JPH06153965A - Microbial strain of genus pseudomonas having recombinant dna and production of lipase using the microorganism - Google Patents

Abstract

PURPOSE:To improve the productivity of lipase by the genetic recombination of a lipase-producing microorganism resistant to surfactant. CONSTITUTION:Lipase gene of Pseudomonas cepacia A-0727 which is a surfactant-resistant lipase-producing microorganism is cloned and a plasmid vector containing introduced lipase gene is prepared. The vector is included in a cell of a microorganism of the genus Pseudomonas and the cell is cultured in a nutrient medium for the Pseudomonas to enable the mass-production of lipase.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a lipase gene, a recombinant DNA containing the lipase gene, a Pseudomonas bacterium containing the same, and a method for producing lipase using the same.

[0002] Lipase is an enzyme that hydrolyzes lipids, and is used not only in the fields of medicine and food, but also in the processing of fats and oils, clinical diagnostic agents,
It is also widely used in industrial fields other than pharmaceuticals and foods such as detergents, asymmetric synthesis and decomposition of organic compounds.

[0003]

2. Description of the Related Art Hitherto, when utilizing lipase in industrial fields other than medicine and food, a lipase produced by the genus Pseudomonas has been mainly used, and further, for example, JP-A-3-87187. Pseudomonas cepacia M-12-33, as described
Pseudomonas sp (Ps) described in JP-A-3-47079
eudomonas sp.) KWI-56 and J. Bacteriol., 173, 559-
567 (1991) described Pseudomonas cepacia (Pseudom
onas cepacia) Pseudomonas such as DSM-3959
recombinant DNA in the lipase produced by
Its productivity is enhanced by using technology.

[0004]

However, these existing lipases have a drawback that they are easily affected by a surfactant, and therefore, they are used for detergents and clinical examinations in which lipase is used in the presence of a surfactant. In the industrial fields such as asymmetric synthesis and decomposition, it is desired to supply a better lipase, that is, a lipase highly resistant to a surfactant at a low cost.

[0005]

[Means for Solving the Problems] Therefore, first, the present inventors sought from the natural world a lipase-producing bacterium highly resistant to a surfactant, and as a result of diligent studies, obtained one strain suitable for the purpose. . The mycological properties of this strain are as follows.

(A) Morphology (1) Bacillus (0.6 × 2.0) μ (2) Alone or short chain (3) Motile, hyperhairy (4) No spores (5) Gram stain: Negative (6) Acidic : Negative (7) Polymorphism: None

(B) Growth state (1) Meat broth agar plate culture: round, convex circle, smooth and thin surface, yellow-white semi-transparent (2) Meat broth agar slope culture: straight, smooth on the periphery, normal growth , Convex circle, light yellowish white and slightly glossy (3) Liquid culture of gravy: growth is normal, thin film is formed on the surface, turbid (4) Gelatin gelatin stab culture: growth is normal, liquefaction is funnel-like (5) litmus Milk: Slightly alkaline, reducing litmus, liquefying but slightly forming a precipitate

(C) Viability (1) MacConkey medium: growth (2) KCN medium: no growth (3) SS agar medium: no growth

(D) Physiological properties (1) Reduction of nitrate: negative (2) Denitrification reaction: negative (3) MR test: negative (4) VP test: negative (5) Indole formation: negative (6) Generation of hydrogen sulfide: Negative (7) Hydrolysis of starch: Negative (8) Utilization of citric acid: Positive (Simmons, citric acid medium) (9) Utilization of inorganic nitrogen source: Nitrate is not used but ammonium is used (10) Pigment formation King (A) medium: light yellow pigment King (B) medium: light brown water-soluble pigment (11) Tween (80) degradation: positive (12) Casein degradation: positive (13) PHB Accumulation: Positive (14) Urease: Negative (15) Oxidase: Positive (16) Catalase: Positive (17) Arginine dihydrolase: Negative (18) Lysine decarboxylase: Positive (19) Ornithine decarboxylase: Sex (20) Acyl amidase: Positive (21) Growth pH: 5.0 to 9.0 (22) Growth temperature: 12 to 41 ° C (23) Attitude toward oxygen: Aerobic (24) OF test: Oxidative ( 25) Acid and gas production from sugars i) Acid production: shown in Table 1.

