-Pantolactone Hydrolase Cultured Fusarium oxysporum cells are disrupted, and centrifuged to isolate chromosomal DNA, followed by decomposition and removal of RNA, in a conventional manner.

15 DNA components are purified by removing proteins therefrom.

Further information on preparation of the materials referred Sto in this application is disclosed, for example, in "Shokubutsu Biotechnology-Jikken Manual (Plant Biotechnology Experiment Manual)", Noson Bunkasha, page 252, the disclosures 20 of which are hereby incorporated by reference.

As a source for DNA, any microorganism which belongs to the genus Fusarium and has an ability of producing D-pantolactone hydrolase may be suitably used. Examples of the microorganism belonging to the genus Fusarium which is 25 applicable here are Fusarium oxysporum IFO 5942, Fusarium semitectam IFO 30200, etc.

Similarly, other microorganisms which belong to a member selected from the group consisting of the genera: Cylindrocarpon, Gibberella, Aspergillus, Penicillium, Rhizopus, Volutella, Gliocladium, Eurotium, Nectoria, Schizophyllum, Myrothecium, Neurospora, Acremonium, Tuberculina, Absidia, Sporothrix, Verticillium or Arthroderma and have the ability to produce D-pantolactone hydrolase may be used as a source for DNA. Examples of such microorganisms are Cylindrocarpon tonkinense IFO 30561, Gibberella fujikuroi IFO 6349, Aspergillus awamori IFO 4033, Penicillium chrysogenum IFO 4626, Rhizopus 1 0oryzae IFO 4706, Volutella buxi IFO 6003, Gliocladium catenulatum IFO 6121, Eurotium chevalieri IFO 4334, Nectria elegans IFO 7187, Schizophyllum commune IFO 4928, Myrothecium roridum IFO 9531, Neurospora crassa IFO 6067, Acremonium fusidioides IFO 6813, Tuberculina persicina IFO 6464, Absidia lichtheimi IFO 4009, Sporothrix schenckii IFO 5983, Verticillium malthousei IFO 6624, Arthroderma uncinatum IFO 7865, etc., wherein "IFO" is Zaidan-Hojin Hakko Kenkyusho (the Institute for Fermentation, Osaka; 17-85, Juso-hon-machi 2-chome, Yodogawa-ku, Osaka 532, Japan) and each number thereafter stands for the number in the Catalog issued by said IFO or the Accession Number'given by IFO.

15 2) Preparation of Probe Synthetic oligonucleotide primers are prepared according to information on amino acid sequences regarding the internal peptide of D-pantolactone hydrolase. For example, 20 synthetic oligonucleotide primers can be prepared according to eo information on amino acid sequences regarding the internal peptide of pure D-pantolactone hydrolase obtained from the microorganism which is selected from those mentioned hereinabove and has an ability of producing D-pantolactone hydrolase. In a typical case, degenerate primers, etc. are designed and prepared based upon information on the amino acid sequence of natural D-pantolactone hydrolase fragments.

Preparation of primers may be carried out by techniques which are known in the art. For example, the primers may be synthesized by means of a phosphodiester method, a phosphotriester method, a phosphoamidite method, etc. using an automatic DNA synthesizer. To be more specific, D-pantolactone hydrolase is purified from the cells obtained by culturing Fusarium oxysporum IFO 5942 in a nutrient medium and fragmented, if necessary, with a peptidase, etc. whereupon the information on an amino acid sequence of the internal peptide of the enzyme is collected. From the information on 1 1 the amino acid sequence obtained as such, preferred synthetic oligonucleotide primers are designed and prepared.

A polymerase chain reaction (PCR) is carried out using a pair of said primers wherein a genomic DNA for D-pantolactone hydrolase is used as a template. The PCR may be carried out by techniques known in the art or by methods substantially equivalent thereto or modified techniques. The reaction may be conducted by the methods disclosed, for example, in R. Saiki, et al., Science, Vol. 230, pp. 1350 (1985); R. Saiki, et al., Science, Vol. 239, pp. 487 (1988); and Henry A. Erlich, PCR Technology, Stockton Press.

The reaction may also be carried out, for example, using a commercially available kit or reagent.

The resulting amplified DNA fragments are sequenced and, after confirming that they contain a sequence which is homologous to that coding for the amino acid sequence of the o internal peptide of the purified enzyme, they are labeled with an isotope and are used for future experiments or the like.

20 Sequencing of nucleotide sequences may be carried out by a dideoxy technique (such as an M13 dideoxy method), a Maxam-Gilbert method, etc. or may be carried out using a commercially available sequencing kit such as a Taq dyeprimer cycle sequencing kit or an automatic nucleotide sequencer 25 such as a fluorescent DNA sequencer. Labeling of probes, etc.

with a radioisotope, etc., may be carried out using a commercially available labeling kit such as a random primed DNA labeling kit (Boehringer Mannheim).

1 2 3) Cloning of D-Pantolactone Hydrolase cDNA a) Preparation of mRNA and Construction of cDNA Library.

Cultured Fusarium oxysporum cells are disrupted, extracted according to an AGPC method to isolate total RNA.

Then mRNA is isolated and purified from the total RNA fraction by a suitable method such as by the use of an oligo dT cellulose column. Although, in an embodiment, mRNA may be isolated with a method known in the art or by the substantially same method as it is or modifications thereof, the isolation and purification of mRNA can be conducted by methods disclosed in, for example, T. Maniatis, et al., "Molecular Cloning", 2nd Ed., Chapter 7, Cold Spring Harbor Laboratory, Cold Spring 15 Harbor, N. T. (1989); L. Grossman, et al. ed., "Methods in Enzymology", Vol. 12, Parts A B, Academic Press, New York (1968); S. L. Berger et al. ed., "Methods in Enzymology", Vol. 152, p. 33 p. 215, Academic Press, New York (1987); Biochemistry, 18, 5294-5299, 1979; etc., the disclosures of 20 which are hereby incorporated by reference. Examples of such mRNA isolating and purifying techniques are a guanidine-cesium chloride method, a guanidine thiocyanate method, a phenol method, etc. If necessary, the resulting total RNA may be subjected to a purification process using an oligo(dT)- 25 cellulose column, etc. to give poly(A) mRNA. As a source for mRNA, any microorganism which belongs to the genus Fusarium and has an ability of producing D-pantolactone hydrolase may be suitably used. Examples of the microorganism belonging to the genus Fusarium which is applicable herein are Fusarium oxysporum IFO 5942, Fusarium semitectam IFO 30200, etc. Similarly, other microorganisms which belong to a member selected from the group consisting of the genera: Cylindrocarpon, Gibberella, Aspergillus, Penicillium, Rhizopus, Volutella, Gliocladium, Eurotium, Nectoria, Schizophyllum, Myrothecium, Neurospora, Acremonium, Tuberculina, Absidia, Sporothrix, Verticillium or Arthroderma and have an ability of producing D-pantolactone hydrolase may be used as a source for 1 3 mRNA. Examples of such microorganisms are Cylindrocarpon tonkinense IFO 30561, Gibberella fujikuroi IFO 6349, Aspergillus awamori IFO 4033, Penicillium chrysogenum IFO 4626, Rhizopus oryzae IFO 4706, Volutella buxi IFO 6003, Gliocladium catenulatum IFO 6121, Eurotium chevalieri IFO 4334, Nectria elegans IFO 7187, Schizophyllum commune IFO 4928, Myrothecium roridum IFO 9531, Neurospora crassa IFO 6067, Acremonium fusidioides IFO 6813, Tuberculina persicina IFO 6464, Absidia lichtheimi IFO 4009, Sporothrix schenckii IFO 5983, Verticillium malthousei IFO 6624, Arthroderma uncinatum IFO 7865, etc.

cDNAs are prepared by using, as a template, the resulting mRNA and a reverse transcriptase, etc. The reverse transcriptase synthesis of cDNA using mRNA may be carried out by standard techniques known in the art, by the substantially same techniques or by modified techniques thereof.

