JPH0714346B2 - Genes involved in maltooligosaccharide production, corresponding recombinant plasmids and recombinant microorganisms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a maltooligosaccharide-forming amylase gene, more specifically, an amylase gene produced mainly from maltopentaose and maltohexaose, and a novel amylase gene containing the gene. Plasmid, more specifically, deoxyribonucleic acid (DN) that carries the genetic information involved in the production of the amylase.
A) A plasmid incorporating the fragment, a novel Escherichia microorganism containing the plasmid, and a novel Bacillus microorganism.

[Conventional technology]

Conventionally, α-amylase, β-amylase, glucoamylase and the like have been known as amylase acting on starch and have been used for the production of glucose (GI) and maltose (G2). However, in recent years, sugars called maltooligosaccharides having a large molecular weight of about 3 to 10 glucose residues have been attracting attention, and their applications in the fields of food and drug industries are being developed.

For example, maltooligosaccharides such as maltotriose (G3), maltotetraose (G4), maltopentaose (G5), and maltohexaose (G6) are sweeteners, excipients and clathrates for the food and pharmaceutical industry. Alternatively, it can be widely used as a raw material for diagnostic reagents for clinical tests.

To date, several types of microorganisms have been searched from the soil for the purpose of producing these maltooligosaccharides, and reported [see “Amylase” Academic Publishing Center, published by Michinori Nakamura (1986)).

However, fermentative production of maltooligosaccharide-forming amylase by these microorganisms is often difficult to separate and purify due to long culture time, difficult scale-up, and small amount of enzyme production.

On the other hand, there has been no research to date on the production of maltooligosaccharide-forming amylase using genetic engineering.

Reference is made to the study of cloning of α-amylase and β-amylase that produces maltose (G2), which is a smaller sugar than maltooligosaccharide, and cyclodextrin-forming enzyme, as a reference.

α-amylase (Studies on cloning of α-amylase of Bacillus subtilis; Yamazaki, H et al. J. Bacteriol., 156
(1983)), β-amylase (β of Bacillus polymyxa
-Study on cloning of amylase; Tetsu Kawazu et al. 1984 Abstracts of Japan Agricultural Chemistry, etc.) and Cyclodextrin synthase (CGTase of Bacillus macerans); Toshiya Ohno, Kunio Yamane et al. Abstracts).

[Problems to be solved by the invention]

MEANS TO SOLVE THE PROBLEM This invention solves the problem of the said prior art, The gene which carries the specific genetic information relevant to the production ability of the maltooligosaccharide synthase, the novel plasmid incorporating this gene, and the maltooligosaccharide synthase transformed by this plasmid. It is intended to provide a novel microorganism that produces bacterium outside the cell.

[Means for solving problems]

A plasmid is an extrachromosomal gene found in the cells of microorganisms, which has recently been used as a means for gene recombination in microorganisms, and its importance in research in the field of the fermentation industry has been increasing.

In recent years, as shown in the examples of amino acids and peptides, research has been conducted on plasmids incorporating DNA that carries the genetic information involved in the specific requirements for the growth of microorganisms and the productivity of metabolites. The plasmid has been introduced into the host microorganism, and a transformant thereof has been obtained.

Therefore, for the purpose of creating a microorganism having a strong malto-oligosaccharide-forming amylase-producing ability, the present inventors have isolated a strain of Bacillus sp. No. 8792) as a gene source, the gene of interest is identified by a genetic engineering method, and the gene is incorporated into a plasmid.
Furthermore, by successfully utilizing the plasmid to transform Escherichia and Bacillus microorganisms, a novel and useful microorganism having malto-oligosaccharide-forming amylase-producing ability was created to complete the present invention.

The Bacillus sp. # 707 strain used as a gene source produces a multi-component amylase and has the property of mainly producing maltotetraose from starch (Japanese Patent Application No. 61-148).
In addition to G4, it also produces amylase that produces maltooligosaccharides other than G4.

The transformant obtained by the genetic engineering method produced a large amount of amylase, which mainly produces maltopentaose and maltohexaose, from starch outside the cells.

The present inventors have identified a gene DNA that carries the genetic information involved in extracellular production of maltooligosaccharide-forming amylase, which is a metabolite of a microorganism belonging to the genus Bacillus,
We succeeded in preparing a new plasmid by inserting and ligating the DNA into a vector. Furthermore, by introducing the plasmid into Escherichia coli HB101 strain and Bacillus subtilis 1A-289 strain, a novel and useful transformant Escherichia coli HB101 (pTUE33
0) and Bacillus dibutylus 1A-289 (pTUB812) were successfully obtained, and the present invention was completed.

The present invention will be described in detail below.

