JPH0239890A - Lipase gene - Google Patents

Abstract

PURPOSE:To industrially improve efficiency by arranging a nucleotide so as to code a lipase derived from a bacterium of the genus Pseudomonas. CONSTITUTION:Microbial cells of Pseudomonas fragi IFO 12049 strain capable of producing a lipase are enzymically treated to provide a chromosomic DNA, which is then digested with a restriction enzyme Sau3A1 to afford a DNA fragment. The resultant DNA fragment is then linked to an Escherichia coli plasmid vector pUC9 to provide (A) a recombinant DNA containing a structural gene of the lipase. The component (A) is subsequently introduced into Escherichia coli JM83 strain to carry out transformation. The resultant transformant is then screened to afford (B) an Escherichia coli JM83 (pFL-1) strain containing a plasmid pFL-1 having the DNA fragment derived from the Pseudomonas fragi IFO 12049 strain. The obtained strain (B) is subsequently cultured in a culture medium to produce the lipase.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a DNA sequence encoding a lipase derived from a bacterium of the genus Pseudomonas, and a recombinant DNA containing the DNA sequence.
and Escherichia coli containing the same, and a method for producing lipase.

[Conventional technology and its problems]

Lipase is an enzyme that hydrolyzes lipids and is used in oil processing, clinical diagnostic reagents, detergents, digestive medicines, etc. In recent years, lipase has also been used in chemical products, especially ester hydrolysis, ester synthesis, which is a method for producing optically active compounds. Alternatively, it is an important enzyme that catalyzes ester conversion.

In addition, Pseudomonas bacteria can hydrolyze esters,
It is known that lipases useful for catalyzing ester synthesis or ester conversion are produced (Japanese Patent Laid-Open No. 166-1989).
．． No. 8911).

The properties of the lipase produced by this Pseudomonas bacterium are interesting for industrial use, but in order to achieve that purpose, even greater substrate specificity (including stereospecificity) is required.
is desired.

However, since its secondary and higher-order structures are unknown, lipases with desired activity could not be obtained without extensive screening or inefficient methods such as multiple mutation treatments.

Furthermore, since lipase is usually produced by the expression of one structural gene contained in the chromosomal DNA of the producing bacterium, it has been difficult to dramatically improve lipase production.

In recent years, with the development of recombinant DNA technology, it has become possible to isolate genes and induce them to be highly active, or to change the nucleotide sequence, and the creation of proteins of commercial interest is being considered or implemented. ing.

In order to improve the conventional inefficient lipase modification method described above, the present inventors cloned the structural gene of lipase present in the chromosome of a Pseudomonas bacterium and determined the lipase coding sequence. By obtaining recombinant DNAs containing such DNA sequences and further using Escherichia coli containing these recombinant DNAs, lipases derived from Pseudosonus bacteria that can be used for industrial purposes or with substantially the same properties can be obtained. Its variant (vari) retains
The purpose was to produce ant).

As a result of intensive research to achieve the above objective, the present inventors have found that
Previously, the cloning of the lipase structural gene present in the chromosome of Pseudomonas fragi strain IP012049 (Japanese Patent Application No. 62-308.638) and the production of lipase using Escherichia coli (Japanese Patent Application No. 123-672-1989) successful,
Subsequently, the present invention was completed by determining the DNA sequence encoding lipase.

[Means for solving problems]

The present invention has the following configurations +1) to (8).

(1) Pseudomonas (
A DNA sequence encoding a lipase derived from a bacterium of the genus Pseudomonas and a DNA sequence encoding a functional equivalent of the nucleotide sequence.

(2) Pseudomonas bacteria is Pseudomonas fragi IFCI
The DNA sequence according to item 1 above, which is the 12049 strain.

(3) A DNA sequence encoding a lipase derived from a bacterium of the genus Pseudosonus, which was confirmed in Section 1 of the library manual, and a DNA sequence encoding a functional equivalent of the nucleotide sequence are incorporated into an E. coli vector. Replicable recombinant DNA.

(4) Pseudomonas bacteria is Pseudomonas fragi IFo 1
The recombinant DNA according to item 3 above, which is the 2049 strain.