Detailed techniques are found in, for example, H. Land et al., "Nucleic Acids Res.", Vol. 9, 2251 (1981); U. Gubler et al., "Gene", Vol. 25, 263-269 (1983); S. L. Berger et al. ed., ~20 "Methods in Enzymology", Vol. 152, p. 307, Academic Press, New York (1987); etc., the disclosures of which are hereby incorporated by reference. The cDNA thus obtained is inserted into a commercially available phage vector or, further, subjected to a packaging by conventional techniques.

25 Then, based upon the cDNA thus prepared, cDNA libraries can be constructed.

b) Cloning of D-Pantolactone Hydrolase cDNA.

The above recombinant phage was transfected into host cells, followed by subjecting to a plaque hybridization to select positive plaques (clones). DNA fragments from the resulting clones are sequenced. The resultant nucleotide sequences are decoded and analyzed in view of an encoded amino acid sequence. As a result of such analyses and investigations, it is confirmed that the target D-pantolactone hydrolase gene is cloned.

1 4 Besides the technique using a phage vector, transformations of host cells including Escherichia coli may be conducted according to techniques known in the art, such as a calcium technique and a rubidium/calcium technique, or the substantially same methods Hanahan, J. Mol. Biol., Vol.

166, p. 557 (1983), etc.).

PCR may be conducted using the prepared cDNA as a template. In an embodiment, the primer obtained in the above 2) can be used.

With respect to a plasmid into which the D-pantolactone hydrolase gene is incorporated, any plasmid may be used as long as said DNA can be expressed in host cells conventionally used in gene engineering techniques (such as procaryotic host cells including Escherichia coli, Bacillus subtilis, etc. and eucaryotic host cells including yeasts).

In such a sequence of the plasmid, it is possible, for example, to incorporate codons suitable for expressing the cloned DNA in 20 selected host cells or to construct restriction enzyme sites.

It is also possible to contain control sequences, promotion sequences, etc. for facilitating the expression of the aimed gene; linkers, adaptors, etc. useful for ligating the aimed gene; sequences useful in controlling resistance to antibiotics 25 or in controlling metabolism or in selection; and the like.

Preferably, suitable promoters may be used. For example, such promoters may include tryptophan (trp) promoter, lactose (lac) promoter, tryptophan-lactose (tac) promoter, lipoprotein (Ipp) promoter, A phage P promoter, etc. in the case of plasmids where Escherichia coli is used as a host; and GAL1, GAL10 promoters, etc. in the case of plasmids where yeast is used as a host.

1 5 Examples of the plasmid suitable for host Escherichia coli are pBR322, pUC18, pUC19, pUC118, pUC119, pSP64, pSP65, pTZ-18R/-18U, pTZ-19R/-19U, pGEM-3, pGEM-4,

TM

pGEM-3Z, pGEM-4Z, pGEM-5Zf(-), pBluescript KSTM (Stratagene), etc. Examples of the plasmid vector suitable for expression in Escherichia coli are pAS, pKK223 (Pharmacia), pMC1403, pMC931, etc. Examples of the plasmid for host yeasts are YIp vector, YEp vector, YRp vector, YCp vector, etc., including pGPD-2, etc. Escherichia coli host cells may include those derived from Escherichia coli K12 strains, such as NM533, XL1-Blue, C600, DH1, HB101 and JM109.

In the gene engineering techniques of the present invention, it is possible to use various restriction enzymes, 15 reverse transcriptases, enzymes for DNA modification and decomposition, used for modifying or converting a DNA fragment to a structure suitable for cloning, DNA polymerases, terminal nucleotidyl transferases, DNA ligases; etc., which are known or common in the art. Examples of the restriction enzyme are 20 those disclosed in R. J. Roberts, "Nucleic Acids Res.", Vol.

13, r165 (1985); S. Linn et al. ed., "Nucleases", p. 109, Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1982; *00 *etc. Examples of the reverse transferase are those derived from mouse Moloney leukemia virus (MMLV), from avian 25 myeloblastosis virus (AMV), etc. Particularly, RNase H-deficient reverse transferase or the like is preferably used. Examples of the DNA polymerase are Escherichia coli DNA polymerase, Klenow fragment which is a derivative of E. coli DNA polymerase, E. coli phage T4 DNA polymerase, E. coli phage T7 DNA polymerase, thermoduric bacteria DNA polymerase, etc.

The terminal nucleotidyl transferase includes TdTase capable of adding a dideoxynucleotide (dNMP) to a 3'-OH terminal, as disclosed in R. Wu et al. ed., "Methods in Enzymology", Vol. 100, p. 96, Academic Press, New York (1983).

The enzyme for modifying and decomposing DNA includes exonuclease, endonuclease, etc. Examples of such enzymes are 1 6 snake toxin phosphodiesterase, spleen phosphodiesterase, E. coli DNA exonuclease I, E. coli DNA exonuclease III, E. coli DNA exonuclease VII, A exonuclease, DNase I, nuclease SI, Micrococcus nuclease, etc. Examples of the DNA ligase are E. coli DNA ligase, T4 DNA ligase, etc.

The vector (or vehicle) which is suitable for cloning DNA genes and constructing DNA libraries includes plasmid,A phage, cosmid, P1 phage, F factor, YAC, etc.

Preferred examples of such vectors are vectors derived from A phage, such as Charon 4A, Charon 21A,A gtlO, A gtll,

TM

A DASHII, A FIXII, 'A EMBL3 and A ZAPIITM (Stratagene), etc.

In addition, based upon the gene nucleotide sequence 15 encoding the D-pantolactone hydrolase of the present invention, methods and means conventionally used in gene engineering techniques enable us to manufacture proteins, such as variants and mutants, wherein a modification is introduced into the amino acid sequence of the D-pantolactone hydrolase in such 20 a manner that one or more amino acid(s) is/are substituted, deleted, inserted, translocated or added.

Examples of the methods and means for such a variation, substitution and modification are those disclosed in Nippon Seikagaku Kai (Biochemical Society of Japan) ed., 25 "Zoku-Seikagaku Jikken Kouza 1, Idensi Kenkyuho II Lectures on Biochemical Experiments (Second Series; Methods for Gene Study p.105 (Susumu Hirose), Tokyo Kagaku Dojin, Japan (1986); Nippon Seikagaku Kai (Biochemical Society of Japan) ed., "Shin-Seikagaku Jikken Kouza 2, Kakusan III (Kumikae DNA Gijutsu) (New Lectures on Biochemical Experiments 2, Nucleic Acids III (Recombinat DNA Technique))", p. 233 (Susumu Hirose), Tokyo Kagaku Dojin, Japan (1992); R. Wu, L. Grossman, ed., "Methods in Enzymology", Vol. 154, p. 350 and p. 367, Academic Press, New York (1987); R. Wu, L.