[0010]

[Table 1]

B) Gas production: Negative The above-mentioned mycological properties are described in the Vergis Manual of
Detergent Native Bacteriology 8th Edition [Bergey's
Manual of Determinative Bacteriology Volume 8 (1974
)], Volume 1 of the Burj's Manual of Systematic Bacteriology [Bergey's Systematic Ba
cteriology 1 (1984)], Robert E (Rober
Gram Negative Organisms An Approach to Identification (Gr.
am-negative organisms an approach to identificatio
n) Guide to presumptive identification [Guide to presumptive identification
(1983)], Cowan's Manual for the identification of med
ical bacteria (1974)], it is consistent with Pseudomonas cepacia. Therefore, this strain is Pseudomonas cepacia (Pseu).
domonas cepacia) A-0727.
No. 72 (FERM-P No13272) has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology.

Next, the present inventors cloned the lipase gene from this strain, found that the lipase gene was a novel gene, and further prepared a plasmid vector into which the lipase gene had been introduced. By retaining it in Pseudomonas, it succeeded in mass production of Pseudomonas in lipase, and it was possible to inexpensively supply a lipase resistant to a surfactant, thus completing the present invention.

The present invention will be described in detail below. (1) Method for preparing chromosomal DNA Pseudomonas cepacia A-0
The 727 strain was treated with LB medium (tryptone 1.0%, yeast extract 0.5
%, Sodium chloride 1.0%) and aerobically incubate at 30 ℃ overnight. After collecting the cells, the method of Saito and Miura [Biochim. B
iophys. Acta., Vol. 72, 619-629 (1963)] and the like to extract chromosomal DNA and purify it to obtain DNA.

(2) Method for Inserting DNA Fragment into Plasmid Vector and Transformation The plasmid vector has a known restriction enzyme cleavage site capable of replicating in the host and has a selectable marker such as drug resistance, for example, a broad vector. Host range plasmid vector RSF101
0, R16679, R1162, etc., using a plasmid vector in which a drug resistance gene such as kanamycin or chloramphenicol is introduced, and the plasmid vector DNA is cleaved with a restriction enzyme such as HindIII, and then cut and purified with the same restriction enzyme. The DNA is ligated by a known method using ligase to obtain a recombinant plasmid, and using the recombinant plasmid, Pseudomonas cepacia (Pseudomonas cepacia), Pseudomonas putida, etc. Transformation is carried out using a method such as the calcium chloride method, the rubidium chloride method or the electroporation method.

(3) Method for Selective Isolation of Transformant Bacterium Having Lipase-Producing Ability Agar medium containing triglyceride such as tributyrin and triolein emulsified with polyvinyl alcohol and containing an antibiotic at a predetermined concentration was used to transform the transformed bacteria. By isolating a strain that forms a large clear zone in the agar medium, a bacterium containing the lipase gene is selectively isolated, and then the lipase gene is contained from the bacterium using a known method such as an alkaline method or a boiling method. Plasmid D
NA is obtained, and lipase is produced using Pseudomonas sp. Obtained by introducing the plasmid DNA of the present invention.

The amino acid sequences of the structural genes of the lipase of the present invention thus obtained were compared with the amino acid sequences of the structural genes of various known lipases derived from Pseudomonas sp.
Shown in. The entire sequence of the lipase structural gene of the present application is shown, and for other lipase structural genes, only the part different from the lipase structural gene of the present application is shown. Amino acids are represented by the one-letter code.

As is clear from FIG. 1, the homology between the lipase gene of the present invention and other lipase genes is derived from Pseudomonas cepacia M-12-33.
95%, Pseudomonas sp.
sp.) From KWI-56 and Pseud
It can be seen that 92.4% are all derived from DSM-3959. However, Pseudomonas cepacia M, which has the highest homology with 95%,
Even when the lipase gene derived from -12-33 is compared with the lipase gene of the present invention, 16 substitutions in the number of amino acids are recognized.

