KR100201263B1 - Gene coding for esterase and novel microorganism containing said gene - Google Patents

Abstract

Esterase coding genes derived from Serratia genus microorganisms, high yielding strains containing esterases containing the genes, recombinant plasmids containing the genes inserted into vector plasmids, novel microorganisms transformed with the recombinant plasmids, And culturing the mutant strain or the new microorganism as described above in a medium, and collecting the esterase produced intracellularly and externally. Variants with high esterase productivity and transformed novel microorganisms can produce a large amount of esterase with high esterase productivity and high purity.

Description

Esterase coding genes and microorganisms containing the genes

1 shows a restriction endonuclease map of plasmid DNA pLIPE101 isolated from recombinant cells in Example 2. FIG.

2 shows a restriction endonuclease map of plasmid DNA, pLIPE111, pLIPE121, pLIPE131, pLIPE141 and pLIPE151, respectively, prepared by deleting plasmid DNA pLIPE101 using restriction endonucleases and DNA ligase.

The present invention relates to an esterase coding gene, a recombinant plasmid containing the gene, a novel microorganism transformed with a recombinant plasmid, and a method for producing an esterase by culturing the microorganism.

Recently, the use of enzymes such as esterases in hydrolysis reactions has often been attempted. For this purpose, esterases derived from animals (eg pig liver, pig pancreas, etc.), and microorganisms [1 example: Arthrobacter globiformis, Geotrichum candidum , Esterases derived from Candida cylindracea, Pseudomonas fluorescens, and the like.

Also, esterase is Rhizopus delemar (see Japanese Patent Publication No. 87175/1991), Pseudomonas fragi [Biochemical and Biophysical Research Communications, Vol. 141, 185-190 (1986), are known to be obtained using recombinant DNA technology.

However, known methods for producing known esterases or esterases have several problems. For example, esterases derived from animals are very expensive. In addition, the esterases derived from the above-mentioned Arthrobacter, Geotrichum, Candida, Pseudomonas, and Rhizopus microorganisms may be active, stable, or mutant. The disadvantage is that the esterase-producing microorganisms are not advantageous or that the productivity is not high.

As a result of various investigations, the present inventors have found that by using recombinant DNA technology in microorganisms of Serratia, excellent target esterase can be produced. That is, the present inventors have succeeded in obtaining an esterase coding gene from a microorganism of the genus Serratia, and by introducing a recombinant plasmid containing the gene into the host microorganism, a transformed microorganism with a markedly increased esterase productivity can be obtained. In addition, it was found that the desired esterase can be produced by adapting the transformed microorganism to an industrial scale.

It is an object of the present invention to provide an esterase coding gene derived from a microorganism of the genus Serratia. Another object of the present invention is to provide a mutant strain having high esterase productivity. Another object of the present invention is to provide a recombinant plasmid prepared by inserting this gene into a vector plasmid. Another object of the present invention is to provide a transgenic microorganism containing the prepared plasmid. It is still another object of the present invention to provide a method for producing esterase by culturing the transgenic microorganism and collecting the esterase produced inside and outside the microbial cells.

The esterase coding genes derived from the microorganisms of the genus Serratina of the present invention are 1839 base pairs of double stranded DNA with an open reading frame, in particular the nucleotides shown in SEQ ID NO: 1 and SEQ ID NO: 2 below. DNA with sequence.

Donor microorganisms of such esterase coding genes include any microorganism belonging to the genus Serratia with esterase productivity. Serratia marsescens Sr41 (FERM BP-487), Serratia liquefaciens ATCC 27592, Serratia marcesense ATCC 13880, Serratia marsesense 14764, Serratia marsense ATCC 19180, Serra Tia Marsense ATCC 21074, Serratia Marsense ATCC 27117, and Serratia Marsense ATCC 21212.