Grossman, ed., "Methods in Enzymology", Vol. 100, p.457 and p. 468, Academic Press, New York (1983); J. A. Wells et al., "Gene", Vol. 34, p. 315 (1985); T. Grundstroem et al., 1 7 "Nucleic Acids Res.", Vol. 13, p. 3305 (1985); J. Taylor et al., "Nucleic Acids Res.", Vol. 13, p. 8765 (1985); R. Wu, ed., "Methods in Enzymology", Vol. 155, p. 568, Academic Press, New York (1987); A. R. Oliphant et al., "Gene", Vol. 44, p.177 (1986); etc., the disclosures of which are hereby incorporated by reference. Examples of such methods and means are techniques utilizing synthetic oligonucleotides for introducing a mutation or variation into a specific site (site-directed mutagenesis techniques), Kunkel techniques, dNTP[a S] techniques (Eckstein method), techniques using sulfurous acid (or bisulfite), nitrous acid (or nitrite), etc. for introducing a mutation or variation into a specific domain or area, etc.

Moreover, the resulting protein according to the 15 present invention may be subjected to chemical techniques whereby an amino acid residue(s) contained therein is(are) 0 modified or may be made into its(their) derivative(s) by subjecting to a partial decomposition or a modification using an enzyme such as peptidase (for example, pepsin, chymotrypsin, 20 papain, bromelain, endopeptidase, exopeptidase, etc.).

It is also possible to express, as fusion proteins, the recombinant proteins of the present invention on the manufacture by means of gene recombinant techniques and then to convert/process the fusion proteins in vivo or in vitro to 25 products having a biological activity substantially equivalent to a natural D-pantolactone hydrolase. A fusion production conventionally used in gene engineering techniques may be used as well. Such a fusion protein may be purified by means of an affinity chromatography, etc. utilizing its fusion part.

Modifications, alterations, etc. of protein structures are found, for example, in Nippon Seikagaku Kai (Biochemical Society of Japan) ed., "Shin-Seikagaku Jikken Kouza 1, Tanpakushitsu VII, Tanpakushitsu Kogaku (New Lectures on Biochemical Experiments 1, Protein VII, Protein Engineering)", Tokyo Kagaku Dojin, Japan (1993), the disclosures of which are hereby incorporated by reference. Such modifications, alterations, etc. may be conducted according to techniques 1 8 disclosed therein, techniques disclosed in references cited therein, and those substantially similar thereto.

Thus, the products according to the present invention may include either proteins wherein one or more amino acid residue(s) is/are different from that/those of the natural one in terms of identity or proteins wherein one or more amino acid residue(s) is/are shifted from the position(s) of the natural one. The products according to the present invention may include deletion analogs wherein one or more amino acid residue(s) specified for the natural D-pantolactone hydrolase is/are deficient therefrom (for example, 1 to 80, preferably 1 to 60, more preferably 1 to 40, still more preferably 1 to and particularly preferably 1 to 10 amino acid residue(s) specified for the natural D-pantolactone hydrolase is/are deficient therefrom); substitution analogs, wherein one or more amino acid residue(s) specified for the natural D-pantolactone hydrolase is/are replaced with other residue(s) (for example 1 to 80, preferably 1 to 60, more preferably 1 to 40, still 20 more preferably 1 to 20 and particularly preferably 1 to amino acid residue(s) specified for the natural D-pantolactone hydrolase is/are replaced with other residue(s)); and addition analogs, wherein one or more amino acid residue(s) is/are added to the sequence specified for the natural 25 D-pantolactone hydrolase (for example 1 to 80, preferably 1 to 60, more preferably 1 to 40, still more preferably 1 to and particularly preferably 1 to 10 amino acid residue(s) is/are added to the amino acid sequence specified for the natural D-pantolactone hydrolase. The products may include proteins wherein a domain structure characteristic to the natural D-pantolactone hydrolase is contained or retained.

Further, the products may include proteins having the same quality in view of D-pantolactone hydrolase activity as the natural D-pantolactone hydrolase.

The products of the present invention may include all of the variants and analogs as mentioned herein above, as long as they have the domain structure which is characteristic to the naturally-occurring D-pantolactone hydrolase. It is also believed that the products of the present invention may include all proteins having a primary structural conformation substantially equivalent to that of the naturally-occurring D-pantolactone hydrolase according to the present invention and those having a portion of the primary structural conformation of naturally-occurring D-pantolactone hydrolase according to the present invention.

It is further believed that the products of the present invention may include proteins sharing all or part of the biological properties of naturally-occurring D-pantolactone S 15 hydrolase or having a biological activity substantially equivalent to that of the natural D-pantolactone hydrolase.

Furthermore, the product of the present invention may include one of the variants which naturally occur. The D-pantolactone hydrolase products of the present invention can be separated, 20 isolated or/and purified as illustrated hereinafter.

Further, the products according to the present invention may include DNA sequences coding for the abovementioned polypeptide and DNA sequences encoding 25 D-pantolactone hydrolase polypeptides (including analogs and derivatives thereof) having all or part of the natural characteristics of the naturally-occurring D-pantolactone hydrolase. Said D-pantolactone hydrolase nucleotide sequences may also be modified (such as inserted, added, deleted and substituted). Thus, the products according to the present invention may include such modified nucleotide sequences as well.

Since the DNA sequences of the present invention provide information on the amino acid sequence of D-pantolactone hydrolase protein which has heretofore been unavailable, utilization of such information is within the 2 0 scope of the present invention as well. Such utilization may include designing of probes for isolation and/or detection of genomic DNA and cDNA coding for D-pantolactone hydrolase or proteins related thereto, of microorganisms, or particularly preferably microorganisms having an ability of producing D-pantolactone hydrolase, such as those belonging to a member selected from the group consisting of the genera: Fusarium, Cylindrocarpon, Gibberella, Aspergillus, Penicillium, Rhizopus, Volutella, Gliocladium, Eurotium, Nectoria, Schizophyllum, Myrothecium, Neurospora, Acremonium, Tuberculina, Absidia, Sporothrix, Verticillium and Arthroderma with an ability of producing D-pantolactone hydrolase.

The DNA sequences of the present invention are *8*6 15 valuable, for example, as probes for isolation and/or detection of genomic DNA and cDNA coding for D-pantolactone hydrolase or proteins related thereto, of microorganisms having an ability of producing D-pantolactone hydrolase, or particularly fotf preferably microorganisms belonging to the above-mentioned 20 genus, including the Fusarium, etc. Isolation of the gene may .too be carried out by utilizing PCR techniques or RT-PCR techniques (PCR using a reverse transcriptase D-Pantolactone hydrolase DNA and its related DNA may be utilized for isolation, detection, etc. of genes related to 25 D-pantolactone hydrolase by means of PCR techniques, RT-PCR techniques or other methods, using a DNA primer obtained by a chemical synthesis as a result of selecting a characteristic domain (or portion) based upon a putative amino acid sequence derived from the cloned and sequenced D-pantolactone hydrolase cDNA sequence and of designing the DNA primer relied on the selected domain (or portion).

As mentioned hereinabove, the present invention provides a process for producing the aimed D-pantolactone hydrolase which comprises importing a recombinant D-pantolactone hydrolase DNA molecule and/or gene into hosts followed by expressing the D-pantolactone hydrolase therein.