(5) The recombinant DNA according to the above item 3 or 4, wherein the E. coli vector is pUJC9.

(6) Escherichia coli transformed with the recombinant DNA described in item 3, 4, or 5 above.

(7) A method for producing lipase, which uses the recombinant DNA described in item 3 above.

(8) A method for producing lipase, which comprises culturing the Escherichia coli described in item 6 of item IIF.

The present invention will be explained in detail below.

The N-recombinant DNA of the present invention is a recombinant DNA obtained by fragmenting the chromosomal DNA of a lipase-producing bacterium of the genus Pseudomonas and inserting the fragment into a multicopy plasmid vector that replicates in Escherichia coli. It can be obtained by selecting those containing the lipase structural gene.

The chromosomal DNA of Pseudomonas bacteria is obtained by lysing the cells of Pseudomonas bacteria treated with lysotium using a protease in the presence of sodium dodecyl sulfate (S05), deproteinizing the lysate by extracting the lysate with phenol, and precipitating it with ethanol. It is adjusted by

Fragmentation of chromosomal DNA is performed using endonucleases that have no base sequence specificity.

5au for fragmentation of Pseudomonas fragi chromosomal DNA
υC as an E. coli plasmid vector using 3^l.
9 (J, νJara and J, Masslng, G
A chromosomal DNA fragment can be introduced into the old cloning site using the method (Ang, Dan 25g (1982)).

Here, pUJC9 is used as a DNA introduction vector for E. coli, but in the present invention, any type can be used as long as it can replicate in E. coli, has an appropriate selection marker, and can be expected to express the foreign gene. No question. For example p11c8. pLIcll, pUJC19, etc. can be used.

Chromosome D of Pseudomonas bacteria digested with restriction enzymes
NA fragments are separated by electrophoresis in an agarose gel, visualized under UV light by ethineum bromide staining, and DNA fragments of the desired size are adsorbed onto DEAE cellulose paper. Is the adsorbent sodium chloride in IM? 8 and recovered by ethanol precipitation.

Introduction of the chromosomal DNA of a Pseudomonas bacterium into a plasmid vector is achieved by cleaving the chromosomal DNA and plasmid vector with a restriction enzyme and joining the two fragments.

The vector cleaved before the ligation is dephosphorylated by bacterial alkaline phosphatase treatment to prevent self-ligation. A chromosomal DNA fragment and a plasmid vector can be ligated using ligase.

E. coli is transformed with the recombinant DNA obtained in the above steps, and E. coli having lipase-producing ability is selected based on the marker of the plasmid vector and the lipase-producing ability. Here, Escherichia coli was tested with appropriate markers provided by insertion of a specific cloning vector, such as JM83i to select for ampicillin-resistant bacteria.
(J, Messin) (,R.

Craa, and P. H., Seeburg, Nucl.
, ^cld, Ras, 2.3N (+9811) can be used. Furthermore, 118 + 01.

E. coli bacteria such as LE 392 can also be used.

Transformation was performed by the well-known competent cell method,
Selection of plasmid vectors using markers is performed by adding about 50 Mg/ml ampicillin to the medium according to the above specific example.

Further, selection based on lipase production ability can be performed by adding about 1% tributyrin to the medium.

That is, when lipase is not produced on the medium, lipids are decomposed to produce fatty acids, and the emulsification state of the lipids changes, forming a circular clear zone around the colony. ,Y,Ha
Shimoto, and Y, Takagi, Bio
chemBiophys,Res,Commun,,1
41, 185 (19861).

The recombinant DNA containing the lipase structural gene obtained in the above step was digested with various restriction enzymes, and a restriction enzyme map was created based on measurement using agarose gel electrophoresis. Subcloning of the body DNA is performed.

That is, the obtained recombinant DNA is cut with a restriction enzyme, DNA fragments of an appropriate size (larger than the expected lipase structural gene, but not too large) are collected, and this is reused as a plasmid for canine ni. Incorporate it into a vector, group 1!8
The molecular weight of the recombinant DNA can be reduced by cutting the recombinant DNA with a restriction enzyme, removing unnecessary NA portions, and recombining the DNA as is.