Donor microorganisms also include mutant strains derived from wild strains such as various amino acids-demand strains, various nucleic acid-demand strains, various vitamins-demand strains, such as Serratia marsense M-1 (FERM BP-4068). This Serratia Marsense M-1 is more than two times higher in esterase productivity than its parent, Serratia Marsense Sr4l, and contains an esterase gene encoded by the SEQ ID NO2 DNA sequence. The mutant M-I was used as the method of Aderbrug et al., Which is the parent strain of Serratia marcesense Sr4l (FERM BP-487). [Reference: Biochemical and Biophysical Research Communications, Vol. 18. 788 (1965)] according to N-matyl-N'-nitro-N-nitrosoguanidine (NTG), and the microorganisms thus treated are cultured in agar medium containing emulsified triglycerides and It can be obtained by collecting colonies around the formed clear zone. In the same way, another variant strain Serratia marcesense G205, which is at least 1.5 times higher in esterase productivity, can be obtained from parent strain Serratia marsense Sr4l.

The vector plasmid into which the esterase coding gene has been inserted includes any plasmid that can be replicated in the transformed cell, but is preferred for antibiotics such as ampicillin, kanamycin, and chloramphenacol with a copy number of 1 to thousands. Preference is given to plasmids which contain resistance markers for and which also contain suitable promoters such as lac, tac or trp. Vector plasmids may also contain plasmid stabilizing genes such as par and parB.

These vector plasmids are described, for example, in pLG339 [Gene, Yol. 18, 332 (1982), pBR322 [Gene, Vol. 2, 95 (1977), pUB18 [Gene, Vol. 33, 103 (1985), pUC19 [Gene,, Vol. 33, 103 (1985)], pHSG298 [Gene, Vol. 61, 63 (1987)], pHSG299 [Gene, Vol. 61, 63 (1987).

The vector plasmids described above may be purchased commercially, or may be prepared using a transparent lysate method (see, for example, Yasuyuki, Procedure for Experiment in Genetic Engineering, page 125, published by Kodansha, 1980), or an alkaline lysis method (maniatis et al., Molecular Cloning, page 368, Cold Spring Harhor Laboratory, USA (1982) by conventional methods such as can be obtained from the microorganisms containing such plasmid.

Host microorganisms (microorganisms for both recombination of plasmids and expression of the desired esterase) can be transformed into recombinant plasmids, which can replicate plasmids, express genes on plasmids and produce functional proteins. If anything is included. Such host microorganisms include, for example, microorganisms belonging to the genus Serratia or Escherichia, in particular from various strains derived therefrom, such as Serratia marsense Sr4l, and Serratia marsense M-1, Serratia marsense TT392. See Journal of Bacteriology, Vol. 161, 1 (1985), or Escherichia coli Kl2DH5 (Maniatis et al., Molecular Cloning, 2nd vol., A1O, Cold Spring Harbor Laboratory, USA (1989)). .

Chromosomal DNA containing esterase coding genes treat microbial cells with lysozymes and additional surfactants (eg, sodium lauryl sulfate, sodium N-lauroyl sarcosinate, etc.) and treat the cells thus treated. After removal of the protein by extraction with an organic coagulant (e.g., phenol, chloroform, diethyl ether, etc.), it can be easily obtained from a microorganism containing the gene by a known method of precipitating DNA with ethane. Journal of Molecular Biology, vol. 3, 208 (1961), and Biochimica et Biophysica Acta, vol. 72. 619 (1963).

Recombinant plasmids comprising chromosomal DNA and vector plasmids containing esterase coding genes are digested with appropriate restriction endonucleases (e.g. EcoRl, BamHl, Hindlll, Sall, etc.) and, if necessary, the product. Can be readily prepared by conventional methods of treatment with terminal transferases or DNA polymerases and then the product with DNA ligase (e.g., T4 DNA ligase, E. coli DNA ligase, etc.). Methods in Enzymology, vol. 68, 41 (1979), and Yasuyuki Takagi, Procedure for Experiment in Genetic Engineering, page 135, published by Kodansha, 1980].

Selection of the desired recombinant plasmid containing the esterase gene from the recombinant plasmid mixture obtained by the above method can be carried out as follows.