-2 1 Thus, in accordance with the present invention, recombinants (transformants) or transfectants which are endowed with the capacity to substantially express the same; and use thereof are provided.

Another aspect of the present invention also relates to nucleic acids, such as DNA and RNA, which enable the expression in eucaryotic or procaryotic host cells, such as Escherichia coli host cells of proteins or salts thereof having a D-pantolactone hydrolase activity; proteins or salts thereof characterized in having a substantially equivalent activity thereto; or polypeptides having all or at least a part of a D-pantolactone hydrolase protein or a salt thereof (more 15 preferably D-pantolactone hydrolase protein originating in Fusarium oxysporum) and having the substantially equivalent activity or the substantially same primary structural conformation.

In addition, such a nucleic acid, particularly DNA, 20 may be: a sequence capable of encoding the amino acid sequence of SEQ ID NO:1 or a sequence complementary thereto; a sequence capable of hybridizing with said DNA sequence or a fragment thereof; and a sequence having a degenerate code capable of hybridizing with the sequence or The characteristics of the present invention reside in eucaryotic or procaryotic host cells, such as Escherichia coli host cells, transformed or transfected with such a nucleic acid, which are endowed with the capacity to express said polypeptide of the present invention.

It may also be possible in accordance with the present invention to obtain a microorganism in which its ability to produce D-pantolactone hydrolase is modified by introducing 2 2 DNA coding for a protein having a D-pantolactone hydrolase activity or a protein having the substantially equivalent activity thereto or (ii) DNA, such as vector, containing said DNA into said microorganism in an expressible manner. Such microorganisms possessing the ability to produce D-pantolactone hydrolase may include microorganisms belonging to a member selected from the group consisting of the genera: Fusarium, Cylindrocarpon, Gibberella, Aspergillus, Penicillium, Rhizopus, Volutella, Gliocladium, Eurotium, Nectoria, Schizophyllum, Myrothecium, Neurospora, Acremonium, Tuberculina, Absidia, Sporothrix, Verticillium and Arthroderma. Examples of such microorganisms are Fusarium oxysporum IFO 5942, Fusarium semitectam IFO 30200, Cylindrocarpon tonkinense IFO 30561, 15 Gibberella fujikuroi IFO 6349, Aspergillus awamori IFO 4033, Penicillium chrysogenum IFO 4626, Rhizopus oryzae IFO 4706, Volutella buxi IFO 6003, Gliocladium catenulatum IFO 6121, Eurotium chevalieri IFO 4334, Nectria elegans IFO 7187, Schizophyllum commune IFO 4928, Myrothecium roridum IFO 9531, 20 Neurospora crassa IFO 6067, Acremonium fusidioides IFO 6813, Tuberculina persicina IFO 6464, Absidia lichtheimi IFO 4009, Sporothrix schenckii IFO 5983, Verticillium malthousei IFO 6624, Arthroderma uncinatum IFO 7865, etc.

Transformation may include techniques in which protoplast cells prepared by the use of a suitable cell wall lytic enzyme are contacted with DNA in the presence of calcium chloride, polyethylene glycol, etc.; electroporation techniques (see: for example, E. Neumann et al., EMBO J, Vol. 1, pp. 841 (1982), etc.); microinjection techniques; shot gun methods for shooting a gene with a gun; etc.

The enzymes can be isolated and prepared by purifying techniques from various materials, such as produced enzyme materials including cell growth culture medium, disrupted cultured cells, transformed cells, etc. The purification may include methods known in the art, including salting out -2 3such as precipitation with ammonium sulfate; gel filtration using Sephadex or the like; ion exchange chromatography technique using, for example, a carrier having a diethylaminoethyl group or a carboxymethyl group; hydrophobic chromatography technique using, for example, a carrier having hydrophobic groups including a butyl group, an octyl group, a phenyl group, etc.; pigment gel chromatography technique; electrophoresis technique; dialysis; ultrafiltration; affinity chromatography technique; high performance liquid chromatography technique; etc.

When the enzyme is obtained as an inclusion body, it may be subjected to a solubilizing treatment using, for example, a denaturing agent, such as guanidine hydrochloride and urea, and, if necessary, in the presence of a reducing agent, such as 2-mercaptoethanol and dithiothreitol, whereupon an activated form of the enzyme is produced.

For enzyme materials, enzyme-producing cells per se may be used instead. Immobilized enzymes may include products prepared by immobilizing the enzyme or enzyme-producing cells 20 according to techniques known in the art. The immobilization can be conducted by carrier bonding techniques, such as a covalent method and an adsorption method, a cross-linking method, an encapsulation, etc. The immobilization can also be conducted using a condensing agent such as glutaraldehyde, 25 hexamethylene diisocyanate and hexamethylene diisothiocyanate if necessary. In addition, monomer techniques in which monomers are gelled in a polymerization, prepolymer techniques in which molecules having bigger size than conventional monomers are polymerized, polymer techniques in which polymers are gelled, etc. may be exemplified. It may include an immobilization using polyacrylamide, an immobilization using natural polymers such as alginic acid, collagen, gelatin, agar and K -carrageenan, an immobilization using synthetic polymers such as photosetting.resins and urethane polymers, etc.

It may be possible to carry out the optical resolution of lactone compounds by an enzymatic asymmetric hydrolysis utilizing a lactone hydrolase (such as a D-pantolactone -2 4hydrolysis using a culture of microorganisms and enzymes), as well as treatment of products obtained thereby in the same manner as disclosed in Unexamined Japanese Patent Publication (KOKAI TOKKYO) Nos. Hei 3-65,198 and Hei 4-144,681.

For example, the transformed microorganisms (transformants) thus obtained are subjected to shaking culture in a liquid medium. The resulting cultured cells are harvested, to which an aqueous solution of D,L-pantolactone (concentrations: 2 to 60%) is added. The mixture is made to react at 10 to 40 0 C for from several hours to one day while adjusting the pH to from 6 to 8. After completion of the reaction, the cells are separated and the unreacted L-pantolactone in the reaction solution is separated by extracting with an organic solvent (preferably an ester such as ethyl acetate, an aromatic hydrocarbon such as benzene or a halogenated hydrocarbon such as chloroform). D-Pantoic acid remaining in the aqueous layer is heated under an acidic condition with hydrochloric acid to conduct a lactonation 'ego 20 followed by extracting with the above-mentioned organic solvent whereupon the resulting D-pantolactone is obtained. As such, processed cells (dried cells, immobilized cells, etc.) of the transformed microorganisms or enzymes and immobilized enzymes obtained from the transformed cells can be used in the same manner as well.

As a result of utilization of various embodiments of the present invention as mentioned hereinabove, it is now possible to provide various technical means, such as means valuable or useful for the synthetic studies concerning an optical resolution of lactone compounds by an enzymatic asymmetric hydrolysis utilizing a lactone hydrolase (for example, D-pantolactone hydrolase) as well as means applicable to other uses. The present invention will be more specifically illustrated by way of the following examples although it is to be understood that the present invention is not limited to such examples but various embodiments within the spirit of this specification are possible.

Incidentally, when nucleotides (bases) and amino acids are indicated by abbreviations in the specification and in the drawings, they depend upon an "IUPAC-IUB Commission on Biochemical Nomenclature" or upon the meanings of the terms which are commonly used in the art. When optical isomers are present in amino acids, an L-isomer is referred to unless otherwise specified.