FIG. 2 shows, as an example, a restriction enzyme map of plasmid pFL-1 containing the structural gene of lipase of Pseudomonas fragi strain IF012049. In addition, plasmid pF1. Plasmids pF1.-1 were inserted with DNA fragments cut with various restriction enzymes of RI, Kan I5, and Fumi1Smal. -2. pFL-25CpFL-2
An example of the presence or absence of clear zone formation in a tributyrin agar medium of Escherichia coli containing ND, pFl, -2AB, and pFL-25M is shown.

DN encoding lipase derived from Pseudomonas bacteria
The DNA region where the A sequence is present can be determined using the dideoxy method by Sangar et al.
F, at al Proc, Natl, Acad, Sc.
i, LIS^7454631977), etc., can be determined using a known method.

The DNA sequence starting with the start codon of TG and ending with the stop codon of TAG described in FIG. 1 related to Section 1 above is
The question is whether it actually corresponds to the lipase produced by E. coli transformed with plasmid pFL-2.
The above deletion experiment and the present inventors have been published in Japanese Patent Application No. 63-123.
This was determined based on the confirmation of approximately 33,000 polypeptides of Molecule 1 expressed in Escherichia coli JM83 (pFL-2) strain in No. 672.

That is, D of the lipase structural gene of Pseudomonas fragi IF012049 strain contained in plasmid pFL-2.
The NA sequence is a sequence that includes a sequence recognized by the restriction enzyme Ndel but does not include a sequence recognized by 5cal, and was determined to be an open reading frame with a molecular weight of approximately 33,000.

Figure 4 shows the chromosomal DNA sequence of plasmid pFL-2 containing the lipase-encoding region of Pseudomonas fragi strain IF012049 (linker of plasmid pUJC9).
(including a portion of the lipase gene) and the amino acid sequence corresponding to the DNA sequence of the lipase gene.

By transforming E. coli with a recombinant DNA containing a lipase structural gene derived from a Pseudomonas bacterium whose molecular weight has been reduced in the subcloning step, E. coli having lipase-producing ability can be obtained again.

The components of E. coli that have this ability to produce lipase are: 5
It is separated down to the polypeptide level by electrophoresis in a DS-polyacrylamide gel. The separated polypeptides are visualized by staining in Coomassie brilliant blue solution.

Expression in E. coli of a lipase structural gene derived from a Pseudomonas bacterium that has been incorporated into a DNA introduction vector for E. coli can be achieved by culturing the E. coli and E. coli containing only the E. coli DNA introduction vector in which no heterologous structural gene has been incorporated. Confirmation is achieved by separating, visualizing, and comparing polypeptides from samples prepared by

For example, Fig. 5 shows E. coli JM83 (pUJC9) strain (
FIG. 3 shows diagrams of polypeptides separated and visualized in a 5DS-polyacrylamide gel for the E. coli JM83 (pFL-2) strain (a) and b).

Comparison of both samples confirms the expression in E. coli of the lipase structural gene of Pseudomonas fragi strain IF012049 derived from plasmid pFL-2.

Furthermore, in order to ensure the highest possible expression level of the heterologous structural gene incorporated into the E. coli vector, it is necessary to associate it with as effective a promoter and operator system as possible.

Various strong promoter-operator systems are known in E. coli. For example, lactose (Iac)
Known operons include tryptophan (trp) operon, outer membrane protein gene (lpp) promoter, and β-lactamase promoter.

These various methods can be used to express the lipase-encoding region of the invention in E. coli. Even more! Φ
Different origins of replication, marker genes, effective promoters, and other structural elements can be combined to achieve similar results.

The transformed E. coli strains obtained in the various cases described above are also within the scope of the present invention. Furthermore, growth conditions can be determined according to the specific characteristics of these E. coli strains so as to produce lipase most efficiently.

[Examples] The present invention will be further explained in detail with reference to Examples, but the present invention is not limited by the Examples.

In the Examples, all restriction enzymes used were those commercially available from Nippon Gene ■.

Example 1 (Pseudomonas fragii strain IF012049 chromosome C
Preparation of NA) 200ml L-medium (1% batatotributone 0.5
% Yeast Powder 0.5% Sodium Chloride Adjusted to pH 7.2) Pseudomonas flagi IFo 12049 strain, which was cultured overnight at 30°C and collected, was added to 100 liters of culture solution.
10mM Tris-HCl buffer (pH 8,0),
Wash once with 10 ml of 30 mM sodium chloride solution,
2i+l IOmM-tris-1lC1&l? i liquid (p
o a, o), suspended in 1mM-EDT^2Na solution.

This suspension had a final concentration of 5hM Tris-1IC1il buffer (pH, o), 50mM-Tris2Na 50mM
EDTA-He1m Soaking solution (pH 8,0) IB/m
1 Lysotium (Seikagaku Kogyo Co., Ltd.) was added to each solution, and the mixture was left at room temperature (25°C) for 1 hour.

Proteiniace K (Merck) was added to this solution in the presence of 0.5% SDS to a concentration of 1100 u/ml, and
The cells were dissolved by gentle shaking at 50°C.

This sample was extracted twice with an equal volume of Tris-saturated phenol and once with an equal volume of diethyl ether, then ethanol precipitated and the precipitate was dissolved in 10+oM Tris IIcI buffer (pt
l 8.0) -1mM-EDTA solution (rl, (hereinafter referred to as T
E) and dialyzed in the same solution at 4°C for 2 days to prepare chromosomal DNA.

Example 2 (Cloning of lipase gene) Pseudomonas fragi IF0 prepared in Example 1
Approximately 7 μg of chromosomal DNA of strain 12049 was partially digested at 37°C with 5 IU of au3A1 for 5 minutes, 15 minutes, 25 minutes, 35 minutes, and 50 minutes, and diluted with EDTA 2Nail solution to a final concentration of 20 μg. After stopping the reaction, each reaction solution was mixed.

This mixed sample was mixed with 40IIM Tris 2 at pH 1-9.
1 sodium acetate, 1 mM EDTA in 2Na.
40a+A was electropulsed for 16 hours in a 7% agarose gel.

A 2 to 6 Kb DNA fragment from the agarose gel was adsorbed onto Whatman DE8I DEAE cellulose paper (Whatman), and the adsorbed material was transferred to a 300 μm I M-N.
The aCI-TE warm solution was eluted, and the eluted material was extracted twice with an equal volume of Tris-saturated phenol and once with an equal volume of diethyl ether, and 2.5 times the volume of ethanol was added to precipitate and collect.

At this time, the amount of recovered material was about 0.7 μg.

This sample was combined with pUJC91. which was dephosphorylated by reaction with 0.6 U of alkaline phosphatase (Takara Shuzo ■) at 50°C for 3 hours after BamH1 digestion. sμg and 86mM Tris-11clil buffer (pH 8,0) 6.6mM-M
gCl2O, 1 ul (147 U
/ul) using T4 DNA ligase.

Using this ligated DNA sample, ~2 x 10'' cells of JM [13 strain] were transformed by the competent cell method, and 50 Mg/ml bixilin (Meiji Seika Co., Ltd.), 1%
Contains tributyrin and 0.0002% X-gal.

The cells were cultured on an agar medium for 16 hours.

Among the approximately 1,500 transformants selected using the marker of the pLlc9 plasmid vector, one strain that formed a clear zone in the culture medium was isolated. This strain was named E. coli JM83 (pFL-1).

Example 3 (Analysis of recombinant DNA) Plasmid pFL-1 was digested with various restriction enzymes, and a restriction enzyme map was created based on measurement using agarose gel electrophoresis. The restriction enzyme map of pFL-1 is shown in FIG.

Based on the restriction enzyme map of plasmid pFL-1, each DNA fragment obtained by cutting with various restriction enzymes was subcloned into pucq, and Escherichia coli strain JM83 was transformed, and the medium containing 1% tributyrin was used in the same manner as in Example 2. The presence or absence of clear zone formation was investigated.

In Figure 3, plasmids pp+ and -t were added to EcoRI, 5
Plasmids into which DNA fragments cut with various restriction enzymes of calNdel, ^pal, and S+l1al were inserted, pFL-2pFL-25C, pFL-2ND, p, respectively.
The presence or absence of clear zone formation in E. coli containing FL-2AB and pFL-25M is shown.

Example 4 (Determination of DNA sequence) Plasmid pFL-2 of Scudomonas fragi IFO1
MI for the part derived from the chromosomal DNA of the 2049 strain
:l Saaquenching it (Takara Shuzo ■)
The DNA sequence was determined using IT (Nippon Gene ■) for DeazaSequencing. As a result, the oven reading frame shown in FIG. 4 was confirmed.

Example 5 (Expression of recombinant DNA in E. coli) E. coli JM8
3 (pUJC9) strain and JM83 (9FL-2) strain in culture medium at 37°C for 16 hours at 50 μg/yen.
The cells were cultured in L liquid medium containing bixilin (Meiji Seika@). Collect the bacteria and add 0.1M phosphoric acid solution (p) 10.7
) and culture! ! Add an equal volume of 2× sample Ml ffi solution (10% glycerol,
5% 2-mercaptoethanol, 2.3% SDS, 0
．． 0525M1-Squirrel-11CI (pl+6.8)
), boiled for 5 minutes and analyzed for polypeptides by 15% 5DS-polyacrylamide gel electrophoresis.

5O5-polyacrylamide gel electrophoresis was performed using Laem
mli et al? (Laemmli, U, to,
Nature 227680 (1970)), and electrodynamics was performed at 20 mA until bromophenol blue reached the bottom of the gel.

In Figure 5, in a 15% 5DS-polyacrylamide gel,
Escherichia coli JM83 (pU[:9) strain (lane a) and JM stained with Coomassie brilliant blue solution
Expression of the lipase structural gene derived from Pseudomonas fragi IF012049 strain contained in plasmid pFL-2 in E. coli JM83 strain (pn, -2) strain (lane b) showing the polypeptide for the -2 strain (lane b). peptide) was confirmed.

[Brief explanation of the drawing]

1 to 5 are explanatory diagrams for explaining the present invention. FIG. 1 shows the DNA sequence of the lipase structural gene derived from Pseudomonas fragi strain IF012049. FIG. 2 shows a restriction enzyme map of plasmid pFL-1. The black part is Pseudomonas flagi rF0120
This is a DNA fragment derived from chromosome CNA of strain 49. Figure 3 shows plasmid PFL-2,9FL-25C,1
) FL-2NO. Plasmid pHG of 9Fl, -28B, pFL-25@
9 shows the presence or absence of clear zone formation in tributyrin agar culture medium. Clear zone + indicates clear zone formation. Figure 4. Chromosomal DNA sequence containing the lipase structural gene of Pseudomonas fragi strain IF012049 of plasmid pFL-2 (Plasmid I) UO3 contains a portion of the linker) and lipase. Figure 5 shows the amino acid sequence corresponding to the DNA sequence of E. coli JM, which was analyzed for polypeptides by 15% 5DS-polyacrylamide gel electrophoresis.
83 (pUJC9) strain (lane a) and JMB3 (p
FL-21 strain (lane b) is shown. Numbers on the left indicate molecules i (X 1.000) of protein markers electrophoresed at the same time. that's all

Claims (8)

[Claims] (1) Pseudomonas (
A DNA sequence encoding a lipase derived from a bacterium of the genus Pseudomonas and a DNA sequence encoding a functional equivalent of the nucleotide sequence. [There is a gene sequence] (2) Bacteria of the genus Pseudomonas are Pseudomonas fragi (
Pseudomonas fragi) IFO1204
The DNA sequence according to claim 1, which is 9 strains. (3) A DNA sequence encoding a lipase derived from a Pseudomonas bacterium identified in claim 1 and a DNA sequence encoding a functional equivalent of the nucleotide sequence in an E. coli vector. Replicable recombinant DNA. (4) Bacteria of the genus Pseudomonas are Pseudomonas fragi (
Pseudomonas fragi) IFO1204
The recombinant DNA according to claim 3, which is 9 strains.
. (5) The recombinant DNA according to claim 3 or 4, wherein the E. coli vector is pUJC9. (6) Escherichia coli transformed with the recombinant DNA according to claim 3, 4, or 5. (7) A method for producing lipase, which comprises using the recombinant DNA according to claim 3. (8) A method for producing lipase, which comprises culturing E. coli according to claim 6.