Microbial cells that lack restriction endonucleases and lack esterase activity (e.g. Escherichia coli Kl2 DH5) are transformed with a mixture of the above recombinant plasmids, and the transformed cells are agar containing emulsified triglycerides. The medium is spread over a nutrient agar medium containing tributyrin emulsified with polyoxyethylene cetyl alcohol ether (Brij 58) and a defined concentration of antibiotics. After 1 to 2 days of incubation at 30 to 37 ° C, colonies of transformants around the large transparent bands are isolated.

Introduction of recombinant plasmids into host microorganisms may be accomplished by, for example, treating host cells with aqueous calcium chloride solution at low temperatures to increase cell permeability and then introducing the recombinant plasmids into host cells [Journal of Molecular Biology, Vol. 53, 159 (1970)], or conventional methods such as electroporation.

The plasmid DNA is then extracted from the transformant by alkaline lysis to obtain a recombinant plasmid containing the esterase coding gene.

In order to efficiently perform transformation, the resulting recombinant plasmid is altered among microorganisms which are the same species as the host microorganisms lacking restriction endonucleases and used for expression of esterases. That is, when the host microorganism used is Serratia marsense Sr4l, the obtained recombinant plasmid is introduced into the Seratia marsense TT392 restriction strain. This modified recombinant plasmid is isolated from the microorganism.

The modified recombinant plasmid is then introduced into the host microorganism to obtain the desired transformant suitable for preparing the desired esterase.

Introduction of recombinant plasmids into host microorganisms can be easily carried out by the Dakagi & Gizumi method [Journal of Bacteriology, Vol. 161. 1 (1985).

Host microorganisms used for transformation include the above microorganisms, but a high esterase productivity strain is preferable. Desired transformants can be obtained by isolating colonies that express antibiotic resistance.

The transforming microorganism obtained by the above-mentioned method is, for example, Ceratia marsense obtained by introducing a recombinant plasmid composed of a 2.8 kb Sa1Il-BamHl DNA fragment containing an esterase gene and pUC19 into Serratia marsense Sr41. TA5025 (FERM BP-4067): There is a Serratia marsense TBE101 obtained by introducing a recombinant plasmid consisting of 4.Okb Sall DNA fragment containing an esterase gene and pBR322 into Serratia marsense Sr4l. All of these transformed strains have the same morphological characteristics as the host microorganism Serratia marsense Sr41.

The production of esterases using the transformed microorganisms is carried out by culturing the microorganisms in the medium and collecting the esterases inside and outside the microbial cells.

The medium used for the production of esterase includes any conventional medium in which microorganisms can grow. Suitable media include carbon sources such as sugars (e.g. glucose, sucrose, molasses, etc.), organic acids (e.g. fumaric acid, citric acid, etc.), alcohols (e.g. glycerol, etc.) or amino acids (e.g. alanine, glutamine, asparagine, etc.) and Nitrogen sources such as inorganic ammonium salts (e.g. ammonium sulfate, ammonium chloride, etc.), urea, peptone, cornsip liquor, yeast extract, casein hydrolysates and the like.

The carbon source is generally contained in an amount of 1 to 15% by weight relative to the total weight of the medium, and the nitrogen source is generally contained in an amount of 0.1 to 2.0% by weight relative to the total weight of the medium. The medium may further optionally contain an inorganic salt (eg phosphate, magnesium salt, potassium salt, calcium salt, etc.) and / or metal ions (eg iron, manganese, copper, zinc, etc.) in an appropriate amount. In the case of a synthetic medium, it may further contain vitamins or amino acids and antibiotics suitable for stabilizing esterase production inducers (e.g. vegetable oils, surfactants, etc.), foam inhibitors, recombinant plasmids in microorganisms. . It is preferable to adjust a medium to pH 5-8.

Cultivation of the transformed microorganism is carried out in a conventional manner. For example, microorganisms are inoculated into a medium and cultured with shaking culture, aerobic culture, stationary culture, and the like. The culture conditions may vary depending on the type of medium and the culture method, but may be any conditions suitable for the growth of microorganisms, and are generally 1 to 2 days at an initial pH of 5 to 8 and 20 to 40 ° C.