The transformant Escherichia coli, designated JM109 (EJM-ESE-1) having a recombinant vector (PFLC40E) into which the enzyme D-pantolabtone hydrolase gene is integrated and obtained in Example 1 mentioned herein below has been deposited as from August 30, 1995 (original deposit date) with the National Institute of Bioscience and Human Technology (NIBH), Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan,,located at 1-3, Higashi 1-chome, Tsukuba-shi, IBARAKI (Zip Code: 305), JAPAN and has been assigned the Accession Number FERM P-15141.

20 The original deposit of the transformant E. coli JM109 (EJM-ESE-1) has been transferred to one under the Budapest Treaty by a request dated August 28, 1996 and is on deposit with the Accession Number FERM BP-5638 under the terms of the Budapest Treaty at NIBH.

EXAMPLES

Described below are examples of the present invention which are provided only for illustrative purposes, and not to limit the scope of the present invention. In light of the present disclosure, numerous embodiments within the scope of the claims will be apparent to those of ordinary skill in the art.

2 6 Example 1 1) Amino Acid Sequencing of Purified Enzyme.

A sample of freeze-dried D-pantolactone hydrolase (14.3 nmol; subunit molecular weight: 60,000) prepared according to Example 1 in Unexamined Japanese Patent Publication (KOKAI TOKKYO) No. Hei 4-144,681 was dissolved in 44g 1 of 50 mM Tris-HCl (pH: 9.0) containing 8M urea and was denatured at 37 0 C for 1 hr. To this solution was added 44u 1 of 50 mM Tris-HC1 (pH: 9.0) whereupon the urea concentration was made 4M. Then 12 g 1 (0.144 nmol; E/S 1/100) of 12 nmol/ml of lysyl endopeptidase (Wako Pure Chemicals, Japan; was added thereto and a digestion was carried out at 30 0 C for 12 hrs. The resulting digested peptide was collected by means S: of a reversed phase column (Nakarai Tesuku, Japan) and analysis S 15 of the amino acid sequence was carried out using a 477A Protein Sequencer (ABI, USA).

Collecting Conditions Column: Cosmosil 5C18-AR (4.6 x 250 mm) 20 Flow Rate: 1 ml/min.

Temperature: Detecting Wave Length: 210 nm Eluting Solution: A, 0.1% TFA (TFA: trifluoroacetic acid) B, 0.1% TFA/80% CH CN 25 Eluting Conditions: Gradient elution of A B Results of the amino acid sequencing was as shown in Figures 1 and 2.

2) Preparation of Genomic DNA.

a) Process for the Extraction of Genomic D-Pantolactone Hydrolase DNA Cultured cells at an anaphase of a logarithmic growth phase were harvested by means of a filtration in vacuo. The cells were placed in liquid nitrogen and finely disrupted using 2 7 a Waring Blender. The cell mixtures which were made fine to some extent were transferred to a mortar and ground together with the addition of liquid nitrogen. This product was suspended in a 2 x CTAB solution CTAB (CTAB: cetyl trimethylammonium bromide; Sigma, USA), 0.1M Tris-HC1 (pH 1.4M NaC1 and 1% PVP (PVP: polyvinylpyrrolidone; Sigma, USA)) kept at 70 0 C and incubated at 65 0 C for 3-4 hours.

The supernatant liquid obtained by centrifugation was successively treated with phenol, phenol/chloroform and chloroform and the resultant solution was then treated with the same volume of isopropanol to precipitate DNA. This DNA paste was washed with 70% ethanol, air-dried and dissolved in a TE buffer (10mM Tris and ImM EDTA; pH RNA was decomposed with ribonuclease A and ribonuclease T1. Then the DNA product was successively treated with phenol, phenol/chloroform and chloroform to remove the protein therefrom. The resultant product was treated with the same volume of isopropanol to precipitate DNA. This DNA was washed with 70% ethanol, air-dried and dissolved in a TE buffer to afford a genome 20 sample.

b) Amplification of D-Pantolactone Hydrolase Gene.

25 Based upon the information on amino acid sequences (Figures 1 and 2) of D-pantolactone hydrolase internal peptides, a sense primer corresponding to a sense strand coding for the N-terminal amino acid sequence and an antisense primer corresponding to an antisense strand for the internal peptide sequence were synthesized (Figure 3).

PCR was carried out under the following conditions using, as a template, a genomic DNA sample of D-pantolactone hydrolase: The PCR was conducted by the techniques mentioned in the art, for example, in R. Saiki, et al., Science, Vol. 230, PP. 1350 (1985); R. Saiki, et al., Science, Vol. 239, pp. 487 -2 8- (1988); PCR Technology, Stockton Press (1989); etc.

As a result of the PCR, amplified DNA fragments with about 1 kb were obtained.

PCR Conditions Genomic DNA: 2.5 u g Sense Primer: 250 pmol (cf. Figure 3) Antisense Primer: 250 pmol (cf. Figure 3) dNTP (2 mM): 5 u 1 Tth Polymerase Buffer (x 10): 5 u 1 Tth DNA Polymerase (Toyobo, Japan): 3 units

H

2 0: Total 50 u 1 15 The cycle for amplification including 92 OC for 1 min., 55 °C for 1 min. and 73 OC for 3 min. was repeated 30 times.

The resulting amplified DNA fragments were subjected a sequencing and the disclosed DNA sequence was decoded to an amino acid sequence whereby a portion corresponding to the partial amino acid sequence of the D-pantolactone hydrolase •internal peptide was found among the decoded amino acid sequences.

S

3) Preparation of cDNA.

a) Preparation of mRNA.

Cultured cells were harvested at a prophase of the logarithmic growth phase, immediately frozen with liquid nitrogen, disrupted and subjected to an AGPC (Acid Guanidinium Thiocyanate Phenol Chloroform Method; see, for example, Jikken Igaku, Vol. 15, p. 99 (1991)) to extract total RNA. The resulting total RNA was subjected to an oligo dT-cellulose column (Pharmacia) for purification to afford a mRNA fraction.

2 9) b) Preparation of cDNA Library.

The resulting mKNA was used as a template for synthesizing cDNA by a cDNA rapido adaptor ligation module (cDNA synthesis module RPN 1256, 1994; Amersham International PLC) and the cDNA was used for construction of cDNA Libraries.

c) Cloning of D-Pantolactone llydrolase cDNA.

The cDNA libraries were infected to host Escherichia coli cells and positive plaques were selected by means of a plaque hybridization. In the plaque hybridization, probes used for selection were prepared by using about 1 kb fragments containing Fusarium oxysporum D-pantolactone hydrolase gene and by labeling the about 1 kb fragments according to a multiprime method. The resulting positive clone was sequenced and the 15 disclosed DNA sequence was decoded to an amino acid sequence whereby it was found that the full length of the above D-pantolactone hydrolase gene was successfully cloned.

As such, the isolated and sequenced DNA has a nucleotide sequence of SEQ ID NO:2. The sequence showing a homology with the amino acid sequence represented by SEQ ID NO:1 encoded by this nucleotide sequence is not present in the Protein Sequence Data Bank of NBRF (National Biomedical Research Foundation). Thus, the DNA having this nucleotide sequence has been found to be entirely novel.