Method for measuring lipase activity 5 ml of a substrate solution prepared by mixing 225 ml of 2% polyvinyl alcohol (PVA) solution and 75 ml of olive oil and 4 ml of 0.2M McIlvaine buffer (pH 7) was placed in a flat-bottomed test tube.
Preheat at ℃ for 5 minutes. To this, add 1 ml of the sample solution, shake well and immediately leave at 37 ° C for 30 minutes. After 30 minutes, add 10 ml of acetone / ethanol mixture (1: 1) and shake well. To this, 10 ml of 0.05N sodium hydroxide solution and 10 ml of acetone / ethanol mixed solution (1: 1) were added, and 2 drops of phenolphthalein reagent solution was further added thereto, while blowing nitrogen gas on the liquid surface while stirring with a stirrer, With 0.05N hydrochloric acid
Titrate to pH 10.00. For the blank value, use purified water instead of the sample solution and perform the same operation. Enzyme titer is 1 per minute
One unit is used when micromoles of fatty acid are produced.

The present invention will be specifically described below with reference to test examples and examples, but the present invention is not limited to these. Test Example 1 Pseudomonas ce
pacia) A-0727 with soybean oil 2.0%, peptone 0.5% (Difco
Company), beef extract 0.3% (manufactured by Difco), KH ₂ PO ₄ 0.1%, MgSO ₄ 7H ₂ O 0.02%, FeSO ₄ 7H ₂
Liquid medium consisting of 0.001% O and 1 drop adecanol, 30
Lipase activity by centrifugation at 3 ℃ for 3 days with shaking
A culture supernatant of 21 u / ml was obtained, and the obtained culture supernatant was diluted and used as an enzyme solution to examine the effects of various surfactants on the lipase produced by this strain. The results are shown in Table 2. . As a control, lipase derived from Pseudomonas cepasia M-12-33 strain was used.

[0020]

[Table 2]

As is clear from Table 2, the lipase (A-0727 lipase) produced by this strain is a control lipase (M-
12-33 lipase) compared to the detergent Triton X-1
It can be seen that the inhibition by 00 and Sodium dodecyl sulfate is small.

[0022]

【Example】

Example 1 (1) Preparation of chromosomal DNA Pseudomonas cepacia A-0
727 strains were aerobically shake-cultured in LB medium at 30 ° C overnight, and after collecting the bacteria, chromosomal DNA was extracted and purified by the DNA extraction method by Saito and Miura method to obtain 5.0 mg of chromosomal DNA. It was

(2) Preparation of vector pFL210 The broad host range plasmid vector RSF1010 (2.75 μg) was digested with the restriction enzyme PstI, the ends were treated with nuclease S1 and Klenow fragment, and the 8.1 Kb fragment was extracted.
A 1.4 Kb fragment containing a kanamycin resistance gene was prepared from 4.63 μg of UC-4K (Pharmacia) by the same procedure, and both were ligated with T4-DNA ligase, and then Escherichia coli C600 strain was subjected to format conversion to obtain a kanamycin resistant strain. A plasmid was prepared from the kanamycin resistant strain, and a PstI-nondegradable plasmid was selected and digested with the restriction enzyme HpaI. The plasmid was prepared from Escherichia coli C600 strain by Maniatis et al., Molecular Cloning a Laboratory Manual [Maniatis et al, Molecular Cloning a laboratory m
anual., 92-94 (1981)].

On the other hand, plasmid pUC19 (Takara Shuzo) 1 μg
Was digested with restriction enzyme HindIII, treated with Klenow fragment, subjected to agarose gel electrophoresis, extracted with 2.7 Kb fragment, ligated with T4-DNA ligase, and transformed into Escherichia coli C600 strain. Ampicillin- and kanamycin-resistant strains were obtained, plasmids were extracted from the resistant strains, digested with restriction enzyme SacI, and self-ligated to prepare plasmid vector pFL200 into which multiple cloning sites were introduced.

Furthermore, the plasmid pBR328 (Boehringer
1 μg (mannheim) was cleaved with restriction enzyme HhaI, treated with nuclease S1 and Klenow fragment,
After agarose gel electrophoresis, an approximately 1.4 Kb fragment containing the chloramphenicol resistance gene was extracted, and the restriction enzyme SmaI was previously extracted.
After mixing with the plasmid pUC18 that had been cleaved with, ligated with T4-DNA ligase, transformed into E. coli C600 strain, selected chloramphenicol resistant strain, extracted plasmid DNA, digested with restriction enzymes BamHI and KpnI, About 1.4 Kb DNA fragment was collected, while 0.5 μg pFL200 also had the same restriction enzyme BamHI,
After digestion with KpnI and mixing with the above DNA fragment, T4-D
After ligation with NA ligase, Escherichia coli C600 strain was transformed to select a chloramphenicol and kanamycin two-drug resistant strain, a plasmid was extracted from the resistant strain, and the plasmid was named pFL210.