Esterases produced in and around cultured cells are collected by conventional methods. For example, esterases contained in the medium may be salted out using inorganic salts, precipitation using organic solvents, adsorption or desorption using ion exchange resins and various column chromatography, gel filtration, use of protein-precipitating agents, or Collected by a combination of these methods. The esterase accumulated in the cells is first destroyed by a physical method such as a frictional decay device (DynoMill) or a chemical method such as treatment with lysozyme, and then the esterase of the cell extractor is removed by the method described above. Collect.

The esterase coding gene of the present invention is not limited to the DNA sequences described in detail herein, but includes any gene having a DNA sequence obtained by modification of sequencing such as insertion, deletion, and substitution. That is, the esterase gene is artificially directly in vitro using synthetic mutant DNA primers designed based on the DNA sequence of the esterase gene described in detail herein, or chemical mutants such as formic acid and hydrazine sulfite. It can be transformed into. In addition, mutant genes can be obtained by treating esterase producers with NTG or UV.

The invention will be illustrated by the following examples which should not be construed as being limited thereto.

In the Examples, the esterase activity is measured by a convenient method (using Lipase Kit S, manufactured by Dainippon Pharmaceutical Co., Lt, d., Japan). Another method consists in enzymatically reacting the product in olive oil (as substrate) at pH 8.0, 37 ° C. for 20 minutes, and then measuring the amount of fatty acid formed. Units of esterase activity are expressed in μmol of fatty acid formed per minute. In addition, the medium used in the Example has the following composition (wherein% is w / v% unless there is particular notice).

LB medium: 1% bactotrypton (manufactured by Difco), 0.5% bacterium yeast extract (manufactured by Difco), and 0.5% sodium chloride

LBG plate medium: 1% bactotriptone (manufactured by Difco). 0.5% bacterium yeast extract (manufactured by Difco). 0.5% sodium chloride, and 1.0% gellan rubber (manufactured by Wako Pure Chemical Industries, Ltd., Japan.).

Tributyrin-containing LBG flat media: 0.5 v / v% tributyrin, 0.5% polyoxyethylene cetyl alcohol ether, and 0.005% ampicillin containing LBG flat media.

Esterase production medium: 1.0% dextrin, 2.0% mist, 0.2% ammonium sulfate, 0.1% sodium dihydrogenphosphate, 0.05% magnesium sulfate hexahydrate, 0.01% calcium chloride dihydrate, 0.001 % Ferrous sulfate heptahydrate, 0.5% Tween 80, and 0.1% choline.

Example 1

(1) preparation of chromosomal DNA containing esterase coding genes;

Seratia marcesense Sr4l (FERM BP-487) is subjected to aerobic shaking culture overnight in LB medium (200 ml) at 30 ° C., and then cells are collected by centrifugation medium. The cells are suspended once in 0.9% aqueous sodium chloride solution (200 ml) and then collected by centrifugation to wash them. The cells thus washed are suspended in 50 mM Tris-HC1-50 mM disodium ethylenediamine tetraacetate aqueous solution (pH 7.5, 200 ml) containing 200 mg of lysozyme, and the mixture is allowed to stand at room temperature for 1 hour.

To the mixture is added sodium lauryl sulphate at a concentration of 0.5%, followed by additional proteinase K, and the mixture is lysed at 50 ° C. for 3 hours to lyse the cells. The mixture was extracted twice with the same amount of panol saturated with 10 mM Tris-HC1-lmM disodium ethylenediamine tetraacetate aqueous solution (pH 8.0) (hereinafter referred to as TE), followed by a mixture of TE-saturated phenol and eastern blood of chloroform After extraction twice with, the resulting aqueous phase is precipitated with ethanol. The precipitate is dissolved in TE to prepare a TE solution comprising 2.5 mg of chromosomal DNA containing an esterase encoding gene.

(2) Preparation of Recombinant Plasmid DNA:

The chromosomal DNA (20 μg) prepared in (1) above was completely digested with restriction endonuclease Sa1I and extracted twice with a mixture of TE-saturated phenol and eastern blood of chloroform. 0.5 volume of 7.5 M ammonium acetate is added to the mixture and centrifuged with 2 volumes of ethanol to recover DNA.

DNA (0.5 μg) of the plasmid vector pUC19 completely digested with the same restriction enzyme was dephosphorylated by treatment with alkaline phosphatase (Takara Shuzo Co., Ltd., Japan, manufactured by 0.4 units) for 1 hour at 56 ° C., The product is extracted twice with an equal volume mixture of TE-saturated phenol and chloroform, to which 0.5 volume of 7.5 M ammonium acetate rate is added, followed by precipitation with 2 volumes of ethanol to recover the vector plasmid DNA. The dephosphorylated plasmid vector thus obtained was mixed with the chromosome .DNA (3 μg) obtained above and ligated with a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.) at 4 ° C. for 16 hours to obtain recombinant plasmid DNA.

(3) transformation with recombinant plasmid and E. coli. Preparation of Colony Banks Using Collie Host

this. Coli DH 5 cells were treated by Hanahan method and described in Journal of Molecular Biology, Vol. 166, 557 (1983)] The transformation is carried out by adding a reaction mixture containing the plasmid DNA obtained in the above (2). The treated cells were spread on LBG plate medium containing ampicillin (50 μg / ml) and incubated overnight at 37 ° C. to transform cells containing recombinant plasmid containing the chromosomal DNA fragment of Serratia marsense Sr4l. (About 20,000 weeks) is obtained.

(4) Isolation and Identification of Transformants Containing Esterase-Encoding Genes When esterase producers are inoculated in tributyrin-containing LBG flat medium, the esterase produced in the medium decomposes tributyrin to produce fatty acids. The triglyceride emulsion around the colonies is deformed thereby to form a circular transparent band around the colonies. By using such a phenomenon, the transformant is screened.

The transformant obtained in (3) (about 20,000 strains) was inoculated in a tributyrin-containing LBG plate medium and incubated overnight at 37 ° C, whereby a monarch forming a zona pellucida from the DNA bank of Sa1I was obtained. Separate.

Inoculation of Seratia marsense Sr4l into tributyrin-containing LBG plate medium and incubation at 37 ° C. overnight showed formation of a zona pellucida. The formation of a zona pellucida may be due to esterase activity since no zona pellucida is formed when E. coli DH 5 carrying coli DH 5 and plasmid vector pUC19 are inoculated into tributyrin-containing LBG plate medium and incubated overnight at 37 ° C.

In addition, transformants capable of forming a zona pellucida were cultured by aerobic shaking culture overnight at 37 ° C. in LB medium (60 ml) containing ampicillin (200 μg / m 1), cells were collected and 200 ml Tris-HCI Suspension in complete layer solution (pH 8.0, 20m1). After disrupting the cells by sonication, the esterase activity of the mixture is measured in a convenient way. As a result, it was observed that the esterase activity was 1.5 × 10 ³ units.

In addition, the obtained mixture is subjected to electrophoresis using SDS polyacrylamide gel. As a result, the product shows a new band at the same position as in the partially purified standard esterase obtained from the culture supernatant of Cerratier marsense Sr-41, which is a product from E. coli DH5 containing only a vector plasmid Does not appear.

Example 2

Analysis of plasmids:

Plasmid DMA is routinely described by Maniatis et al., Molecular Cloning, page 368, Cold Spring Harbor Lahoratory, U.S.A. (1982)], which were prepared from the transformant cells obtained in Example 1- (4). Agarose gel electrophoresis after cleavage with restriction endonucleases. As a result, it was confirmed that this plasmid (hereinafter referred to as pLIPE101) contained a Sa1I DNA fragment of about 4.0 kb.

A restriction endonuclease map of about 4.0 kb of recombinant DNA fragment contained in pLIPE101 is shown in FIG.

Approximately 4.0 kb of the Sa1I DNA fragment of plasmid pLIPE101 was digested with various restriction endonucleases, and each DNA fragment was subcloned into plasmid vector pUC19. Transform the coli. Transformants were cultured in tributyrin-containing LBG plate medium in the same manner as above and observed whether a zona pellucida was formed.

The restriction endonuclease map of each DNA fragment is shown in FIG. E. transformed with a plasmid (pLIPE111) containing a Sa1I-BamHl DNA fragment of about 2.8 kb. In the strain transformed with plasmid (pLIPE121) containing Coli DH5 and a Sa1I-BstPI DNA fragment of about 2.6 kb, formation of a zona pellucida and esterase activity was observed (measured by a convenient method). However, a plasmid containing about 1.9 kb of Sa1I-Pvu1I DNA fragment (pLIPE131), a plasmid containing about 1.7 kb of Sall-BstPI DNA fragment (pLIPE141), and a plasmid containing about 3.6 kb of Clal-Sall DNA fragment. In E. coli DH5 transformed with (pLIPE151), neither formation of a zona pellucida nor esterase activity was observed.

(Measured in a convenient way).

From the above results, it was found that the esterase coding gene derived from Serratia marcesense Sr4l was present in the Sall-BstPI fragment of about 2.6 kb.

Example 3

Analysis of clone DNA:

[Determination of Nucleotide Sequences]

Various deletion plasmids are prepared by treatment with the recombinant plasmid pLIPE121 Kilobase Deletion Kit (manufactured by Kilobase Deletion Kit Takara Shuzo Co., Ltd). The primers were annealed to the plasmid thus obtained, and complementary chain [(α -32p) dCTP (14.8x10 ⁶ Bq) was prepared using cleno fragments of DNA plymerase according to dideoxy chain termination method such as Sanger et al. / pmol, labeled 74 × 10 ⁴ Bq)] and [Ref. Proc. Natl. Acad. Sci. USA, Vol. 74,5463 (1977)], its nucleotide sequence is determined based on 8% urea-modified polyacrylamide gel and electrophoretic data using radiographs.

As a result, the DNA sequence of the esterase coding gene derived from Serratia marsense Sr4l is composed of 1839 base pairs from the start codon ATG to the codon GCC.

In addition, amino acid sequences determined from nucleotides are shown in SEQ ID NO: 3. The N-terminal Met of this amino acid sequence was not found at the N-terminus of the amino acid sequence of the purified enzyme product of Serratia and derived esterases, a protein produced in the cell by methionine aminofabtidase present in the cell. This is due to the removal of N-terminal methionine from. Moreover, although the esterase derived from Serratia is an extracellular enzyme, it does not have a signal peptide generally present at the N-terminus of the secreted protein.

Example 4

Certia marcesense Sr4l (FERM BP-487) was incubated overnight in nutrient medium (0.5% glucose, 1.0% fabton, 0.3% meat extract, 1.0% yeast extract, and 0.5% sodium chloride, pH 7.0, 3 ml), The obtained culture solution (0.1 ml) was inoculated in the same fresh nutrient medium (3 ml) as described above, which was incubated at 30 ° C. for 3 hours, to which 0.1 mg / ml of NTG was added, followed by further 30 minutes of incubation. do. The culture broth is centrifuged and the cells obtained are washed three times with physiological saline (by centrifugation). The cells thus obtained are suspended in physiological saline and spread on LBG plates containing 0.5% tributyrin 0.5%, glucose 2% and EDTA 2 mM in amounts of 1-1000 cells / 1 plate. After 1 hour of incubation at 30 ° C., colonies forming a large transparent zone are collected to obtain a Serratia marsense M-1 strain (FERM P-12833). The thus obtained mutant strain (Platinum Platinum) was inoculated into esterase-producing medium containing ampicillin (500 μg / μl) and reciprocally shake cultured at 30 ° C. for 20 hours (shaking butterfly 7 cm, 120 rpm). The culture broth is centrifuged to obtain a supernatant with esterase activity of about 80 × 10 ³ units / ml (measured by convenient method). This strain is about 2.5 times higher in esterase productivity than the host parent Ceratia marcesense Sr4l.

In the same manner as described in Examples 1 to 3, chromosomal DNA is isolated from Serratia marsense M-1 described above and the sequence is analyzed. As a result, it has a nucleotide sequence as shown in SEQ ID NO: 2. As is evident from the above, the sequence is identical to the DNA sequence obtained from Serratia marcesense Sr4l, except that the ninth base is different, ie, it consists of 1839 base pairs from the start codon ATG to the codon GCC. The amino acid sequence determined therefrom is determined from SEQ NO: 1. Same as inferred.