It was found that, in the cDNA where the nucleotides were sequenced, a part of the N-terminal region was lacked and there was no initiation codon therein. Therefore, an initiation codon was artificially incorporated into the cDNA by a PCR technique to construct a vector for expressing the gene (PFLC 40E). The technique used to clone the 5' end of the molecule was RACE (rapid amplification of DNA ends), and this proved essential to overcome the practical difficulties described above which were encountered in cloning the genes. Moreover, based on a comparison between the cDNA and genomic DNA there have been two sites in which 29a the nucleotide sequences are different (other, of course, than merely through the presence of introns in the genomic DNA) which suggests that RNA editing takes place in the organisms which express D-pantolactone hydrase.

Sense and antisense oligonucleotide primers having the restriction enzyme sites as shown in Figure 4 were synthesized. PCR was carried out utilizing those primers under the following condLtions: •go o• •go• ooo ••go I:\Mz-P.\Yeep\ Spec t, P S944 L dcoc 27, 09 /00 3 0 The PCR was conducted by the techniques mentioned in the art, for example, in R. Saiki, et al., Science Vol. 230, pp. 1350 (1985); R. Saiki, et al., Science, Vol. 239, pp. 487 (1988); and PCR Technology, Stockton Press (1989).

PCR Conditions Total DNA (cDNA): 10 g Sense Primer: 0.1 nmol (cf. Figure 4) Antisense Primer: 0.1 nmol (cf. Figure 4) dNTP (2 mM): 10 u 1 Tth Polymerase Buffer (x 10): 10 g 1 Tth DNA Polymerase: 4 units

H

2

O:

Total 100 U 1 e* The cycle for amplification including 94 OC for 1 min., 55 OC for 1 min. and 75 OC for 3 min. was repeated 30 times.

The PCR products prepared as such had each S 20 restriction enzyme EcoRI and XbaI sites at their both terminals.

Therefore, each of them was treated with EcoRI (Takara Shuzo, Japan) and XbaI (Takara Shuzo, Japan) followed by a ligation with pUC18 (Takara Ligation Kit; Takara Shuzo, Japan) whereby the expression vector (PFLC40E) was constructed.

Then said vector was transfected into E. coli JM 109 competent cells according to a technique as mentioned in "Molecular Cloning", Second Edition, 1989, edited by J.

Sambrook, et al., Cold Spring Harbor Laboratory Press, to transform host cells. The target transformants were selected on a 2 x YT medium tryptone, 1% yeast extract and NaC1) containing 50 mg/liter ampicillin. The transformation was done according to a calcium chloride technique.

The transformant E. coli prepared as such was precultured in a test tube containing 10 ml of the abovementioned 2 x YT medium containing 50 mg/liter ampicillin and then the resulting precultured solution (100u 1 in total) was 3 1 used as seed cells for checking culture time, culture temperature and periods for adding isopropyl-8 -thiogalactopyranoside (IPTG) in 100 ml of main culture broths having the same composition as the preculture broth.

Results of the culture is shown in Table 1.

Upon the construction of large-scale expression systems in E. coli, the lactonase proteins Dpantolactone hydrase proteins) have been recognized to be expressed in a large amount at usual culturing temperatures such as 37'C and 28"C but a ratio of active D-pantolactone hydrase proteins is extremely poor at such temperatures.

Unexpectedly, when at the culture temperature of it is possible to isolate recombinant D-pantolactone "hydrase proteins as active ones.

15 After the cultivation, the resulting harvested cells were disrupted by ultra-sonication and centrifuged to afford a supernatant. The resultant supernatant was measured in-view of D-pantolactone hydrolase activity.

The specific activity was 2.25 U/mg at an optimal condition. Enzymatic activities of the recombinant proteins were assayed in view of D-pantolactone hydrolase under the following conditions: The enzymatic activity capable of hydrolyzing 1 u mol of D-pantolactone per minute was defined as one unit 25 To 200u 1 of 10% D-pantolactone solution in 0.5M PIPES buffer (pH 7.0) was added 50 a 1 of an enzyme solution and the mixture was made to react at 30 OC for 120 minutes followed by adding 250 1 of 2 mM EDTA in methanol to quench the reaction.

After completion of the reaction, the liquid reaction mixture was subjected to an HPLC (Nucleosil 5C18 4.6 x 150 mm; eluent: 10% methanol; flow rate: 1 ml/minute; detection wavelength: 230 nm) to determine the hydrolysis. For example, where the hydrolysis is the enzymatic activity/ml of -2 the enzyme solution corresponds to 1.6 x 10 U/ml.

The transformant E. coli JM109, transformed with was cultured in a 2 x YT medium. IPTG was added thereto to make its final concentration 2mM.

-3 2- 4** Table 1 Time for Supply- Culturing Culturing Tem- Specific Actiing IPTG (hr) Time (hr) perature (OC) vity(units/mg) 0 6 28 0.86 0 12 28 1.94 4 7 28 1.33 4 12 28 2.25 0 6 37 1.05 0 12 37 1.73 4 7 37 1.31 4 12 37 1.67 IPTG was added to the 2 x YT medium together with the 15 initiation of the culture.

IPTG was added to the 2 x YT medium after four hours from the initiation of the culture.

20 As a result of an SDS-PAGE, a deep band with an expected molecular weight was detected for an insoluble fraction of the centrifuged precipitate. Therefore, the band was subjected to a blotting and the sample was investigated in view of an N-terminal amino acid sequence by an Edman degradation technique whereby its N-terminal amino acid sequence was found to be identical with that of D-pantolactone hydrolase.

Accordingly, it is likely that, although the recombinant D-pantolactone hydrolase was in part expressed as a soluble form in this E. coli expression system for expressing the D-pantolactone hydrolase cDNA, most of the recombinant D-pantolactone hydrolase is expressed as an inclusion body.

The transformant Escherichia coli, designated JM109 (EJM-ESE-1), having a recombinant vector (PFLC40E) into which the above-mentioned enzyme D-pantolactone hydrolase gene is integrated has been deposited and stored with the National Institute of Bioscience and Human Technology (NIBH), 3 3 Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan, located at 1-3, Higashi 1-chome, Tsukuba-shi, IBARAKI (Zip Code: 305), JAPAN.

The transformant E. coli JM109 (EJM-ESE-1) has been assigned the Accession Number FERM BP-5638 by NIBH. A request for transferring the original deposit (Accession Number FERM P-15141 deposited on August 30, 1995) to one under the Budapest Treaty was submitted on August 28, 1996.

J

33a Example 2: Expression in A. oryzae A. oryzae/pNALC22 Plasmid pFLC40E which was already cloned in E. coli in advance was isolated and cut with restriction enzymes, EcoRI and XbaI to give a DNA fragment containing Lactonase cDNA D-pantolactone hydrolase cDNA) which was blunted followed by insertion into PmaCI site at the downstream of P-No8142 promoter in expression vector pNAN8142 (to form pNALC22). The pNALC22 was expressed in E. coli microorganisms, and established using as an indicator its Amp resistance.

Next, the cloned pNALC22 vector was introduced into A. oryzae for transformation. The transformed cells were selected on Czapeck medium.

Mycelia and spores were harvested from the culture of A. oryzae/pNALC22 and tested for enzyme activity (Tables3 4 Simultaneously, various lactonase-active microorganisms (F.

oxysporum AKU3702, E. coli JM109/pFLC40E) were also tested.