I. Host The host microorganisms used in the present invention are the following two types.

(A) A foreign gene DNA is added to a plasmid (vector DNA) having a trait capable of growing in cultured cells, such as colicin E ₁ factor known as a plasmid of the genus Escherichia.
As a microorganism belonging to the genus Bacillus, for example Bacillus sp.
A microorganism belonging to the genus Escherichia represented by Escherichia coli, which can contain a plasmid into which a DNA fragment prepared from # 707 bacterium (Microtechnology Research Institute No. 8792) has been incorporated. Examples include Escherichia coli HB101 and C600.

(B) pUB110 known as a Bacillus plasmid
, Etc. into a plasmid (vector DNA) that can be propagated in the cultured cells as a foreign gene DNA, for example, a microorganism such as Bacillus sp. # 707 (Microtechnology Research Institute No. 8792).
A microorganism belonging to the genus Bacillus represented by Bacillus subtilis, which can contain a plasmid incorporating a DNA fragment prepared from Bacillus subtilis 1A-28
Examples are microorganisms having amy E gene such as 9, NA64.

II. DNA-donating bacterium As an example of a microorganism containing a DNA that carries the genetic information involved in the extracellular production of maltooligosaccharide-forming amylase used in the present invention, Bacillus sp. The first
No. 8792) can be given.

Bacillus sp. # 707 is a microorganism isolated from soil collected in Ichihara City, Chiba Prefecture, and is known as "Aerobic Sporeforming Bac".
teria "(United State Department of Agriculture, No
v.1952 by NRSmith, REGordon & FEClark)] and "Burger's Manual of Detaminative Bacteriology"["Bargry's Manual of Datermi
As a result of classification according to the classification method according to "native Bacteriology" (1957)], it was a new alkalophilic bacterium belonging to the genus Bacillus, which is an aerobic spore bacterium.

Bacillus stearothermophila among known bacterial species except that Bacillus sp. # 707 is an alkaliphilic bacterium with a growth pH range of 9 to 12 and that it is positive for Forges-Proscauer test (VP test) Su (Bacillus stearot
hermophilus) is the closest species to Bacillus sp.

III. About Vector DNA and Transformation of Escherichia Microorganisms A DNA fragment prepared from Bacillus sp. # 707, which is incorporated into the vector DNA, is an extracellular form of maltooligosaccharide-forming amylase which is a metabolite of the Bacillus bacterium. The above-mentioned vector DNA, which is DNA that carries the genetic information involved in production.
As for, in addition to naturally occurring ones, those lacking a portion of DNA other than the portion necessary for proliferation may be used, for example, ColE ₁ strain, pMB9 strain, pBR322
And the pSC101 system.

In addition, any known method can be applied to the method for incorporating the DNA fragment into the vector DNA. For example, select and treat an appropriate restriction enzyme (Endonuclease) to cut at a specific site in DNA, then mix with a vector DNA that has been treated with a restriction enzyme that forms a complementary nucleotide sequence at the cutting site and religate with ligase. A method is used. The thus obtained DNA fragment-vector DNA conjugate is introduced into the cells of a microorganism of the genus Escherichia, which is a recipient bacterium, by a transformation method, and is propagated until it becomes stable as a genetic trait. A transformant having both the traits of and vector DNA is obtained.

The transformants obtained in the examples described below are created by the following two-step operation. However, the operation of can be omitted, and DNA fragments prepared from Bacillus sp.
It is also possible to integrate it into vector DNA such as BR322.

As a bacteriophage vector DNA, DNA of λD69 phage [Silhavy TJ, Berman ML, Enquist LW: “E
xperiments with Gene Fusions "(Cold Spring Harbar
See Laboratory (1984) p.247. This is a 38.8 kb DNA having one restriction enzyme cleavage site by the restriction enzyme BamHI. A restriction enzyme map is shown in Fig. 3], and a DNA fragment carrying genetic information involved in extracellular production of maltooligosaccharide-forming amylase prepared from Bacillus sp. Method [(Horn,
B., Murray, K.:Proc.Natl.Acad.Sci.US, 74 .3259 (197
7)] The novel transduced phage particles λD69-T
Prepare U38.

Using pBR322 as the plasmid vector DNA, the DNA fragment prepared from the transduced phage particles was incorporated into the vector DNA, and the resulting mixture was competent cell transformation method (Lederburg, EM, Cohen, SN; J.
Bacteriol. 119 , 1072 (1974)) was introduced into the body of a recipient microorganism, Escherichia, and propagated until it became stable as a genetic trait, resulting in a transformant having a vector DNA trait. Was created,
This is Escherichia coli HB101 (pTUE330) [Escheric
It is called hia coli HB101 (pTUE330). The restriction enzyme map of the plasmid of pTUE330 of Escherichia coli HB101 (pTUE330) is shown in FIG.

