JPH05168468A - Substabtially pure microorganism for mass-producing monoglyceride lipase - Google Patents

Abstract

PURPOSE:To obtain a new microorganism useful for mass-producing a monoglyceride lipase. CONSTITUTION:The objective substantially pure microorganism e.g. Bacillus sp. H-257 strain (FERM P-12656) is belonging toe Escherichia coli transformed with a recombinant plasmid having a DNA capable of expressing the monoglyceride lipase and can produce the monoglyceride lipase. A genetic DNA capable of expressing the monoglyceride lipase is screened from a chromosomic DNA library derived from a microorganism capable of producing the monoglyceride lipase and used to construct an expression vector, which is then transduced into a microorganism of the genus Escherichia to create a transformed microorganism.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a substantially pure microorganism which produces a monoglyceride lipase, a substantially pure D expressing a polypeptide having a monoglyceride lipase activity.
The present invention relates to a method for producing NA and monoglyceride lipase. More specifically, the present invention, for example, the activity measurement of lipase in serum, food and drink, monoglyceride lipase useful as an enzyme for analysis of monoglyceride in body fluids, Escherichia coli for efficiently producing by genetic recombination technology. (Escherichia co
li), a transformed microorganism belonging to Bacillus sp. H
-257 produces a monoglyceride lipase gene DNA having a nucleotide sequence encoding the amino acid sequence of monoglyceride lipase, and a method for efficiently producing monoglyceride lipase by culturing the transformed microorganism.

[0002]

BACKGROUND OF THE INVENTION Monoglyceride lipase is a reaction formula

[Chemical 1] As shown in, it has a property of acting on a substrate monoglyceride and decomposing it into glycerol and a fatty acid. Conventionally known monoglyceride lipase is known to act not only on monoglyceride but also on diglyceride and triglyceride [ "Enzyme Handbook", page 424, published by Asakura Shoten in 1982]. Further, a strain derived from a strain belonging to the genus Staphylococcus has been found (Japanese Patent Laid-Open No. 55-425).
32), this enzyme acts as a substrate specificity on triglycerides as well as monoglycerides. By the way, as a lipase activity measuring method, for example, a turbidimetric method using triolein, a coloring method using a synthetic substrate, a method of titrating decomposed fatty acids, and a natural substrate 1,2-diglyceride are used as synthetic substrates. A method of enzymatically measuring the product is known. Among these methods, the method of enzymatically measuring the product using 1,2-diglyceride as a synthetic substrate is advantageous in that the operation is simple, the measuring time is short, and the measuring is accurate. When using monoglyceride lipase in this enzymatic method, conventional monoglyceride lipase acts not only on monoglyceride but also on diglyceride and triglyceride, so for example, a method for measuring only monoglyceride released from glyceride synthetic substrate in lipase activity measurement In addition, it cannot be used, and in the analysis of monoglycerides in foods and drinks and body fluids, conventional monoglyceride lipases also act on coexisting diglycerides and triglycerides, so that accurate analysis values cannot be obtained. Therefore, the present inventors
After repeated research on monoglyceride lipase, which has excellent substrate selectivity, we first studied Bacillus stearothermophilus (B
acillus stearothermophill
It was found that a monoglyceride lipase produced by a certain strain belonging to us) acts only on monoglyceride and does not substantially act on diglyceride and triglyceride (Japanese Patent Laid-Open No. 63-245672). However, although this monoglyceride lipase has excellent substrate selectivity and is used for measuring monoglyceride and for measuring lipase activity as a clinical test reagent, there is a problem in productivity with the conventional production method, and the quality is constant. It has been desired to develop a method for producing the monoglyceride lipase with high productivity.

[0003]

Under the circumstances, the present invention is excellent in substrate selectivity useful for measurement of lipase activity in serum, analysis of food and drink, and monoglyceride in body fluid. The object of the present invention is to develop a microorganism that efficiently produces monoglyceride lipase, and to provide a method for mass-producing the enzyme having a constant quality by using the microorganism.

[0004]

Means for Solving the Problems As a result of intensive studies to achieve the above-mentioned object, the present inventors have expressed the enzyme from a chromosomal DNA library derived from a microorganism producing the monoglyceride lipase. After screening the gene DNA and constructing an expression vector using this DNA, it is introduced into a microorganism of the genus Escherichia to produce a transformed microorganism, which is then cultured in a medium to efficiently mass-produce the monoglyceride lipase. Therefore, the present invention has been completed based on this finding. That is, the present invention relates to E. coli transformed with a recombinant plasmid having DNA expressing monoglyceride lipase. A substantially pure microorganism that produces a monoglyceride lipase characterized by being a microorganism belonging to E. coli, and a polypeptide having a monoglyceride lipase activity characterized by having a base sequence encoding the amino acid sequence shown in FIG. A method for producing monoglyceride lipase, which comprises culturing substantially pure DNA to be expressed and the transformed microorganism belonging to the genus Escherichia in a medium, and then collecting monoglyceride lipase from the culture. Is.

The present invention will be described in detail below. In the present invention, transformed E. coli producing monoglyceride lipase. DNA for expressing monoglyceride lipase used for producing microorganism belonging to E. coli
Can be obtained by screening from a chromosomal DNA library derived from a microorganism that produces the enzyme. The screening method is not particularly limited, and a method conventionally used in gene recombination technology can be used. For example, first, the chromosomal DNA of a microorganism that produces monoglyceride lipase is separated and purified, then cleaved with an appropriate restriction enzyme, and ligated to a cloning vector to prepare a chromosomal DNA library. On the other hand, using a gene code table, a nucleotide sequence of a part is assumed from the amino acid sequence of the enzyme, an oligonucleotide having a length of 15 to 25 nucleotides corresponding to the nucleotide sequence is synthesized, and is labeled with an isotope or a non-radioactive compound. To create. Next, using this oligonucleotide probe, a gene DN expressing monoglyceride lipase is selected from the above-mentioned chromosomal library.
By picking up A, the gene DNA of interest is screened.

In the present invention, Bacillus sp. H is used as a microorganism producing the above-mentioned monoglyceride lipase.
-257 (Bacillus sp. H-257) is preferably used. This Bacillus SP H-257
Strain (collected and isolated from wheat field soil in Kawabe Town, Kawabe District, Kagoshima Prefecture)
The mycological properties of are as follows. (1) Characteristics of growth In a normal agar slant medium, it grows linearly and satisfactorily. Pale yellowish white, translucent, wet, no soluble pigment produced. In addition, it forms circular and flat colonies on ordinary agar flat medium. Light yellowish white, translucent, smooth surrounding, no soluble pigment produced. Furthermore, the liquid medium with peptone water is cloudy uniformly, but the growth is weak. It is unchanged in BCP milk medium. (2) Characteristics of morphology It is a rod-shaped bacterium with a rounded end, which is straight or slightly bent, and is short-chained singly or twice in a row. Spores are formed at the ends or sub-ends.
Exercise with periflagellates. Size is 0.6-0.8x2-5μ
m, spores are 0.5 to 0.8 × 1 μm, spores are oval to oval in shape, and the cells do not swell.

(3) Physiological and biochemical shapes Gram stain + KOH reaction-Capsule formation-Acidic acid stain-OF test (Hugh-Leifson medium) No change OF test (modified medium) No change Growth under aerobic conditions + Anaerobic growth + Growth temperature 52 ° C + 40 ° C-Salt resistance 0.5% + 1.0% -Decomposition of gelatin + Decomposition of starch (+) Decomposition of casein-Decomposition of aesculin + Decomposition of cellulose-Arginine Degradation-Tyrosine degradation-Catalase production (+) Oxidase production + Urease production (SSR) -Indole production-Hydrogen sulfide production-Acetoin production-MR test-Reduction of nitrate + Denitrification reaction + Simmons medium availability Quenching Acid salt-maleate-malate-gluconate-propionate-malonate-koha Salt-Availability in Kristensen medium Citrate + maleate + malate + gluconate (+) propionate-malonate-succinate + gas production from glucose-acid from each sugar Production of adonitol-L (+) arabinose-cellobiose-dulcitol-meso-erythritol-fructose-galactose-glucose-glycerin + inositol-inulin-lactose-mannitol + melezitol-melezibose-raffinose-L (+) rhamnose-saline. -L sorbose-sorbitol-starch-saccharose + trehalose (+) xylose-From the above mycological properties, this strain belongs to the genus Bacillus because it is a facultative anaerobic bacterium that forms spores. Next, Table 1 shows a comparison of various properties between the present strain and four other bacterial strains showing the same properties as the present strain with respect to the properties of producing no acid, producing no acetoin, and reducing nitrate from glucose.

[0008]

[Table 1]

(Note) +: Positive,-: Negative, d: Depends on the strain From the results of the comparison test in Table 1, no strain was found to be the same as the H-257 strain of this strain. Therefore, this strain was identified as Bacillus sp. H-257 strain (FERM P-12656). Next, from this chromosomal DNA library of Bacillus sp. H-257, the gene D expressing the enzyme was expressed.
Explaining a specific example of the method for screening NA, first, chromosomal DNA 100-2000 of the microorganism is described.
After extracting about μg by a commonly used method, about 1 to 10 μg thereof is cleaved with a restriction enzyme ClaI to obtain, for example, a plasmid pAC of a cloning vector.
YC184 was ligated to the ClaI site, and then this recombinant DNA was introduced into a host microorganism to clone the chromosomal DNA to obtain chromosome D consisting of 10 ⁴ to 10 ⁵ clones.
Create an NA library. The host microorganism used at this time is not particularly limited as long as the recombinant DNA is stable and capable of autonomously growing, and those used for ordinary gene recombination such as Escherichia coli. (E. coli) and the like are preferably used.

As a method for introducing the recombinant DNA into the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, the recombinant DNA may be introduced in the presence of calcium ions, or it may be competent. The cell method may be used, and in the case of a microorganism belonging to the genus Bacillus, the competent cell method or the protoplast method may be used, or the microinjection method may be used. Desired recombinant DNA for host microorganism
Regarding the selection of the presence or absence of introduction, the host microorganism may be cultured in a selective medium based on the drug resistance marker of the vector constituting the recombinant DNA, and the host microorganism that grows may be selected.

On the other hand, the N-terminal side amino acid sequence of the purified preparation of monoglyceride lipase and the amino acid sequence of the lysyl endopeptidase-treated fragment were determined, and based on this, 15-2
After synthesizing various oligonucleotides having a base length of about 5 and labeling with, for example, an isotope, a radioactive oligonucleotide probe is prepared. Then, using this radioactive oligonucleotide probe, according to the method conventionally used, the chromosome D
A gene DNA that expresses monoglyceride lipase is screened from the NA library. In the present invention, the target gene DNA is the one that is picked up with a radioactive oligonucleotide probe based on the amino acid sequence of the fragment treated with lysyl endopeptidase. It has been found. Next, from the transformed host microorganism containing the gene DNA of interest, for example, Maniatis
and a recombinant plasmid (pM containing DNA expressing monoglyceride lipase).
GLP31) can be prepared. A schematic diagram showing the constitution of this plasmid is shown in FIG. In addition, Bacillus sp. H-257 chromosomal DNA in the plasmid
The entire base sequence and the restriction enzyme map of the origin site are as shown in SEQ ID NO: 1 of the sequence listing and FIG.

Next, as described above, an expression vector incorporating a gene DNA for expressing monoglyceride lipase is constructed from the chromosomal DNA library. As the expression vector, a vector constructed for gene recombination from a phage or a plasmid capable of autonomously growing in a host microorganism is suitable. Examples of the former phage include E. coli. When E. coli is used as the host microorganism,
λgt · λC, λgt · λB, etc. are used. Also,
As the plasmid, E. Since E. coli is used as a host microorganism, for example, pBR322, pBR325, pACYC
184, pUC12, pUC13, pUC18, pUC
19, pUC118 or the like is used. The method for incorporating the monoglyceride lipase gene DNA into these vectors is not particularly limited, and a conventionally used method can be used. For example, using a suitable restriction enzyme, the recombinant plasmid containing the monoglyceride lipase gene DNA and the expression vector are treated to obtain a DNA fragment containing the monoglyceride lipase gene and a vector fragment, respectively, and then the respective cohesive ends are After annealing, an expression vector can be obtained by ligating with an appropriate DNA ligase. In some cases, the recombinant plasmid pMGLP31 can be directly used as an expression vector. In the examples described below, the recombinant plasmid p was used as an expression vector.
MGLP31 was used.

The expression vector constructed in this manner was transformed into E. coli. When introduced into a microorganism belonging to E. coli and transformed into the host microorganism, a substantially pure microorganism producing monoglyceride lipase can be obtained. The method of introducing the expression vector is not particularly limited, and it may be introduced in the presence of calcium ions, or may be introduced using the competent cell method. The selection as to whether or not the expression vector is introduced into the host microorganism may be carried out by culturing the microorganism in a selective medium based on the drug resistance marker of the vector and selecting a microorganism that grows. In the present invention, E. coli transformed with the recombinant plasmid pMGLP31. The microorganism belonging to E. coli is Escherichia coli DH1-pMGLP31 (FERM P-12657).
Is named.

Cultivation of the transformed microorganism thus obtained can be carried out in a medium containing nutrients such as carbon and nitrogen sources necessary for the growth of the microorganism and inorganic components. Examples of the carbon source include glucose, starch, sucrose, molasses, dextrin, etc., and examples of the nitrogen source include peptone, meat extract, casein hydrolyzate, corn steep liquor, nitrate, ammonium salt, etc. Examples of the salt include salts containing cations such as sodium, potassium, calcium, magnesium, cobalt, zinc, manganese and iron and anions such as chlorine, sulfuric acid and phosphoric acid.

The culturing method is not particularly limited and may be a known method such as aeration and agitation culture, shaking culture, rotary culture, stationary culture, etc., usually at 20 to 50 ° C., preferably 25 to 42 ° C., more preferably Is around 37 ° C,
A method of culturing for about 48 hours is used. After culturing in this way, cells are collected by means such as centrifugation, then cells are disrupted by autolysis, French press, ultrasonic treatment, etc. to obtain an extract. By examining the enzymatic activity of monoglyceride lipase, it was confirmed that the transformed microorganism has the ability to produce monoglyceride lipase. Therefore, the gene DNA expressing the monoglyceride lipase used in the present invention is SEQ ID NO: 1 in Sequence Listing 1.
It is clear from the above that it has a base sequence encoding the amino acid sequence shown in FIG. 1 and the base sequence is the sequence shown in FIG.

In order to separate and purify the enzyme from the extract, for example, salting out, desalting, adsorption / desorption treatment with an ion exchange resin, and the like, followed by gel adsorption chromatography, gel filtration and electrophoresis. It may be purified by the following methods. The monoglyceride lipase thus obtained does not substantially act on diglyceride and triglyceride, but has a catalytic action of acting only on monoglyceride and effectively decomposing into glycerol and fatty acid. Therefore, it is useful as an enzyme for measuring lipase activity in serum, for analyzing monoglycerides in foods and drinks, and body fluids. The symbols for base sequences and symbols for amino acid sequences described in the specification of the present invention are based on conventional abbreviations in the art, and examples thereof are listed below. Also,
All amino acids shall be in the L form.

DNA: deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine Ala: alanine Arg: arginine Asn: asparagine Asp: aspartic acid Cys: cysteine Gln: glutamine Glu: glutamic acid Gly: glycine Hisle: histidine Leu: Leucine Lys: Lysine Met: Methionine Phe: Phenylalanine Pro: Proline Ser: Serine Thr: Threonine Trp: Tryptophan Tyr: Tyrosine Val: Valine

[0018]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 966 base pairs Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomic DNA Origin of organism Name: Bacillus sp. Strain name: H-257 Name: Monoglyceride lipase gene

Sequence GTCAACGGCT TGTTTCACGA ATGGTGGCGG TCATGGTTGT CGATGTGGTA ACGGCGTCCG 60 ATCAAAAAAG GAAAAAAGAG GTGCGGCGGC GAATATAGTG AGAGGAACGG ATTTGACTAG 120 ATAAGGGGGA GAGGAAG ATG AGC A GLA GTC GTG CTC GTG CTC GTG CCC GTG CTC GTG CTC GTG CTC TAC GCC GAA AAC GGG CCG GTC GGG GTG CTG CTC GTG CAC GGA 221 Pro Phe Tyr Ala Glu Asn Gly Pro Val Gly Val Leu Leu Val His Gly 15 20 25 TTC ACC GGC ACG CCC CAC AGC ATG CGC CCG CTC GCT GAA GCG TAT GCG 269 Phe Thr Gly Thr Pro His Ser Met Arg Pro Leu Ala Glu Ala Tyr Ala 30 35 40 AAA GCC GGC TAT ACC GTT TGC CTG CCG CGC TTA AAA GGG CAC GGA ACG 317 Lys Ala Gly Tyr Thr Val Cys Leu Pro Arg Leu Lys Gly His Gly Thr 45 50 55 60 CAT TAC GAA GAC ATG GAA CGG ACG ACG TTC CAC GAT TGG GTC GCC TCG 365 His Tyr Glu Asp Met Glu Arg Thr Thr Phe His Asp Trp Val Ala Ser 65 70 75 GTC GAA GAA GGA TAT GGA TGG CTG AAA CAA CGA TGC CAA ACC ATT TTT 413 Val Glu Glu Gly Tyr Gly Trp Leu Lys Gln Arg Cys Gln Thr Ile Phe 80 85 90 G TC ACC GGG CTG TCG ATG GGC GGG ACG CTC ACG CTT TAT TTG GCG GAA 461 Val Thr Gly Leu Ser Met Gly Gly Thr Leu Thr Leu Tyr Leu Ala Glu 95 100 105 CAT CAC CCA GAC ATC TGC GGC ATC GTG CCG ATT AAC GCC GCT GTC GAC 509 His His Pro Asp Ile Cys Gly Ile Val Pro Ile Asn Ala Ala Val Asp 110 115 120 ATC CCG GCC ATC GCC GCC GGG ATG ACG GGC GGG GGC GAG CTG CCG AGG 557 Ile Pro Ala Ile Ala Ala Gly Met Thr Gly Gly Gly Glu Leu Pro Arg 125 130 135 140 TAT CTG GAT TCG ATC GGT TCG GAC TTG AAA AAT CCG GAT GTG AAA GAG 605 Tyr Leu Asp Ser Ile Gly Ser Asp Leu Lys Asn Pro Asp Val Lys Glu 145 150 155 CTG GCA TAC GAG AAA ACG CCG ACC GCT TCG CTT CTT CAG CTG GCT AGG 653 Leu Ala Tyr Glu Lys Thr Pro Thr Ala Ser Leu Leu Gln Leu Ala Arg 160 165 170 CTG ATG GCA CAG ACA AAA GCG AAA CTC GAT CGC ATC GTC TGT CCG GCG 701 Leu Met Ala Gln Thr Lys Ala Lys Leu Asp Arg Ile Val Cys Pro Ala 175 180 185 TTG ATT TTT GTC TCC GAC GAA GAT CAC GTC GTG CCG CCG GGA AAC GCC 749 Leu Ile Phe Val Ser Asp Glu Asp His Val Val Pro Pro Gly Asn Ala 19 0 195 200 GAC ATC ATC TTT CAA GGC ATT TCA TCG ACG GAG AAA GAG ATC GTC CGC 797 Asp Ile Ile Phe Gln Gly Ile Ser Ser Thr Glu Lys Glu Ile Val Arg 205 210 215 220 CTC CGA AAC AGC TAC CAT GTG GCG ACG CTC GAT TAC GAC CAA CCG ATG 845 Leu Arg Asn Ser Tyr His Val Ala Thr Leu Asp Tyr Asp Gln Pro Met 225 230 235 ATT ATT GAA CGG TCT CTC GAA TTT TTC GCC AAG CAC GCC GGA TAAAACGAAT 897 Ile Ile Glu Arg Ser Leu Glu Phe Phe Ala Lys His Ala Gly 240 245 250 CGGTATATGG CTGATCCTGT TCAAAGGAGA GGCCATTTTT TGTTTGTCGG TATTTTTGTG 957 AAAAAATTA 966

[0020]

EXAMPLES Next, the present invention will be explained in more detail by reference examples and examples, but the present invention is not limited to these examples. Reference Example 1 Separation of chromosomal DNA Bacillus sp. H-257 (FERM P-126
56) After culturing the strain in 200 ml of normal broth medium [18 g / liter, normal broth “Eiken” (manufactured by Eiken Chemical Co., Ltd.)] at 50 ° C. for one day and night, this culture solution was subjected to high-speed cooling centrifuge ( Tommy CX-250 type), 6500
The cells were collected by centrifugation at rpm (7660G) for 10 minutes. Then, the cells were treated with 50 mM Tris-hydrochloric acid (pH 8.0), 50 mM EDTA (pH 8.0) and 15%.
Suspend in 20 ml of sucrose solution and make lysozyme to a final concentration of 2 mg / ml [Seikagaku Corporation.
[Production] was added and treated at 37 ° C. for 30 minutes to destroy the cell wall of the strain. Next, 1 ml of a 10% sodium lauryl sulfate (manufactured by Sigma) aqueous solution was added thereto, and the mixture was treated at 37 ° C. for 20 minutes, and then chloroform / phenol (1 /
1) Add 21 ml of the mixed solution, stir, and then 10,000 rpm (12
The mixture was centrifuged at 080 G) for 10 minutes and the aqueous layer was collected. This aqueous layer was gently overlaid with 2 volumes of ethanol, and the DNA was sprinkled on the glass rod while gently stirring with a glass rod to separate the DNA, and then 10 mM Tris-hydrochloric acid (pH 8.
Dissolved in 20 ml of a solution consisting of 0) and 1 mM EDTA,
Then add an equal amount of chloroform / phenol (1 /
1) The mixed solution was added and treated in the same manner as above to separate the aqueous layer. Then, twice the amount of ethanol was added to this aqueous layer to separate the DNA again by the above-mentioned method, and then 2m of a solution containing 10mM Tris-hydrochloric acid (pH 8.0) and 1mM EDTA was added.
dissolved in l.

Reference Example 2 Isolation of pACYC184 Plasmid DNA Escherichia coli pM carrying pACYC184
191 ["Journal of Bacterior (J. Ba
Cteriol) ", Volume 134, Page 1141 (1
981); ATCC37033] as BHI medium [Brain heart infusion medium (manufactured by Difco)]
After shaking culture at 1 liter, the turbidity was OD660 = 1.
Once grown to 0, spectinomycin was added to give a final concentration of 300 μg / ml and an additional 37
Shaking was continued for 16 hours or more at ℃. Then 6500rp
Collect the cells by centrifuging at m for 10 minutes, then lysozyme SD
S method and cesium chloride-ethidium bromide method ["Molecular Cloning: Cold Cloning Harbor (Co
ld Spring Harbor ”, pp. 86-94 (1982)], and plasmid DNA was prepared.

Reference Example 3 Preparation of Bacillus Gene Library 5 μg of Bacillus chromosome obtained in Reference Example 1 was treated with 30 units of restriction endonuclease ClaI [manufactured by Takara Shuzo Co., Ltd.], 50 mM Tris-acetic acid (pH 7.5), 100 mM NaC
Cleavage was carried out for 2 hours at 37 ° C. in the presence of 10 μg / ml of a mixture consisting of 1, 10 mM MgCl ₂ and 1 mM DTT. On the other hand, the vector plasmid pA obtained in Reference Example 2
2 μg of CYC184 was added with the restriction endonuclease Cl
After treating with 20 units of aI in the same manner as above, 1 unit of alkaline phosphatase [Takara Shuzo Co., Ltd.] was further added and treated at 65 ° C. for 2 hours. Next, the two kinds of DNA solutions obtained as described above were mixed, an equal amount of a chloroform / phenol mixed solution was added to the mixed solution, the mixture was treated, and the aqueous layer was separated by centrifugation. .. Next, to this aqueous layer, 1/10 amount of 3M sodium acetate solution was added, and further 2 times amount of ethanol was added to perform centrifugation treatment to precipitate DNA, and then, it was dried under reduced pressure. This DNA was added to 10 mM Tris-hydrochloric acid (pH 8.0).
And 66 mM Tris-hydrochloric acid (pH 7.6), 6.6 mM MgC.
Ligation was carried out at 16 ° C. for 16 hours using 100 units of T4 DNA ligase [Takara Shuzo Co., Ltd.] in the presence of l ₂ , 10 mM DTT and 660 μM ATP (Boehringer Mannheim). Then, this is Shige
The method of sada ["Cell Engineering" Vol. 2, 616-626]
Page (1983)] as a competent cell. ^{coliDH1 (ATCC33849) [F -,} r
ecA1, endA1, gyrA96, thi-1, h
sdR17 (r _k ⁻ , m _k ⁺ ), SupE44, relA
1, λ ^-] [ "Morekyuraru Cloning: Cold
Spring Harbor (Molecular Clon)
ing: Cold Spring Harbor ”, pages 504 to 506 (1982)], and chloramphenicol 30 μg.
Cultivated in BHI agar medium containing / ml at 37 ° C for 24 hours,
About 27,000 transformed microorganisms were obtained and used as a Bacillus gene library.

Reference Example 4 Preparation of Radioactive Oligonucleotide Probe When the amino acid sequence of the N-terminal side of the purified monoglyceride lipase preparation and the amino acid sequence of the lysyl endopeptidase-treated fragment were examined, the sequences shown in FIGS. 5 and 6 were determined. .. Based on this information, the base sequence was predicted from the 5'end side of the gene. Since there are various possibilities for this predicted base sequence, two oligonucleotide sequences with high possibility were designed and tested. This oligonucleotide is a DNA synthesizer [Cyclone (Cyclone (Cyclone (Phaset)], based on the method of Earl Lessinger et al. [J. Am. Chem. Soc.] Vol. 98, page 3655]. Biosearch Co., Ltd.)]. That is, lysyl endopeptidase-treated "K6" fragment amino acid sequence 5th residue His to 10th residue Glu and 1
DNA corresponding to 4th residue Phe to 19th residue Ala
From the combination of 17-mer 16Mix and 17-mer 32Mi
x was designed and the oligonucleotides shown in FIGS. 7 and 8 were prepared and designated as oligonucleotides (1) and (2), respectively. 50 ng of the thus obtained oligonucleotide was added to T4 polynucleotide kinase buffer [50 mM Tris-hydrochloric acid (pH 8.0), 10 mM MgC].
l ₂ , 10 mM _2- mercaptoethanol] and 370 kilobecquerel of ³² P-ATP (manufactured by Amersham Japan).
In the presence of T4 polynucleotide kinase [TOYOBO
8.5 units were used and reacted at 37 ° C. for 30 minutes to incorporate isotope ³² P to prepare a radioactive oligonucleotide probe.

Example 1 Screening of DNA containing monoglyceride lipase gene On the Bacillus gene library obtained in Reference Example 3, that is, on chloramphenicol resistant colonies on plate agar medium, nylon membrane filter [Magnagraph nylon (micron separation) was used. Manufactured by K.K.), and a part of the colony cells is transferred onto the filter, and the filter is immersed in an alkaline denaturing solution (0.5 M NaCl-containing 0.5N-NaOH solution) for 5 minutes for further neutralization. Liquid [0.5 M Tris-hydrochloric acid (pH 7.0) and 3 M Na
Cl mixed solution] for 5 minutes and then dried. Next, this filter was heated at 80 ° C. for 2 hours to immobilize the plasmid DNA contained in the cells on the filter. In addition, the filter was filtered through 1.8M NaCl, 0.18M sodium citrate, 0.05% sodium diphosphate, 0.1% sodium lauryl sulfate, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1%. It was immersed in a prehybridization solution containing% BSA and 0.01% salmon sperm DNA, and prehybridization was carried out at 37 ° C for one day. After that, the filter is set to 1.8 M NaCl,
0.18 M sodium citrate, 0.05% sodium diphosphate, 0.1% ficoll, 0.1% polyvinylpyrrolidone, 0.1% BSA and 0.002% E.I. After immersing in a hybridization solution containing transfer RNA derived from E. coli, the radioactive oligonucleotide probe obtained in Reference Example 4 was added, and hybridization was carried out at 37 ° C. for 24 hours. After the hybridization, the filter was washed 3 times with a washing solution containing 1.8 M NaCl, 0.18 M sodium citrate, and 0.05% sodium diphosphate, and then immersed in a washing solution at 42 ° C. for 10 minutes to wash off excess probe. .. Then, after air-drying the filter, X-ray film [Fuji Photo Film Co., Ltd.
Manufactured by NewRXO-H], and protected from light at -80 ° C for 24
Timed autoradiography was performed. Then, when the film was developed, colonies showing a positive signal were confirmed only in those using the synthetic oligonucleotide (1). The colony was transformed into a transformant E. coli containing a DNA encoding a monoglyceride lipase. coli DH1
-PMGLP31 (FERM P-12657).

Example 2 Extraction of Recombinant Plasmid Recombinant Escherichia coli harboring pMGLP31 was cultured in BHI medium at 37 ° C. for a whole day and night, followed by the method of T. Mananinis et al. [“Molecular Cloning: Cold Spring Harbor ( Molecular Cloni
ng: ColdSpring Harbor) "No. 86
~ 94 (1982)], the recombinant plasmid pMGLP31 containing the DNA encoding the monoglyceride lipase was extracted. A schematic diagram showing the constitution of this plasmid is shown in FIG. The site derived from the Bacillus chromosome in the plasmid was determined by the dideoxy method ["Science (Scie
No.), 214, 1205-1210 (1981)], the nucleotide sequence was determined, and it was confirmed that the total DNA encoding the monoglyceride lipase was contained and the entire nucleotide sequence was determined. The sequence shown in SEQ ID NO: 1 in the sequence listing was confirmed. Conventionally known N-terminal amino acid sequence 3 of monoglyceride lipase
The 0 residue completely matched with the amino acid sequence considered from the above-mentioned nucleotide sequence, and also completely matched with the sequence of the 61 amino acid residue portion of the lysyl endopeptidase-treated fragment in the same frame.

Example 3 E. Expression of activity of monoglyceride lipase in E. coli 5 μg of plasmid pMGLP31 obtained in Example 2
Was transformed in the same manner as in Reference Example 3, plated on BHI agar medium containing 30 μg / ml of chloramphenicol, and cultured at 37 ° C. overnight. Then, the transformed microorganism that emerged was treated with BHI containing 30 μg / ml of chloramphenicol.
After culturing at 37 ° C for one day in the medium, the culture solution is 1500
The precipitate was recovered by centrifugation at 0 rpm for 1 minute. To this precipitate, the same amount of 10 mM Tris-hydrochloric acid (pH 8.
0) was added and ultrasonic disruption was performed. 20μ of this crushed liquid
aliquots of 0.2 M pipese-NaOH buffer (pH 7.
3) 100 μl, 0.3% 4-aminoantipyrine 50
μl, 0.3% TOOS 50 μl, 45 units / ml peroxidase 50 μl, 0.2 M MgCl ₂ 2.5 μl,
After adding 0.2 M ATP 2.5 μl, 25 units / ml glycerol kinase 10 μl, 1000 units / ml glycerophosphate oxidase 15 μl, water 120 μl and 10 mM monolaurin 50 μl, and reacting at 37 ° C. for 10 minutes,
Add 2.0 ml of 0.5% sodium dodecyl sulfonate,
By stopping the reaction and measuring the absorbance at 550 nm,
The monoglyceride lipase activity was quantified. The T
OOS means N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine. Also, for comparison, E. coli transformed with only pACYC184. The disrupted liquid of E. coli was treated in the same manner as described above, and the activity of monoglyceride lipase was measured. As a result, the activity with pMGLP31 was 1.5 μ / ml, but the activity with pACYC184 was 0. From this, it can be seen that a transformant having monoglyceride lipase activity was obtained.
This transformant was transformed into E. coli DH1-pMGLP3
1 (FERM P-12657). The obtained monoglyceride lipase had a molecular weight of about 27,000 as determined by gel filtration.

Example 4 Obtaining Enzyme from Recombinant Microorganism According to the method of Example 3, 5 liters of Jafamentor (stirring 300 rpm, 37 ° C., aeration 5 liters / minute)
The bacterial cells obtained in 1. were suspended in 2 liters of 10 mM Tris-hydrochloric acid (pH 7.5) and sonicated at 0 ° C., followed by 15,0
After centrifugation at 00 rpm for 10 minutes, 1,680 ml of supernatant was obtained. Then, 3,760 ml of cold acetone was added to this supernatant, and the resulting precipitate was collected by centrifugation at 5,000 rpm for 10 minutes and 10 mM containing 10% ammonium sulfate.
The enzyme was dissolved in 480 ml of Tris-hydrochloric acid (pH 7.5) and passed through a column (5.5 × 7 cm) packed with octyl-sepharose (Pharmacia) to adsorb the enzyme. Next, the unadsorbed component was washed with 10 mM Tris-hydrochloric acid (pH 7.5) containing 10% ammonium sulfate, and further with 10 mM Tris-hydrochloric acid (pH 7.5), and then 3% Triton X-10.
The adsorbed enzyme was eluted with 400 ml of 10 mM Tris-hydrochloric acid (pH 7.5) containing 0. 400 ml of this enzyme solution was concentrated to 50 ml using an ultrafiltration membrane manufactured by Amicon, and cold acetone 1 was added.
00 ml was added, and the resulting precipitate was mixed with 10 mM Tris-
The product was dissolved in 30 ml of hydrochloric acid (pH 7.5) and dialyzed against 1 liter of 10 mM Tris-hydrochloric acid (pH 7.5) to give a freeze-dried preparation (18.6 units / mg, 340 mg).

Various properties of the purified enzyme thus obtained were examined. The results are shown below. (1) Action Monoglyceride + H ₂ O → glycerol + fatty acid (monoglyceride may be either α- or β-monoglyceride) (2) Molecular weight: 25,000 ± 2,000 (Pharmacia Co., Super Rose 12 (Measurement using a gel filtration agent using a column) (3) Optimum pH: dimethyl glutaric acid-sodium hydroxide (○-○), pH 5.5 as a buffer solution according to the enzyme activity measuring method described later, pH 4 to 6. ~ 7.0 is MES-sodium hydroxide (●-●), pH 7.0-8.5 is Tris-hydrochloric acid (△-
△), pH 9-9.5 is glycine-sodium hydroxide (▲
-▲) was used. The relationship between pH and relative activity is shown graphically in FIG. As can be seen from this figure, the optimum pH was 7-9. (4) Optimum temperature: Tris-hydrochloric acid buffer solution (pH 7.5) was used, and after the reaction at each temperature, the amount of glycerol produced was enzymatically measured. The relationship between temperature and relative activity is shown graphically in FIG. As can be seen from this figure, the optimum temperature was 75 ° C.

(5) pH stability The purified enzyme solution (5.0 U / ml) was added to 10 mM dimethyl glutaric acid-sodium hydroxide buffer solution (pH 4.5 to 7, ○-).
○), Pipes-sodium hydroxide buffer (pH 6.5-
7, ●-●, Tris-HCl buffer (pH 7-9, △-
△), glycine-sodium hydroxide buffer (pH 9-1)
0, ▲-▲) and treated at 70 ° C for 10 minutes, and the residual activity was measured. The relationship between pH and residual activity is shown graphically in FIG. As can be seen from this figure, this enzyme was stable in the pH range of 7-10. (6) Thermostability The purified enzyme solution (5.0 U / ml) was prepared with 10 mM Tris-hydrochloric acid buffer solution (pH 7.5), and the residual activity after heat treatment at each temperature for 10 minutes was measured. The relationship between temperature and residual activity is shown graphically in FIG. As can be seen from this figure, it was stable up to 70 ° C. (7) Isoelectric point: pH 4.7 ± 0.4 (measured by the focus electrophoresis method using carrier amphrite)

(8) Substrate specificity As shown below, it shows the maximum activity for α-monolaurin, but is substantially effective for 1,2-dilinolein, 1,3-dilinolein, egg yolk lecithin-derived diglyceride and triglyceride. Did not work. Substrate lipase activity (%) α-monoglyceride monoacetin 10.1 monobutyrin 47.2 monocaprin 94.2 monolaurin 100.0 monomyristin 84.5 monopalmitin 63.3 monostearin 45.3 β-monoglyceride monoolein 57.2 diglyceride Derived from egg yolk lecithin 0 1,2-dilinolein 0 1,3-dilinolein 0 triolein 0

(9) Effects of Surfactants and Metal Ions As shown below, there was almost no inhibition by surfactants such as Triton X-100 and cholic acid, and calcium and magnesium ions. Reagent concentration Relative activity (%) No addition-100.0 Triton X-100 0.1% 95.3 0.5% 90.8 Cholic acid 1 mM 96.7 5 mM 99.5 CaCl ₂ 1 mM 100.0 MgCl ₂ 1 mM 100.3

The enzyme activity was measured as follows. Enzyme activity measurement method 0.2M Pipese-NaOH buffer (pH 7.3) 0.1 ml 0.3% 4-aminoantipyrine 0.05 ml 0.2% TOOS (previously mentioned) 0.05 ml (45 U / ml) peroxidase 0.05 ml 20 mM MgCl ₂ 0.025 ml 20 mM ATP 0.025 ml (25 U / ml) glycerol kinase 0.01 ml (1000 U / ml) glycerophosphate oxidase 0.015 ml Purified water 0.075 ml

50 ml of 10 mM monolaurin (0.5% Triton X-100 aqueous solution) was added to 0.4 ml of the reaction solution having the above composition.
1 is added and preheated at 37 ° C. for 2 to 3 minutes, and 50 μl of the enzyme solution is added to start the reaction. Exactly 10 minutes later, 0.
The reaction was stopped by adding 2.5 ml of 5% sodium dodecyl sulfate. The wavelength of colorimetric measurement by spectroscopic analysis is 550 nm,
Enzyme activity is defined as 1 unit (1 Unit, 1 U) of activity that produces 1 μmol of glycerol per minute. Further, the calculation of the enzyme activity (titer) by this enzyme activity measuring method follows the following formula. Enzyme activity (U / ml) = ΔA550 × 2.95 / 18.0 × 1,000 / 50 × 1/10 [ΔA550: absorbance at wavelength of 550 nm, 2.9
5: Total amount of color developing solution (ml), 18.0: Hydrogen peroxide milli-molecular extinction coefficient (cm ² / μmol), 50: Amount of enzyme solution used (μ
l), 10: reaction time (min)] Further, by treating the transformant in the same manner as in Example 2, the recombinant plasmid pMGLP3 having the structure shown in the schematic diagram of FIG. 4 is obtained.
1 was extracted. The productivity of monoglyceride lipase of Bacillus sp. H-257 strain having monoglyceride lipase activity was low and the activity value was 0.2 U / ml.
This recombinant E. E. coli DH1-pMGLP31 was found to have a high activity of 1.5 U / ml, and it was confirmed that the method of the present invention is an effective method for producing monoglyceride lipase.

[0034]

According to the present invention, Bacillus sp. H-
A gene DNA expressing monoglyceride lipase was screened from a chromosomal DNA library derived from 257, and a recombinant plasmid of an expression vector constructed using this was cloned into E. coli. By introducing a microorganism belonging to E. coli, the obtained transformed microorganism can efficiently produce monoglyceride lipase. Further, according to the present invention, since the entire amino acid sequence of monoglyceride lipase and the nucleotide sequence of the gene DNA encoding this amino acid sequence can be determined, protein engineering such as exchange of substrate specificity of the enzyme and improvement of heat resistance becomes possible. It was

[Brief description of drawings]

FIG. 1 is a diagram showing an amino acid sequence of a monoglyceride lipase protein.

FIG. 2 is a diagram showing the nucleotide sequence of monoglyceride lipase gene DNA encoding the amino acid sequence of FIG.

FIG. 3 is a restriction map of chromosomal DNA derived from Bacillus sp. H-257 in pMGLP31.

FIG. 4 is a schematic diagram showing the structure of plasmid pMGLP31.

FIG. 5 is a view showing an amino acid sequence on the N-terminal side of a purified monoglyceride lipase preparation.

FIG. 6 is a diagram showing an amino acid sequence of a lysyl endopeptidase-treated fragment of monoglyceride lipase.
In addition, X is an unknown amino acid.

FIG. 7 is a diagram showing a nucleotide sequence of an oligonucleotide used for preparing an oligonucleotide probe.

FIG. 8 is a diagram showing a base sequence of another oligonucleotide used for preparing an oligonucleotide probe.

FIG. 9 is a diagram showing a construction flow of a plasmid pMGLP31.

FIG. 10 is a graph showing an example of the relationship between pH and relative activity of the monoglyceride lipase obtained in the present invention.

FIG. 11 is a graph showing an example of the relationship between temperature and relative activity of the monoglyceride lipase obtained in the present invention.

FIG. 12 is a graph showing an example of the relationship between pH and residual activity of the monoglyceride lipase obtained in the present invention.

FIG. 13 is a graph showing an example of the relationship between the temperature and the residual activity of the monoglyceride lipase obtained in the present invention.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display area C12R 1:19) (C12N 15/55 C12R 1:07)

Claims (7)

[Claims] 1. A DNA expressing a monoglyceride lipase.
A substantially pure microorganism producing monoglyceride lipase, which is a microorganism belonging to Escherichia coli transformed with a recombinant plasmid having: 2. The microorganism according to claim 1, wherein the transformed microorganism belonging to Escherichia coli is transformed with the plasmid pMGLP31. 3. A microorganism belonging to Escherichia coli transformed with the plasmid pMGLP31 is Escherichia coli DH1-pMGLP31 (FER).
The microorganism according to claim 2, which is MP-12657). 4. A substantially pure DNA expressing a polypeptide having a monoglyceride lipase activity, which has a base sequence encoding the amino acid sequence shown in FIG. 5. The DNA according to claim 4, which has a nucleotide sequence represented by FIG. 6. A DNA expressing a monoglyceride lipase.
A monoglyceride lipase characterized by comprising culturing in a medium a substantially pure microorganism that produces a monoglyceride lipase, which is a microorganism belonging to the genus Escherichia transformed with a recombinant plasmid having, and then collecting the monoglyceride lipase from the culture. Manufacturing method. 7. The transformed microorganism belonging to the genus Escherichia is Escherichia coli DH1-pMGLP.
31 (FERM P-12657), The manufacturing method of the monoglyceride lipase of Claim 6.