A. oryzae/pMELC252 Plasmid pFLC40E which was already cloned in E. coli in advance was isolated and used as a template for PCR to form Lactonase cDNA to both ends of which an SalI site was added. This DNA fragment was inserted into SalI site at the downstream of melO promoter in expression vector pMENB (to form pMELC252). The pMELC252 was expressed in E. coli microorganisms, and established using as an indicator its Amp S" resistance.

Next, the cloned pMELC252 vector was introduced into A. oryzae for transformation. The transformed cells were cultured on Czapeck medium.

Mycelia and spores were harvested from the culture of A. oryzae/pMELC252 and tested for enzyme activity (Tables 3 4 As a result, it has been observed that the A. oryzae/pMELC2 5 2 has the property of hydrolyzing D-pantolactone (D-PL).

33b medium Each microorganism cell was cultivated in the following medium: medium for Fusarium Glucose Polypeptone Yeast Extract Corn Steep Liquor 1 0. 5 0. 5 0. 5 (pH 6) Medium for Aspergillus mycelium DP Medium Dextrin Polypeptone KH PO 0 2 4 NaNO 3 0 MgSO 4'7H20 0 Medium for Aspergillus spore Czapeck medium Glucose NaNO 3 0 KH PO 0 2 4 KCl 0 MgSO *7H 20 0 FeSO '7H 0 0 4 2 Agar 1 2 .5 .1 .05 (pH not adjusted) a

S

5 S S

S

2 .2 .1 .05 .05 .001 .5 (pH 5. 'was 00 0 0.0.

0000 .000 Bran Medium Grain Bran Glucose Yeast Extract HO0 2 40 g 0. 2 g 0.05g 24 g Ce) Medium for E. coli 2 x YT medium LB medium 33c Cultivation Fusarium Fusarium cells were sub-cultured in a test tube (medium: 5 ml) at 28 0 C for 2 days under 300 rpm, then cultured in a 2L Sakaguchi's flask (medium: 500 ml) at 28 0 C for 7 days under 150 rpm, and harvested.

Aspergillus Aspergillus cells were sub-cultured in a test tube (DP medium: 5 ml) at 28 0 C for 2 days under 300 rpm, then cultured in a 2L Sakaguchi's flask (DP medium: 500 ml) at 28 0

C

for 7 days under 150 rpm, and filtered by suction to afford mycelia.

Aspergillus cells were also grown on a Czapeck slant.

Next, to the culture was added water at an amount approximately equal to the medium and the mixture was stirred, filtered with gauze to give a filtrate which was centrifuged to yield spores.

The resultant spores were suspended in sterilized water, added to a bran medium, allowed to stand at 28°C for 10 days for cultivation, filtered with gauze to give a filtrate which was centrifuged to yield spores.

S

5@ E. coli E. coli cells were sub-cultured in a test tube (2 x YT medium: 5ml, containing 40u g/mL of ampicillin) at 28 0

C

for 12 hours, then cultured in a 2L Sakaguchi's flask (LB medium: 500 ml, containing 40 u g/mL of ampicillin) at 28 0 C for 12 hours, and harvested, provided that IPTG was added so as to bring the final IPTG concentration to 1 mM at 4 hours after the initiation of the second cultivation step.

0 The results are shown in Table 2 @5CC 33d Assay Each enzymatic activity was tested as follows: Reaction mixtures each containing IM Tris-HCl (pH 7.4), 2% w/v D- or L-PL and 50mg of microorganism cells were prepared to bring each volume to 1ml. The mixture was shaken at 28°C for 2 hours. The reaction was stopped by adding 2ml of MeOH to the reaction mixture. Each sample was measured by HPLC.

Microorganism cells which was measured for wet weight in advance were dried at 100"C overnight. Each (dry weight/wet weight) was calculated.

e

ST?

-u- 3, 33e, Table 2 microorganism medium wet Cell weight (g) A. oryzae/pNALC22 DP 38.7 A. oryzae/pMELC252 DP 40.9 A. oryzae/pNAN8142 DP 34.6 A. oryzae/pMENB DP 32.3 A. oryzae/pNALC22 grain bran 8.73 A. oryzae/pMELC252 grain bran 7.02 A. oryzae/pNAN8142 grain bran 4.20 A. oryzae/pMENB grain bran 2.64 E. coli JM1O9/pFLC4OE 2.84 E. coli JM109/pUC18 1.34 F. oxysporum AKU3702 43.0 33f Table 3 Substrate: D-pantolactone Hydrolysis Hydrolysis Hydrolysis microorganism Rate Rate Rate M% (%/wet mg) Cdry mg) A. oryzae/pNALC22 64.8 1.2 11.2 A. oryzae/pNAN8l42 8.9 0.2 1.2 Mycelium A. oryzae/pMELC252 84.6 1.7 12.7 A. oryzae/pMENB 8.5 0.2 1.2 A. oryzae/pNALC22 76.0 1.5 7.1 A. oryzae/pNAN8142 2.1 0.0 0.2 Spore A. oryzae/pMELC252 79.9 1.6 7.7 A. oryzae/pMENB 7.7 0.2 0.7 E. coli JM1O9/pFLC4OE 84.0 1.7 10.3 E. coli JM1O9/pUC18 0.0 0.0 0.0 F. oxysporum AKU3702 52.4 1.1 6.2 Control 0- E. coli JM1O9/pUC18 is a host cell, a cell which has not yet transformed with a D-pantolactone hydrolase gene.

33g Table 4 Substrate: L-pantolactone Hydrolysis Hydrolysis Hydrolysis Microorganism Rate Rate Rate (%/wet rag) (%/dry mag) A. oryzae/pNALC22 13.0 0.27 2.38 A. oryzae/pNAN8142 3.9 0.08 0.56 mycelium A. oryzae/pMELC252 0.0 0.00 0.00 A. oryzae/pMENB 0.0 0.00 0.00 A. oryzae/pNALC22 5.4 0.11 0.52 A. oryzae/pNAN8l42 6.3 0.13 0.62 Spore A. oryzae/pMELC252 5.6 0.11 0.54 A. oryzae/pMENB 4.2 0.08 0.38 E. coli JM1O9/pFLC4OE 0.0 0.00 0.00 E. coli JM1O9/pUC18 2.2 0.04 0.23 F. oxysporum AKU3702 0.0 0.00 0.00 Control 0- 33h INDUSTRIAL APPLICABILITY The present invention discloses gene structures coding for naturally-occurring D-pantolactone hydrolase (such as natural D-pantolactone hydrolase originating in Fusarium oxysporum) or for proteins having a substantially equivalent activity thereto. Thus, significant developments can be expected in applications, including uses of host cells which are transformed with DNA containing the nucleotide sequence coding for said protein, processes for the preparation of said protein using said host cells and manufacturing processes for producing D-pantolactone using such proteins and host cells. In addition, it is possible to afford a significant increase in the enzymatic activity by modification of the D-pantolactone hydrolase per se.