(3) Insertion of DNA fragment into plasmid vector 4.7 μg of the above chromosomal DNA was taken, restriction enzyme HindIII was added, and the mixture was reacted at 37 ° C. for 5 minutes to partially cut it, while
Add restriction enzyme HindIII to 2.0 μg of plasmid pFL210, and
Completely cleaved plasmid DNA after reaction for 2 hours at ℃
Alkaline phosphatase was added to the mixture to dephosphorylate, and the cleaved chromosomal DNA and plasmid vector DNA were mixed, DNA ligase was added, and the mixture was reacted overnight to ligate the DNA chains.

(4) Transformation with plasmid Since a halo-deficient strain is convenient for the host when the lipase gene is selected and isolated on the agar medium containing triolein, Pseudomonas cepacia is used.
A halo-free mutant strain HWIO obtained by treating M-12-33 strain (FERM-P No.9871) with nitrosoguanidine was used.

Further, the present inventors have used for transformation of the strain,
It has been found that the electroporation method is very effective, and was carried out similarly in the present invention.

That is, the strain was cultured in 20 ml of LB medium at 30 ° C. until the logarithmic growth phase (OD ₆₆₀ = 0.4), and after cooling,
Collect the cells by centrifugation at 10,000 rpm for 5 minutes, and collect 10 ml of cells.
Of 272 mM sucrose and 0.7 mM sodium phosphate buffer (pH 7.4), centrifuge again, and then use the same buffer.
Suspend in 0.8 ml, add ligated recombinant DNA,
A pulse of V / cm was applied, 5.6 ml of LB medium was added, and the mixture was shake-cultured at 30 ° C. for 2 hours to obtain a transformant.

The recombinant DNA thus ligated was transformed into HWIO strain by electroporation to give 100 μg /
Twelve halo-forming strains were obtained by plating on a selective medium containing ml chloramphenicol and 0.2% triolein.

(5) Identification of a transformant containing the lipase gene A plasmid was extracted from the 12 halo-forming strains, cleaved with restriction enzymes HindIII and EcoRI, and the inserted DNA fragment was analyzed by agarose electrophoresis. Shows insertion of only about 9 Kb DNA fragment, and several D in EcoRI.
It was shown to be divided into two types consisting of NA fragments. That is, it was confirmed from the results of constructing restriction enzyme maps of these two types of plasmids that they were two types of plasmids into which only unique DNA fragments having different directions were inserted.

Two kinds of plasmids were designated as pLPA1 and pLPA2, respectively.
The restriction map of pLPA1 is shown in FIG.

(Analysis of lipase gene) A DNA fragment of about 9 Kb, which is considered to contain the lipase gene, was cleaved with various restriction enzymes, and each DNA fragment was subcloned into pFL210 to obtain Pseudomonas cepacia.
cia) HW10 strain was transformed, and the presence or absence of halo formation in triolein medium was examined in the same manner as above.

2.5 Kb BglII-StuI for lipase production
It has been shown that a DNA fragment larger than the fragment is required, and the present inventors have determined that the entire base sequence of the present DNA fragment is a DNA sequencer.
373A [Applied Biosystems
stems)). And this DN
It was found that the A fragment contained the lipase gene, and the deduced amino acid sequence of the lipase is shown in SEQ ID NO: 1 in the sequence listing.

(6) Production of lipase Pseudomonas cepacia HW10 strain was transformed with the plasmid pLPA1 containing the lipase gene, and the obtained transformant Pseudomonas cepacia HW10 (pLPA1) was transformed into Soybean oil 2.0
%, 0.5% peptone (Difco Co.), beef Ikusutorakuto 0.3% (Difco _{Co.), KH 2 PO 4 0.1%} , MgSO 4 · 7H
₂ O 0.02%, FeSO ₄ .7H ₂ O 0.001%, and one drop of adecanol in a liquid medium at 30 ° C. for 3 days with shaking, and then centrifuged to obtain a culture supernatant with a lipase activity of 430 u / ml. The ability of the transformant to produce the enzyme was determined by the parent strain Pseudomonas cepacia A-07.
It was about 20 times higher than the lipase production of 27 strains. ).

[0036]

EFFECT OF THE INVENTION The present invention is a lipase-producing strain Pseudomonas derived from nature and resistant to surfactants.
The lipase gene of Pseudomonas cepacia A-0727 was cloned, the gene was introduced into a plasmid, and transformed into a Pseudomonas genus, which was useful in a wide range of industrial fields by culturing this transformant strain. It is possible to inexpensively provide various surfactant-resistant lipases.

[0037]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 320 Sequence type: Amino acid sequence Ala Asp Asn Tyr Ala Ala Thr Arg Tyr Pro Ile Ile Leu Val His 15 Gly Leu Thr Gly Thr Asp Lys Tyr Ala Gly Val Leu Asp Tyr Trp 30 Tyr Gly Ile Gln Glu Asn Leu Gln Gln His Gly Ala Thr Val Tyr 45 Val Ala Asn Leu Ser Gly Phe Gln Ser Asp Asp Gly Pro Asn Gly 60 Arg Gly Glu Gln Leu Leu Ala Tyr Val Lys Thr Val Leu Ala Ala 75 Thr Gly Ala Thr Lys Val Asn Leu Val Gly His Ser Gln Gly Gly 90 Leu Thr Ser Arg Tyr Val Ala Ala Val Ala Pro Asp Leu Val Ala 105 Ser Val Thr Thr Ile Gly Thr Pro His Arg Gly Ser Glu Phe Ala 120 Asp Phe Val Gln Gly Val Leu Ala Tyr Asp Pro Thr Gly Leu Ser 135 Ser Thr Val Ile Ala Ala Phe Val Asn Val Phe Gly Ile Leu Thr 150 Ser Ser Ser His Asn Thr Asn Gln Asp Ala Leu Ala Ala Leu Lys 165 Thr Leu Thr Thr Ala Gln Ala Ala Thr Tyr Asn Gln Asn Tyr Pro 180 Ser Ala Gly Leu Gly Ala Ser Gly Ser Cys Gln Thr Gly Ala Pro 195 Thr Glu Thr Val Gly Gly Asn Thr His Leu Leu Tyr Ser Trp Ala 210 Gly Thr Ala Ile Gln Pro Thr Phe Ser Val Leu Gly Val Thr Gly 225 Ala Thr Asp Thr Ser Thr Ile Pro Leu Val Asp Pro Ala Asn Val 240 Leu Asp Leu Ser Thr Leu Ala Leu Leu Gly Thr Gly Thr Val Met 255 Ile Asn Arg Ala Ser Gly Gln Asn Asp Gly Leu Val Ser Lys Cys 270 Ser Ala Leu Tyr Gly Lys Val Leu Ser Thr Ser Tyr Lys Trp Asn 285 His Ile Asp Glu Ile Asn Gln Leu Leu Gly Val Arg Gly Ala Tyr 300 Ala Glu Asp Pro Val Ala Val Ile Arg Thr His Ala Asn Arg Leu 315 Lys Leu Ala Gly Val 320

[Brief description of drawings]

FIG. 1 is a diagram comparing the amino acid sequence of the lipase structural gene of the present invention with the amino acid sequences of other lipase structural genes, wherein (1) is derived from Pseudomonas cepacia A-0727 of the present application, (2)
Is Pseudomonas cepaci
a) derived from M-12-33, (3) derived from Pseudomonas sp. KWI-56, (4)
Is Pseudomonas cepaci
a) Those derived from DSM-3959 are shown respectively.

FIG. 2 shows a restriction map of the plasmid pLPA 1 of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1:38) (C12N 1/21 C12R 1:38) (C12N 9/20 C12R 1:38)

Claims (4)

[Claims] 1. A lipase gene encoding the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. 2. A recombinant DNA in which a lipase gene encoding the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing is incorporated into a plasmid. 3. A recombinant DNA, which is expressed in Pseudomonas cells and is incorporated into a plasmid vector capable of growing in Pseudomonas cells, the lipase gene encoding the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. The introduced Pseudomonas sp. 4. A Pseudomonas strain into which a recombinant DNA introduced by incorporating a lipase gene encoding the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing into a plasmid vector capable of growing in Pseudomonas cells is cultured,
A method for producing lipase, which comprises producing lipase in a culture and collecting the lipase.