As is clear from FIG. 4, this plasmid was used as pBR322.
A circular DNA molecule in which a DNA fragment carrying the genetic information involved in the extracellular production of the maltooligosaccharide-forming amylase of Bacillus sp. # 707 is incorporated into the BamHI cleavage site of the restriction enzyme cleavage site of the plasmid DNA, that is, , A novel 7.2 Kb consisting of pBR322 plasmid DNA and DNA of approximately 2,800 base pairs.
It is a circular DNA molecule.

Next, the bacteriology of Escherichia coli HB101 (pTUE330)
Characteristic is that Escherichia coli HB101 strain is a DNA recipient
Properties of ampicillin, malto-oligosaccharide-forming amino acid
Identical except for the productivity of the enzyme, [Molecular Clon
ing A Laboratory Manual, p.504 (1982), inherited form
Quality: F^-hsds20 (r , m ), Rec A13, ara-14, proA2, laY1,
galK2, rpsL20 (Smr), xy1-5, mt1-1, supE44, λ⁻〕,other
As a characteristic of pTUE330 plasmid,
PTUE33 which carries genetic information of oligosaccharide-forming amylase-producing ability
0 plasmid also produces amylase outside the cells,
Its characteristic is that it is added with a characteristic to be accumulated.

The extracellular production of maltooligosaccharide-forming amylase by Escherichia coli HB101 (pTUE330) was observed about 12 hours after the start of the culture, and its production was sustained for a long time, as shown in the Examples below. (See FIG. 1). On the other hand, the extracellular production of maltooligosaccharide-forming amylase by Bacillus sp. # 707 reaches a maximum after 4 days from the start of culture. The amylase relative activity of the above transformant was about 5 times the extracellular activity (indicated by E ₀ in the figure) as compared with Bacillus sp. The binding activity was shown to be about 3-fold higher (indicated by Ei in the figure).

Therefore, the present invention provides the Escherichia coli HB101 (pTU
E330), that is, novel and useful in terms of creating and providing a microorganism that has the ability to produce an enzyme protein extracellularly and which can produce maltooligosaccharide-forming amylase extremely advantageously in a short time. It is equipped with nature.

This means that the DNA of about 2,800 base pairs contained in the pTUE330 plasmid obtained by the present invention imparts the extracellular productivity of metabolites to the host.

Further, the amylase produced by the DNA-donating bacterium Bacillus sp. # 707 mainly produces maltotetraose (G4) from starch, but the E. coli transformant Escherichia coli HB10
1 (pTUE330) mainly produced maltopentaose (G5) and (G6) maltohexaose.

IV. Regarding vector DNA and transformation of Bacillus microorganisms Next, in order to introduce the DNA fragment carrying the genetic information of the maltooligosaccharide-forming amylase into the Bacillus host microorganism, the rapid alkaline method and CsCl-ethidium bromide equilibrium density gradient centrifugation. PTUE33 from E. coli transformants by isolation method
0 The plasmid is purified, and the plasmid is again treated with an appropriate restriction enzyme to cut at a specific site of the DNA to prepare a DNA fragment. The vector DNA may be one obtained by extracting a naturally occurring one, or one in which a part of the DNA other than the portion necessary for growth is partially deleted, for example, the pUB110 strain, pC194.
System, pE194 system, ρ11, Φ105 system, etc.

In addition, any known method can be applied to the method for incorporating the DNA fragment into the vector DNA. For example, a method is used in which the above vector is treated with a restriction enzyme that forms a complementary nucleotide sequence at the cleavage site, mixed with the DNA fragment, and religated with ligase.

The DNA fragment-vector DNA thus obtained was subjected to a protoplast transformation method [Chang, S.'Cohen,
SN,:. Mol.Gen.Genet 168, 111 (1979) ] by introducing into cells of the microorganism of the genus Bacillus is a recipient bacterium, when grown to stabilize the inheritance, traits desirable genetic trait and vector DNA A transformant having the following is obtained. A DNA fragment prepared from the Escherichia coli plasmid pTUE330 is incorporated into, for example, a plasmid vector pUB110 to obtain a novel DNA fragment.
The pTUB812 plasmid was cloned into Bacillus subtilis strain 1A-289 [Catalog of Strains 2nd edition (1982): The Bacil
lus Genetic Stock Center, (The Ohio Stste Uni.Dep
of Microbiology), Genetic traits: aroI906, met B5, sacA32
1, amy E], and a new microorganism was created by the protoplast transformation method. This was Bacillus subtilis 1A.
-289 (pTUB812) 〔Bacillus subtilis 1A-289 (pTUB81
2)] is called. Bacillus subtilis 1A-289
The restriction enzyme map of the pTUB812 plasmid of (pTUB812) is as shown in FIG.

As is clear from FIG. 5, this plasmid
DNA circular molecule in which a DNA fragment of maltooligosaccharide synthase gene carrying the genetic information involved in secretory production of maltooligosaccharide synthase of Bacillus sp. That is, pUB110 plasmid DNA and DNA of about 2,800 base pairs.
It is a novel circular DNA molecule of 7.3 Kb.

Next, the mycological properties of the Bacillus subtilis 1A-289 (pTUB812) are as follows.
The characteristics of the A-289 strain are the same as those of the A-289 strain except for kanamycin resistance and maltooligosaccharide-forming enzyme productivity (Catalog of Str
ains 2nd edition (1982): The Bacillus Genetic Stoc
k Center, (The Ohio State Uni.Dep of Microbiology,
Genetic traits: aroI906, met B5, sacA321, amy E), characteristics of pTUB812 plasmid, that is, pTUB812 plasmid carrying the genetic information of maltooligosaccharide synthase producing ability to secrete maltooligosaccharide synthase extracellularly and accumulate it. The feature is that it is added.

The secretory production of maltooligosaccharide-forming enzyme by Bacillus subtilis 1A-289 (pTUB812) was observed about 12 hours after the start of culturing, and its production was sustained for a long time, as shown in the Examples below. (See FIG. 1). In contrast,
The extracellular production of maltooligosaccharide-forming amylase by Bacillus sp. # 707 reaches a maximum after 4 days from the start of culture.
The relative activity of amylase in the above transformants was
The value increased to about 70 times higher than that of sp. # 707 (indicated by B in the figure). Therefore, the present invention provides the Bacillus subtilis
1A-289 (pTUB812), which has novel and usefulness in terms of creating and providing a microorganism capable of secreting and producing a maltooligosaccharide-forming enzyme extremely advantageously from a microorganism that has never been capable of producing amylase Is.

This means that the DNA of about 2,800 base pairs contained in the pTUB812 plasmid obtained by the present invention enhances extracellular productivity of host metabolites.

Further, amylase produced by the DNA-donating bacterium Bacillus sp. # 707 mainly produces maltotetraose (G4) from starch, but the above Bacillus subtilis transformant Bacillus subtilis 1A is produced.
-289 (pTUB812) is mainly maltopentaose (G5)
And maltohexaose (G6) were produced.

The plasmid, that is, Escherichia coli plasmid pTUE330 and Bacillus subtilis plasmid pTUB812 containing a gene carrying the genetic information involved in the extracellular production of maltooligosaccharide-forming amylase,
As shown in Fig. 6, both insert DNAs (Bacillus sp.
The size of the DNA fragment derived from the 707 chromosome is 2800.
It was bp, and its restriction enzyme maps were in agreement. The genes that carry the genetic information involved in the extracellular production of maltooligosaccharide-forming amylase are shown in FIG.

This plasmid pTUB812 contains a radioisotope [α-3
2P] dCTP labeled and chromosomal DN of Bacillus sp.
A, Ec of Escherichia coli HB101 chromosomal DNA and Bacillus subtilis 1A-289 chromosomal DNA used as hosts
As a result of Southern hybridization (Southern, EM, J. Mol. Biol., 98 (1975)) with the complete degradation product by oRI, the plasmid hybridizes only with the chromosome of the DNA donor Bacillus sp. Then, it was confirmed that the gene carrying the genetic information involved in the extracellular production of the amylase contained in the plasmid was derived from Bacillus sp. # 707 (shown in FIG. 8).

Example (1) The method for separating a gene of the present invention and the plasmid pTUE33 are described below.
0 and pTUB812 construction method and a method for preparing transformant strains Escherichia coli HB101 (pTUE330) and Bacillus subtilis 1A-289 (pTUB812) containing the plasmid are described in detail based on Examples, and the trait is further described. The effect of the converted strain will also be described.

Preparation of chromosomal DNA fragment containing genetic information of malto-oligosaccharide-forming amylase Alkaliphilic Bacillus sp. # 707 (Microtechnological Research Institute No. 8792), which has the ability to produce and accumulate malto-oligosaccharide-forming amylase extracellularly 100 ml of medium [1 starch per 1
5g, peptone 10g, yeast extract 5g, K ₂ HPO ₄ 1g, MgSO ₄・ 7H
₂ O 0.2 g and a composition adjusted to pH 10.0 with sodium carbonate 10 g) at 40 ° C. for 12 hours with shaking culture, and after collecting cells in the late logarithmic growth phase, Saito, Miura method (Saito, H. , Miura, K.: B
iochem.Biophys.Acta, 72 , 619, (1964)) was used to extract and purify DNA to obtain about 5 mg of chromosomal DNA.

Insertion of chromosomal DNA fragment into phage DNA Take 300 μg of the chromosomal DNA prepared above and add restriction endonuclease Sau 3AI (Takara Shuzo Co., Ltd.) to
It was reacted for 0 minutes and partially cut. Further, a chromosomal DNA fragment of about 7 to 12 Kb was separated and purified by a sucrose density gradient ultracentrifugation method to prepare a DNA fragment a. DN of bacteriophage λD69
A (obtained from the Institute of Medical Science, University of Tokyo) 20 μg was completely digested with the restriction endonuclease BamHI (manufactured by Takara Shuzo Co., Ltd.) and a part thereof was mixed with the DNA fragment a to obtain DNA derived from T4 phage. 1 by ligase (Takara Shuzo Co., Ltd.)
In vitro packaging kit (manufactured by Amersham International) after ligating DNA strands at 0 ° C for 24 hours
Was used as transducing phage particles. Λ medium containing 1% starch [Bactotryptone (Difco) 1 per 1
Composition containing 0 g and NaCl 12.5 g] Escherichia on an agar plate
Kori SM32 strain (sold by the Institute of Medical Science, The University of Tokyo.
Genetic traits: SA500, his, pyrD, lons100, gal, strA) were grown at 37 ° C and infected with the above-mentioned transduced Bacillus particles. Was used to select transducing phage λD69-TU38 particles having amylase activity. The PEG6000 method [KRYamamoto, BM Alberts, R. Be, after culturing the transduced phage particles with the Escherichia coli SM32 strain in λ medium 1
nzinger, L, Lowhorne & G. Treiber: Virology, 40 , 734 (1
970)] and dialysis with 50% formamide to extract and purify the transduced phage DNA, and approximately 150 μg of DNA of approximately 20 Kb.
Got

Insertion of Transduced Phage DNA Fragment into E. coli Plasmid Vector Take 50 μg of the DNA prepared above, add Sau3AI, and
The mixture was reacted at 30 ° C. for 30 minutes and partially cut. Further, a DNA fragment b was prepared by separating and purifying a transducing phage DNA fragment of about 2 to 9 Kb by a sucrose density gradient ultracentrifugation method. On the other hand, pBR322 plasmid DNA (Bethesda Research Laboratories (USA)) having tetracycline resistance (Tetr) and ampicillin resistance (Ampr) used as an E. coli plasmid vector was completely digested with BamHI and heat treated at 65 ° C for 10 minutes. And then treated with BAP (; Bacterial Alkaline Phosphatase), mixed with the DNA fragment b, and treated with T4 DNA ligase at 10 ° C.
A ligation reaction of DNA chains was performed for 24 hours to construct a plasmid DNA incorporating a DNA fragment.

Transformation of Escherichia coli with a plasmid carrying genetic information that imparts or enhances extracellular productivity of maltooligosaccharide-forming amylase Escherichia coli HB101, which is a hybrid strain of Escherichia coli K-12 strain and Escherichia coli HB101, in L medium ( 50 g of tryptone (Difco) 10 g, yeast exci (Difco) 5 g, NaCl 5 g per 1 adjusted to pH 7.0)
The cells were inoculated into 1 liter, shake-cultured at 37 ° C., grown to the late logarithmic growth phase, and then collected. Under ice-cooling, the final concentration was 0.
The cells were sequentially suspended in a solution of 1M CaCl ₂ to prepare competent cells. A solution of the plasmid DNA obtained in (3) was added to this cell suspension, and the mixture was reacted for 30 minutes under ice-cooling, and heat shock was applied at 42 ° C. for 2 minutes to incorporate the plasmid DNA prepared above into the cells.

Then, this cell suspension is separately inoculated into the L medium,
Transformation reaction by shaking culture at 37 ° C for 60 minutes [Lederburg, E.
M., Cohen, SN; J. Bacteriol. 119 , 1072 (1974)], and the resulting transformants were subjected to LSAmp agar plates (10 g of tryptone (Difco), 5 g of yeast extract (Difco), 10 g of starch). , NaCl 5g, ampicillin 50mg, agar 15g)
11 Escherichia coli HB101 transformants having ampicillin resistance and having amylase activity (positive for colonies forming a ring by iodine starch reaction on colonies on plate) were cultured at 37 ° C overnight. Got the stock.

About 11 transformant strains obtained in Example (1), LAmp medium [composition containing 10 g of tryptone (Difco), 5 g of yeast extract (Difco), 5 g of NaCl, and 50 mg of ampicillin per 500 ml] was shaken at 37 ° C. for 6 hours. 50 μg / ml after culturing
Chloramphenicol is added so that the concentration becomes
It was cultured for 16 hours. Rapid alkaline method (Birnboim, HCand J.Doly.Nucleic Ac
ids Res. 7 , 1513 (1979)) to obtain a crude plasmid fraction, and further about 150 μg of each purified plasmid DNA was prepared by CsCl-ethidium bromide equilibrium density gradient centrifugation.

Furthermore, as a result of making a restriction enzyme map of the plasmid and comparing the sizes of the insert DNAs contained, the smallest one was found.
It was pTUE330 containing a 2.8 Kb insert (shown in Figure 4). The largest insert of the 11 plasmids was 6 Kb. The transformation containing the plasmid pTUE330 is called Escherichia coli HB101 (pTUE330).

The pTUE330 plasmid was cloned into Sau in the same manner as described above.
After cutting with 3AI, a DNA fragment of about 2 to 9 Kb was separated and purified to prepare. On the other hand, pUB110 plasmid DNA [Bethesda Rese which is used as a Bacillus subtilis plasmid vector and has kanamycin resistance (Kmr) and ampicillin resistance (Ampr)
arch Laboratories (USA)] was treated in the same manner as described above to incorporate the above DNA fragment into the plasmid DNA.
Was built.

Transformation of Bacillus subtilis with a plasmid carrying genetic information that imparts or enhances extracellular productivity of maltooligosaccharide-forming amylase Using the solution of plasmid DNA obtained above, protoplasts using Bacillus subtilis 1A-289 strain as a host Transformation method [(Chang, S., Cohen, SN: Mol. Gen. Genet. 168 , 11
1, (1979)] and introduced the above-mentioned plasmid DNA into cells, and starch 1%, kanamycin 150 μg / ml, agar
DM3 medium containing 0.8% (5% casamino acid 100 ml, 1 M sodium citrate (pH 7.3) 500 ml, 3.5% K ₂ HPO ₄ 50 ml, 1.5% K ₂
HPO ₄ 50ml, 10% yeast extract 50ml, 20% glucose 25m
l, 1M MgCl _{2 (} 20 ml, containing ₂ % bovine serum albumin 5 ml) and cultured at 37 ° C. to obtain 17 strains of Bacillus subtilis resistant to kanamycin and having amylase activity. The strain with the highest amylase activity is called Bacillus subtilis 1A-289 (pTUB812). Next, with respect to the above 17 transformants, LGkm medium [tryptone (Difc
o) Composition containing 10 g, yeast extract (Difco) 5 g, NaCl 5 g, glucose 2 g, and kanamycin 10 mg] 5 After shaking culture at 37 ° C. for 6 hours with shaking, the cells prepared by centrifugation were subjected to a crude alkali method by the rapid alkali method. To obtain the plasmid fraction,
About 70 μg of each purified plasmid was prepared by CsCl-ethidium bromide equilibrium density gradient centrifugation. Further, the plasmid was purified by agarose gel electrophoresis and adsorbed on hydroxyapatite to recover the plasmid (HFTaba
k, RAFlavell, Nucleic Acids Res. 5 , 2321 (1978).

Next, when a restriction enzyme map of the plasmid was created, the insert DNA of Bacillus subtilis plasmid pTUB812 was found to have the same size as the insert DNA of E. coli plasmid pTUE330 (both 280
0 bp) and the restriction enzyme cleavage site matched (Figs. 5 and 6).
(Shown in the figure).

Example (2) The transformant obtained in Example (1), Escherichia coli HB101 (pTUE330), was mixed with LAmp medium (10 g of tryptone (Difco), 5 g of yeast extract (Difco), NaCl5).
g, composition containing 50 mg of ampicillin] A shake culture was carried out at 37 ° C. in a 500 ml Sakaguchi flask containing 100 ml. The cell growth (cell mass) was measured by the absorbance (OD) at 660 nm. The amylase activity was based on the indestructible method. (Fuwa, H., J. Biochem. 41
1954)) That is, the amylase activity was 0.2% after adding 2 ml of 1% soluble starch solution dissolved in M / 10 glycine-caustic soda buffer (pH 8.0) to 1 ml of enzyme solution and reacting at 40 ° C. for 5 minutes.
The enzymatic reaction was stopped by adding 5 ml of a 2 × 10 ⁻⁴ N iodine 0.15N hydrochloric acid solution to the mixture, and 700 nm absorbance was measured to obtain the value from the following formula.

Here, T and To represent the absorbance of the sample and the absorbance of the blank.

The amount of bacterial cells of the transformant reaches the maximum 12 hours after the start of culture,
The extracellular amylase activity and intracellular amylase activity (including the activity of amylase bound to bacterial cells) reached a maximum at approximately 36 hours. The amylase produced is very stable (shown in Figure 1).

This E. coli transformant Escherichia coli HB101 (pTUE3
30) produces about 8 times (the sum of about 3 times the intracellular enzyme and about 5 times the extracellular enzyme) of the amylase activity of the Bacillus sp. Generally, most of the enzymes produced by Escherichia coli are intracellular enzymes, but the transformant obtained by this method produces 60% or more of the amylase produced outside the cells, that is, in the culture supernatant.

For comparison, the microorganism Bacillus sp.
Extracellular amylase activity was measured by culturing 707 bacteria (Microtech Lab. No. 8792). Inoculate 50 ml of a medium (pH 10.0) containing starch 15 g, polypeptone 10 g, yeast extract 5 g, K ₂ HPO ₄ 1 g, MgSO ₄ .7H ₂ O 0.2 g, and sodium carbonate 10 g per 1 to 40
The cells were cultured with shaking at ℃. The cell amount reached the maximum 12 hours after the start of the culture, and the extracellular enzyme activity peaked on the 4th day.

The transformant obtained in Example (1), Bacillus subtilis 1A-289 (pTUB812), was treated with LGKm medium [10 g of tryptone (Difco), 5 g of yeast extract (Difco), N].
Composition containing aCl5g, glucose 2g, kanamycin 10mg] 100
Culture was carried out with shaking in a Sakaguchi flask containing 500 ml at 37 ° C. Cell growth (cell mass) was measured by absorbance at 660 nm (0D). The amylase activity was determined in the same manner as in the case of the Escherichia coli transformed strain indicated by. The amount of bacterial cells in the transformant reached a maximum 24 hours after the start of the culture, and the extracellular amylase activity reached a maximum in approximately 48 hours. The amylase produced is very stable. This Bacillus subtilis transformant Bacillus subtilis is compared with the amylase activity of Bacillus sp.
The extracellular amylase of 1A-289 (pTUB812) increased to about 70-fold activity.

Example (3) Escherichia coli transformed strain, Escherichia coli HB101 (pTU
E330) and Bacillus subtilis transformants, Bacillus subtilis 1A
-289 (pTUB812) was centrifuged in the same manner as in Example (2) for 48 hours after the start of the culture, and ammonium sulfate was added to 100 ml of the culture supernatant prepared by sterilization and salting out. The precipitate was dissolved in water and dialyzed against distilled water overnight to give an amylase enzyme solution. 2 ml of gelatinized 2% corn starch aqueous solution
0.1 ml of the above enzyme solution was added to and reacted at 50 ° C. for 24 hours to prepare a saccharified product.

By using a column (8φ x 250 mm) packed with silica-based ODS-NH ₂ resin by high performance liquid chromatography, the saccharified product prepared by the above method was separated with an eluent of acetonitrile: water (65:35), and the differential The products, which were detected by a refractometer and coincided with the retention time of each sugar in the reagent, were identified and quantified, and the results are shown in the table.

An example of a chromatogram of a saccharified product is shown in FIG.

〔The invention's effect〕

Genes responsible for the genetic information involved in the production of maltooligosaccharide-forming amylase of the present invention, a plasmid incorporating this by a genetic engineering method is prepared, for example, a microorganism belonging to the genus Escherichia and a microorganism belonging to the genus Bacillus contains the By converting, it is possible to create a novel microorganism that produces an amylase that produces malto-oligosaccharides, especially maltopentaose (G5) and maltohexaose (G6) -based saccharification products from starch in a short culture time. Moreover, since the above-mentioned microorganism produces the amylase in a large amount outside the microbial cell, it becomes possible to industrially and efficiently produce the large amount of the amylase by using this microorganism.

[Brief description of drawings]

FIG. 1 shows the Escherichia coli HB101 (p used in the present invention.
TUE330), Bacillus subtilis 1A-289 (pTUB812), and Bacillus sp. # 707 that is a DNA donor bacterium are graphs showing time-dependent changes in amylase activity. Each is shown as E, B, and P, and each value shows 100% activity of B at 48 hours after the start of culture
Is shown as relative activity. E ₀ represents extracellular activity, and Ei represents intracellular activity (including bacterial cell-bound amylase activity). FIG. 2 is a graph showing the change over time in the bacterial concentration of the same. FIG. 3 is a restriction enzyme digestion diagram of bacteriophage λD69. FIG. 4 is a restriction enzyme map of a plasmid (pTUE330) of Escherichia coli HB101 (pTUE330) bacterium, where the open part is derived from pBR322 plasmid DNA and the black part is Bacillus sp. # 707 chromosomal DNA fragment ( (Malto-oligosaccharide-forming amylase extracellularly produced gene DNA fragment). FIG. 5 is a restriction enzyme map of the plasmid (pTUB812) of Bacillus subtilis 1A-289 (pTUB812) bacterium, the open part is derived from pUB110 plasmid DNA, and the black part is Bacillus sp. A DNA fragment (malto-oligosaccharide-forming amylase extracellularly produced gene DNA fragment) is shown. FIG. 6 is a detailed restriction enzyme map of the plasmids pTUE330 and pTUB812. The solid line is derived from the plasmid DNA, and the open part is the chromosomal DNA fragment of Bacillus sp. # 707. FIG. 7 is a liquid chromatogram of a starch degradation product produced by amylase produced by Escherichia coli HB101 (pTUE330), Bacillus subtilis 1A-289 (pTUB812) and the DNA-donating bacterium Bacillus sp. # 707. Denote by E, B and P respectively. FIG. 8 shows the agarose gel electrophoresis pattern (A) of the chromosomal DNA used for Southern hybridization and the result of hybridization (S). M is λDNA / Hind III used as a marker, P, Hb and He are DNA donor Bacillus sp. # 707, Escherichia used as a host
E.coli HB101 and Bacillus subtilis 1A-289 are completely digested EcoRI of chromosomal DNA.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C12R 1:19) (C12N 1/21 C12R 1:07) (C12N 9/30 C12R 1:21) (C12N 9/30 C12R 1:07) (72) Inventor Shinichi Suzuki, 9 Hachiman Kaigan-dori, Ichihara-shi, Chiba Oji Corns Starch Co., Ltd. Central Research Institute No. Oji Paper Co., Ltd. Central Research Laboratory

Claims (15)

[Claims] 1. A Bacillus sp.
No.), is about 2.8 kb in size, and has the following restriction enzyme map, which imparts or enhances the productivity of amylase produced mainly from maltopentaose and maltohexaose. Gene that carries the gene information. 2. A plasmid which gives an extracellular productivity to a host belonging to the genus Escherichia, wherein the restriction enzyme cleavage site is derived from Bacillus sp. Is about 2.8 kb, characterized by having the following restriction enzyme map, the gene carrying the genetic information that imparts or enhances the productivity of amylase produced mainly from maltopentaose and maltohexaose. Integrated plasmid. 3. The plasmid according to claim 2, wherein the plasmid is pTUE330. 4. The plasmid according to claim 2, wherein the host belonging to the genus Escherichia is Escherichia coli. 5. The plasmid according to claim 2, wherein the plasmid vector is DNA of pBR322 plasmid. 6. The plasmid according to claim 2, wherein the restriction enzyme cleavage site is a BamHI cleavage site. 7. A plasmid using a microorganism belonging to the genus Bacillus as a host, the Bacillus s at the restriction enzyme cleavage site.
A maltopentaose and a maltohexaose, which are derived from the p. # 707 bacterium (Microtechnology Research Institute, No. 8792), are about 2.8 kb in size and have the following restriction enzyme maps. A plasmid incorporating a gene that carries genetic information that imparts or enhances the productivity of amylase produced as the main component. 8. The plasmid according to claim 7, wherein the plasmid is pTUB812. 9. The plasmid according to claim 7, wherein the host is Bacillus subtilis. 10. The plasmid according to claim 7, wherein the plasmid vector is DNA of pUB110 plasmid. 11. The plasmid according to claim 7, wherein the restriction enzyme cleavage site is a BamHI cleavage site. 12. A plasmid which gives a host belonging to the genus Escherichia an extracellular productivity, which is derived from Bacillus sp. # 707 (Ministry of Industrial Science and Technology No. 8792) at the restriction enzyme cleavage site and has a large size. Is about 2.8 kb, characterized by having the following restriction enzyme map, the gene carrying the genetic information that imparts or enhances the productivity of amylase produced mainly from maltopentaose and maltohexaose. A microorganism belonging to the genus Escherichia transformed with the incorporated plasmid. 13. The microorganism is Escherichia coli HB101 (pTU
The microorganism belonging to the genus Escherichia according to claim 12, which is E330). 14. A plasmid that uses a microorganism belonging to the genus Bacillus as a host, and has a Bacillus at the restriction enzyme cleavage site.
Maltopentaose and maltohexaose, which are derived from sp. # 707 bacterium (Microtechnology Research Institute, No. 8792) and have a size of about 2.8 kb and have the following restriction enzyme maps: A microorganism belonging to the genus Bacillus transformed with a plasmid into which a gene carrying genetic information that imparts or enhances the productivity of amylase produced as a main component is incorporated. 15. The microorganism is Bacillus subtilis 1A-289.
The microorganism belonging to the genus Bacillus according to claim 14, which is (pTUB812).