Example 5

Preparation of Esterase High Productivity Strains:

The plasmid pLIPElll was introduced into a restriction endonuclease-deficient strain, Serratia marcesense TT392 to obtain a transformant. Modified pLIPElll plasmid DNA using Serratia marsense is extracted from the cells of the transformant by alkaline lysis. Subsequently, Serratia marsense Sr4l cells are transformed with the plasmid DNA obtained above by electroporation to obtain transformant Serratia marsenses TA5025 (FERM P-12607). The resulting transformant (Platinum Platinum) was inoculated into an esterase-producing medium containing ampicillin (500 µg / m1) and reciprocated shaking culture at 30 ° C. for 20 hours (shaking butterfly 7 cm, 120 rpm). Centrifugation of the culture broth yields a supernatant of about 1.9 x 10 ⁵ units / ml esterase (measured by convenient method). This strain has about 6 times higher esterase productivity than the host strain Serratia marsense Sr4l.

Example 6

Production of Esterase: Esterase-containing medium (20 L) containing ampicillin (500 μg / mL) was added to 30 Put into a volume of ego fermenter and sterilize. Prior to this, the Serratia marsense TA5025 is incubated in the same medium by reciprocating shaking culture at 30 ° C. for 20 hours in the same manner as above. The preculture broth (200 ml) thus obtained was inoculated in the medium and 200 rpm. Incubate under aeration at 30 ° C. for 18 hours under agitation of 0.5vvm. Cells were removed by centrifugation of the culture broth, and the supernatant (18 L) was concentrated with an ultrafilter (AIL-1010, manufactured by Asahi Chemical Industry Co .. Lt, d.) To obtain esterase activity of 1.7 x 10 ⁶ units / A crude enzyme solution (200 ml) is obtained (measured by a convenient method).

Example 7

Production of esterases:

Esterase-Production Medium (20ℓ) 30 Put into a volume of ego fermenter and sterilize. Previously, Serratia marcesense M-1 is incubated in the same medium by reciprocating shaking culture at 30 ° C. for 20 hours in the same manner as above. The preculture broth thus obtained is inoculated in the above medium and incubated under aeration at 30 ° C. under stirring at 200 rpm for 0.5 hours at 0.5 vpm. The cells are removed by centrifugation of the culture broth, and the supernatant (18 ) Was concentrated using an ultrafilter (AIL-1010, manufactured by Asahi Chemical Industry Co., Ltd.) to obtain a crude enzyme solution (2000m1) having an esterase activity of 7.9 × 10 ⁵ units / ml (measured by a convenient method). ).

Transformants and mutants containing the recombinant plasmid inserted with the esterase coding gene of the present invention are excellent in esterase productivity, and can be obtained in large quantities with the desired esterase with high purity by culturing the transformant. have.

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Claims (9)

An esterase coding gene derived from a Serratia genus microorganism having the DNA sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. A recombinant plasmid comprising a vector plasmid containing an esterase coding gene derived from a Serratia genus microorganism having the DNA sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. A transformant containing a recombinant plasmid comprising a vector plasmid containing an esterase coding gene derived from Serratia microorganism having the DNA sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. The transformant of claim 3, wherein the host microorganism is a Serratia microorganism or an Escherichia microorganism. Transformant Serratia marsense TA5025 (FERM BP-4067). Culture the microorganism transformed with the recombinant plasmid (the plasmid comprises a vector plasmid containing an esterase coding gene derived from a Serratia microorganism having the DNA sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2) Esterase production method characterized in that the collection of the esterase produced in and outside the cells. A microorganism of the genus Serratia containing a gene having the DNA sequence shown in SEQ ID NO: 2. Esterase production method characterized in that the culture of the microorganisms mentioned in claim 7 in the medium, and collects the esterase produced in and around the cells. Variant Serratia marsense M-1 (FERM BP-4068).