3 -34- SEQUENCE LISTING INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 380 TYPE: Amino acid TOPOLOGY: Linear (ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Ala Lys Leu Pro Ser Thr Ala Gin Ile Ile Asp Gin Lys Ser Phe Asn 1 5 10 Val Leu Lys Asp Val Pro Pro Pro Ala Val Ala Asn Asp Ser Leu Val 25 Phe Thr Trp Pro Gly Val Thr Glu Glu Ser Leu Val Glu Lys Pro Phe 40 His Val Tyr Asp Glu Glu Phe Tyr Asp Val Ile Gly Lys Asp Pro Ser 50 55 Leu Thr Leu Ile Ala Thr Ser Asp Thr Asp Pro Ile Phe His Glu Ala 70 75 Val Val Trp Tyr Pro Pro Thr Glu Glu Val Phe Phe Val Gin Asn Ala 85 90 Gly Ala Pro Ala Ala Gly Thr Gly Leu Asn Lys Ser Ser Ile Ile Gin 100 105 110 Lys Ile Ser Leu Lys Glu Ala Asp Ala Val Arg Lys Gly Lys Gin Asp 115 120 125 Glu Val Lys Val Thr Val Val Asp Ser Asn Pro Gin Val Ile Asn Pro 130 135 140 Asn Gly Gly Thr Tyr Tyr Lys Gly Asn Ile Ile Phe Ala Gly Glu Gly 145 150 155 160 Gin Gly Asp Asp Val Pro Ser Ala Leu Tyr Leu Met Asn Pro Leu Pro 165 170 175 Pro Tyr Asn Thr Thr Thr Leu Leu Asn Asn Tyr Phe Gly Arg Gin Phe 180 185 190 Asn Ser Leu Asn Asp Val Gly Ile Asn Pro Arg Asn Gly Asp Leu Tyr 195 200 205 3 5 Phe Thr Asp Thr Leu Tyr Gly Tyr Leu Gin Asp Phe Arg Pro Val Pro 210 215 120 Gly Leu Arg Asn Gin Val Tyr Arg Tyr Asn Phe Asp Thr Gly Ala Val 225 230 235 240 Thr Val Val Ala Asp Asp Phe Thr Leu Pro Asn .Gly Ile Gly Phe Gly 245 250 255 Pro Asp Gly Lys Lys Val Tyr Val Thr Asp Thr Gly Ile Ala Leu Gly 260 265 270 Phe Tyr Gly Arg Asn Leu Ser Ser Pro Ala Ser Val Tyr Ser Phe Asp 275 280 285 Val Asn Gin Asp Gly Thr Leu Gin Asn Arg Lys Thr Phe Ala Tyr Val 290 295 300 Ala Ser Phe Ile Pro Asp Gly Val His Thr Asp Ser Lys Gly Arg Val S* 305 310 315 320 Tyr Ala Gly Cys Gly Asp Gly Val His Val Trp Asn Pro Ser Gly Lys 325 330 335 Leu Ile Gly Lys Ile Tyr Thr Gly Thr Val Ala Aja Asn Phe Gin Phe 340 345 350 Ala Gly Lys Gly Arg Met Ile Ile Thr Gly Gin Thr Lys Leu Phe Tyr 355 360 365 Val Thr Leu Gly Ala Ser Gly Pro Lys Leu Tyr Asp 370 375 380 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 1140 TYPE: Nucleic acid STRANDEDNESS: Double TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Fusarium oxysporum IFO 5942 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GCTAAGCTTCCTTCTACGGCTCAGATTATTGATCAGAAGTCGTTCAATGTCTTGAAGGAT GTGCCACCTCCTGCAGTGGCCAATGACTCTCTGGTGTTCACTTGGCCTGGTGTAACTGAG 120 GAGTCTCTTGTTGAGAAGCCTTTCCATGTCTACGATGAAGAGTTTTACGATGTAATTGGA 180 36 AAAGACCCCTCTTTGACCCTCATCGCAACATCGGACACCGACCCAATCTTCCATGAGGCT 240 GTCGTATGGTATCCTCCTACTGAAGAGGTGTTCTTTGTGCAGAATGCTGGCGCTCCTGCC 300 GCAGGCACTGGCTTGAACAAGTCTTCCATCATTCAGAAGATTTCCCTCAAGGAGGCCGAT 360 GCTGTTCGCAAGGGCAAGCAGGATGAGGTCAAGGTCACAGTTGTTGACTCGAACCCTCAG 420 GTTATCAACCCAAATGGTGGTACTTACTACAAGGGCAACATCATCTTCGCTGGTGAGGGC 480 CAAGGCGACGATGTTCCCTCTGCGCTGTACCTCATGAACCCTCTCCCTCCTTACAACACC 540 ACCACCCTTCTCAACAACTACTTCGGTCGCCAGTTCAACTCCCTCAACGACGTCGGTATC 600 AACCCCAGGAACGGTGACCTGTACTTCACCGATACCCTCTACGGATATCTCCAAGACTTC 660 CGTCCTGTTCCTGGTCTGCGAAACCAGGTCTATCGTTACAACTTTGACACTGGCGCTGTC 720 ACTGTTGTGGCTGATGACTTTACCCTTCCCAACGGTATTGGGTTTGGCCCCGACGGCAAG 780 AAGGTTTATGTCACCGACACTGGCATCGCTCTCGGTTTCTACGGTCGCAACCTCTCTTCT 840 *CCCGCTTCTGTTTACTCTTTCGACGTGAACCAGGACGGTACTCTTCAGAACCGCAAGACC 900 TTTGCTTATGTTGCCTCATTCATCCCCGATGGTGTCCACACTGACTCCAAGGGTCGTGTT 960 TATGCTGGCTGCGGTGATGGTGTCCATGTCTGGAACCCCTCTGGCAAGCTCATCGGCAAG 1020 ATCTACACCGGAACGGTTGCTGCTAACTTCCAGTTTGCTGGTAAGGGAAGGATGATAATT 1080 ACTGGACAGACGAAGTTGTTCTATGTCACTCTAGGGGCTTCGGGTCCCAAGCTCTATGAT 1140

Claims (11)

1. A recombinant peptide having D-pantolactone hydrolase activity and an amino acid sequence represented by SEQ ID NO:1 or an amino acid sequence represented by SEQ ID NO:1 but in which one or more amino acids is/are substituted, deleted, inserted, translocated or added, or a salt thereof.

2. The recombinant peptide according to claim 1, wherein said recombinant peptide having D-pantolactone hydrolase activity is encoded by a cDNA corresponding to a mRNA isolated from a microorganism of the genus Fusarium.

3. The recombinant peptide according to either of claims 1 or 2, which is produced by expressing an exogenous DNA sequence in prokaryotic host cells.

4. The recombinant peptide according to either one of claims 1 or 2, which is produced by expressing an exogenous DNA sequence in eucaryotic host cells. S*

5. The recombinant peptide according to any of claims 1 to 4, being a peptide of SEQ ID NO:1 from which one or more 20 amino acids has been deleted, or a salt thereof.

6. An isolated nucleic acid having a nucleotide sequence encoding a peptide according to any of claims 1 to

7. The isolated nucleic acid according to claim 6, which has a nucleotide sequence having a portion corresponding to an open reading frame in the nucleotide sequence of SEQ ID NO:2, or a homologue or fragment thereof

8. A vector incorporating a nucleic acid according to claim 6 or 7.

9. A transformant in which a vector according to claim 8 30 is harbored.

10. A process for producing a recombinant peptide as defined in any of claims 1 to 5, which comprises: culturing the transformant according to claim 9 in a nutrient medium suitable for growing said transformant to produce the peptide.

11. A process for producing D-pantolactone, which Scomprises: carrying out an optical resolution of D,L-pantolactone in the presence of a recombinant peptide according to any of claims 1 to 5 or (ii) the transformant according to claim 9. Dated this 8 t h day of May 2002 DAIICHI FINE CHEMICAL CO., LTD. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia