WO1995014783A1

WO1995014783A1 - Lipase gene and variant lipase

Info

Publication number: WO1995014783A1
Application number: PCT/JP1994/001965
Authority: WO
Inventors: Tadashi Yoneda; Yoshiaki Miyota; Kei Ohno; Junji Sasuga
Original assignee: Showa Denko K.K.
Priority date: 1993-11-24
Filing date: 1994-11-21
Publication date: 1995-06-01
Also published as: JPH07143883A

Abstract

A lipase gene isolated from a chromosome DNA of Pseudomonas mendocian SD702; a variant lipase gene obtained by the variation of the above gene; and a variant lipase coded for by the above variant gene and having physical or chemical properties changed thereby. The invention gene serves to facilitate production and modification of lipase LP or a variant lipase thereof. The modification of lipase LP permits production of a variant lipase that has a new amino acid sequence and is more suitable to industrial and other application than the lipase LP, thus providing a lipase useful in the fields of detergent, food processing, papermaking and so forth.

Description

Description: Lipase gene and mutant lipase

Technical field

The present invention relates to a gene encoding a novel lipase useful in the fields of detergents, food processing, papermaking industry and the like, a nucleotide sequence thereof, and a mutation of a gene that changes the physical or chemical properties of the lipase and a mutant gene thereof It relates to the encoded variant lipase. Background art

Lipase, a lipolytic enzyme, is an enzyme for food processing for flavor formation in dairy products, a medical enzyme as a digestive agent, a diagnostic enzyme for measuring blood lipids, and the hydrolysis and modification of fats and oils. O It is widely used as an industrial enzyme for decomposing and removing lipid stains, especially as a component of detergent compositions.

Known lipase-producing microorganisms include the genus Eudomonas, the genus Alcaligenes, the genus Mucor, the genus Candida, and the genus Humicola. Among these, several have obtained lipase genes. Among them, a large number of lipase genes of microorganisms belonging to the genus Pseudomonas have been obtained. Known so far include Pseudomona fragi (JP-A-62-228279, JP-A-2-39890), Pseudomonas cepacia (JP-A-3-87187, JP-A-3-47079), Pseudomonas putida シュー Pseudomonas putuari lignes (Pseudomonas pseudoalcal igenes) (JP-A-3-500845), Pseudomonas aeruginosa (J. Gen. Microbiol. (1992) 138, 1325-1335), Pseudomonas sp. (Pseudomonas sp.) 1 0 9 (J. Biol. Chem. (1991) 266, 18135-18140), Pseudomonas gluiae (Appl. Envir. Microbiol. (1992) 58, 12, 3787-3791) .

Lipases secreted and produced by bacteria of the genus Pseudomonas are used in industrial fields such as detergents, food processing, and paper manufacturing. Lipases used in these fields need to be stable to operating conditions such as temperature, pH, oxygen, solvent, and pressure. In particular, lipase as an adjuvant to be added to detergents to enhance its cleaning effect has high pH, temperature, oxygen, oxidizing agents and other additives in detergents in the alkaline region. Stability is required. As a method for obtaining such chemically stable properties, it is known to change the amino acid sequence of the enzyme. Examples include improvements to proteases and oxidizing agents in JP 4-500608. The present applicant has found a novel lipase (hereinafter abbreviated as lipase LP) having excellent properties used in industrial fields such as detergents and having the amino acid sequence described in SEQ ID NO: 1, and has previously filed a patent. An application has been filed (JP-A-6-38746). Purpose of the invention

An object of the present invention is to change the gene encoding the lipase LP, its nucleotide sequence, and the physical or chemical properties of the lipase LP to those more suitable for industrial use and the like. An object of the present invention is to provide a mutant lipase and a gene whose nucleotide sequence has been changed so as to encode the mutant lipase. Disclosure of the invention

In order to achieve the above object, the present inventors are one of the bacteria belonging to the genus Pseudomonas which produces lipase LP. Obtained lipase gene from Pseudomonas mendocina SD702 deposited with National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology did. The Pseudomonas mendocina SD702 strain was deposited on May 1, 1992, at the depositary institution as described above, and has a deposit number of FERMP-129244. The Budapest Treaty on the International Recognition of the Deposit of the Microorganism for the Purchasing of the International Recognition of the Deposit of Microorganisms in Patent Procedures was granted on May 12, 1993, (PATENT PPROCEDURE) and have been assigned a deposit number of FERMBP—4291.

Furthermore, due to a specific mutation in the gene derived from the strain SD702, the nucleotide sequence is changed so as to encode a mutant lipase in which one or more amino acids in the amino acid sequence of lipase LP have been substituted with another amino acid. The modified gene was obtained. After introducing this gene into a host bacterium and culturing the resulting transformant, various lipases were obtained by a usual separation method. Among these lipases, it was confirmed that the physical or chemical properties of lipase LP changed, and that mutant lipase more useful in industrial fields such as detergent applications could be obtained. The present invention has been completed.

That is, the present invention

1) a gene that encodes the lipase described in SEQ ID NO: 1 and includes all or part of the nucleotide sequence described in SEQ ID NO: 2; 2) the gene of 1 above, which comprises a portion of the nucleotide sequence set forth in SEQ ID NO: 2;

3) the gene according to 1 above, wherein the gene is a lipase gene containing the entire nucleotide sequence of SEQ ID NO: 2;

4) a mutant lipase in which at least one amino acid residue in the amino acid sequence described in SEQ ID NO: 1 has been substituted with another amino acid residue;

5) The amino acid sequence described in SEQ ID NO: 1 or the following position in the amino acid sequence having at least 50% homology with the amino acid sequence: 18, 21, 25, 31 , 43, 60, 75, 79, 93, 104, 107, 125, 142, 145, 148, 155, 156, 159, 164, 183, 198 , 203, 211, 216, 222, 223, 224, 242, 246, 247, 2557, 26, 26, 27, and 292 A mutant lipase in which at least one amino acid residue has been replaced with another amino acid residue;

6) The following substitutions: G1y18A1a; Met21Leu; Asp25Leu; Asp25Arg; Asn31Asp; Asp43Asn; A 1 a60Va1; Ile75Leu; Gly79Pro; Thr93Va1; Va1104Ile; Vall07Ala; Glyl25Gln Glyl 45 Ser; Thrl 48 Ala; Glyl 55 Ser; Serl 56 Gly; Thrl 59 Gly; Alal 64 Ser; Phel 8 3 Tyr; Serl98 Lys; Arg203Ser; Thr210Ser; Val216Phe; Leu222A1a; Leu223Phe; M Agr 246His; Met247 Leu; Met257 Leu; Thr266Va1; Leu267P et 2 24 Leu; he; Asp2776Ser; and Giy292Ser, the mutant strain according to the above 5, which comprises at least one of the following: Eighty- ^zero

7) a gene comprising all or part of a nucleotide sequence encoding the mutant lipase according to any of 4 to 6 above,

8) The following positions in the amino acid sequence described in SEQ ID NO: 1: 18, 21, 25, 31, 43, 60, 75, 79, 93, 104, 107 ,

1 2 5, 1 42, 1 4 5, 1 48, 1 5 5, 1 5 6, 1 5 9, 1 64, 1 8 3, 1 9 8, 2 0 3, 2 1 0, 2 1 6, 2 At least one of the amino acid residues of 2, 2, 2, 3, 224, 242, 246, 247, 25 7, 2 66, 2 67, 2 76, and 2 92 A gene in which the corresponding site in the nucleotide sequence set forth in SEQ ID NO: 2 has been changed so as to encode a lipase replaced with another amino acid residue; and

9) Substitution of the following amino acid residues in the amino acid sequence described in SEQ ID NO: 1: Glyl8Ala; Met21Leu; Asp25Leu; Asp25Arg; Asn A1a60Va1; Ile75Leu; Gly79Pro; Thr93Val; Va1104Ile; V All 0 7 A la; G lyl 25 G ln; Ilel 42 Leu; G lyl 45 Ser; T hrl 48 A la; G 1 y 1 55 Ser; Serl 56 G ly; T hrl 59 Gly; Alal 64 Ser; Phel 83 Tyr; Serl 98 Lys; Arg 203 Ser; Thr 210 Ser; Val 211 Pa He; M 224 L eu; Arg 242 T hr; Arg 246His; Met 247 L eu; M et 257 L eu; At least one of Thr266Va1, Leu267Phe, Asp276Ser; and Gly2992Ser should be encoded. The above-described gene wherein the corresponding site of the nucleotide sequence set forth in SEQ ID NO: 2 has been changed is provided. It is intended to. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of plasmid pSDL23. Arrows in the figure indicate the positions of lipase structural genes. Detailed description of the invention

In the claims and description, the amino acid substitution and the amino acid substituted mutant lipase are represented as follows. That is, the name of the original amino acid residue to be replaced by the mutation (three-letter notation), the position of substitution in the amino acid sequence, and the name of the amino acid residue after the substitution (three-letter notation). It is indicated by notation. For example, a mutant in which the glycine at position 18 has been replaced with alanine is denoted Gly18Ala. In the present invention, a lipase-encoding gene can be isolated from chromosomal DNA by a known method such as colony hybridization method or formation of a clear zone on a plate medium.

That is, in the colony hybridization method, first, a chromosomal DNA library is prepared. Next, when the amino acid sequence of all or a part of the lipase is known, a homologous oligonucleotide probe is prepared, and colony hybridization is carried out using the probe to obtain a gene encoding lipase. Can be isolated.

Alternatively, in the clear zone formation method, a chromosomal DNA library is prepared using a lipase-producing bacterium and cultured on agar containing a lipase substrate. Bacteria that have a chromosomal DNA fragment containing the lipase gene form a clear zone around the colony where the lipase is degraded by the substrate, and use that to encode the lipase. Can be isolated.

In the present invention, the amino acid sequence of lipase LP represented by SEQ ID NO: 1 Amino acid sequence having at least 50% or more homology with the sequence or its amino acid sequence, in which one or more amino acids have been substituted with another amino acid.Nucleotide sequence has been modified to encode lipase. As a method for obtaining a transcribed gene, a gene containing a nucleotide sequence encoding lipase LP may be substituted with a nucleotide sequence that causes amino acid substitution at a target site in the amino acid sequence of lipase LP. Any method can be used as long as it can cause mutation.

One of the methods for causing such mutations at a high frequency is a site-specific mutation method. Site-directed mutagenesis refers to a method in which a wild-type enzyme gene is transformed into type II and a synthetic oligonucleotide having a base substitution at the target site of mutation is used as a mutagen (. J. Zoller, M. Smith, "Method in Enzymology"). vol.100, R. Wu, L. Grossmann, K. Moldave ed., P468, Academic Press (1983)) and the Kunkel method (Kunkel, TA (1985) Proc. Natl. Acad. USA., 82, 488) and improved methods such as the gapped duplex method (Karmer, W. et al (1984) Nucl. Acids Res., 12, 9441). Methods using polymerase 'chain' reaction (PCR) (Mullis, K., Faloona, F., Scharf, S., Saiki, R /. Horn, G. and Erlich, H., (1986) Cold Spring Harber Symp .: 263)

As another method for causing the mutation, there is a random mutation method in which amino acid substitution is performed without particularly specifying the target site of the mutation. Several methods are known for this. For example, there is a method of introducing a plasmid into which a lipase gene has been incorporated into a mutagenized strain of Escherichia coli. For example, a method of performing a polymerase chain reaction (PCR) reaction in the presence of magnesium ions, manganese ions, etc. (Zhou, Y., Zhang, X., Ebright, RH (1991) Nucleic Acid Research 19, 6052, Tindall, KR, Kunkel, TA (1988) Biochemistry 27, 6008-6013) There is a method (Liao, X., Wise, JA (1990) Gene 107-111) in which a DNA amplification reaction is performed using a DNA polymerase.

Further, for example, there is a method using a mutagen such as nitrous acid, formic acid, hydrazine or the like (Myers, RM, Lerman, L.S., Maniatis, T. (1985) Science 229, 242-247). The method of causing mutation is not limited to these, and any other method may be used. For example, a method for obtaining the gene according to the present invention from a strain in which a desired mutation has occurred due to a natural or artificial mutation such as ultraviolet light or the like can be considered.

As the type III for mutation, a lipase gene obtained by incorporating a lipase gene into an Escherichia coli vector such as a pUC system can be used. The mutant lipase gene obtained as described above is introduced into a host bacterium. For example, when a genus Pseudomonas is used as a host, a method for stably maintaining the gene outside the chromosome by using a broad host range plasmid such as RSF11010, or a method for replicating a gene that cannot be replicated in the host bacterium. And integrate it into the chromosome.

When Pseudomonas bacteria are used as a host, mutant lipase is secreted into the culture medium. Separation and purification of the mutant lipase from the culture can be performed according to a conventional method. For example, ammonium sulfate is added to the culture solution, the mutant lipase is roughly fractionated, ammonium sulfate is removed by dialysis, and then fractionated on a CM cellulose column until it becomes a single band by SDS polyacrylamide gel electrophoresis. Purify the mutant lipase. The method for producing and purifying the mutant lipase is not limited to the above-mentioned method, but may be other methods.

The mutant lipase of the present invention can have a stable structure, enhanced Z or function, and improved properties such as thermostability and specific activity by changing its physical or chemical properties. Structural stabilization involves changing amino acid residues in a direction that enhances hydrophobic interactions inside the structure, that reduces the positive charge of the surface layer, that stabilizes the secondary structure, and that stabilizes the loop structure. Is achieved by

Preferred positions of the amino acid sequence for stabilizing the structure are: 18, 31, 60, 79, 93, 104, 125, 145, 155, 164, 183, 20 3, 2 16, 2 23, 242, 246, 267, and 292.

Specific examples of preferred substitutions include: Glyl8Ala; Asn31Asp; A1a60Va1; Gly79Pro; Thr93Val; le; Glyl25Gln; Glyl45Ser; Glyl55Ser; Alal64Ser; Phel83Tyr; Arg203Ser; Val216 Phe; Leu223Phe; Arg242Thr; Arg246His; Leu267Phe, Gly292Ser where at least one of the substitutions is made Is mentioned.

In addition, the enhancement of the function is to increase the positive charge near the active center, to reduce the height of the amino acid residue, to prevent modification of the loop structure, and to reduce the interaction of the loop structure. Achieved by changing the acid residue.

Preferred amino acid sequence positions for enhanced function are: 21, 25, 43, 75, 107, 142, 148, 156, 159, 198, 2110, 22 2, 2 24, 2 47, 2 57, 2 66, and 2 76.

Specific examples of preferred substitutions include: Met21Leu; Asp25Leu; Asp25Arg; Asp43Asn; Ile75Leu; Val107Ala ; I lel 42 Leu; T hrl 48 Ala; Serl 56 Gly; Thrl 59 Gly; Serl 98 Lys; Thr 2 10 Ser; Leu 22 2 A la Met224Leu; Met247Leu; Met257Leu; Thr2666Val; Asp2776Ser substitution; At least one of them has been done.

When the above substitution of an amino acid residue is applied to a lipase having an amino acid sequence having at least 50% homology with the amino acid sequence of SEQ ID NO: 1, the substitution position is as follows. When the amino acid sequence of the lipase having homology with the amino acid sequence described in SEQ ID NO: 1 is juxtaposed, while providing a deletion position as necessary so that the homology of the two amino acid sequences is maximized. Is represented by the amino acid position shown in SEQ ID NO: 1. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described with reference to examples, but the present invention is not limited to the following examples.

Example 1: Preparation of chromosomal DNA

Pseudomonas mendocina SD702 strain was inoculated into 3 Oml of L medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride) and incubated at 37. After culturing for 1 晚, the supernatant was removed by centrifugation to obtain bacterial cells. This was suspended in 5 ml of a 50 mM Tris-HCl buffer (pH 8) containing 0.4 M sodium chloride and 10 mM EDTA. To this, 2.5 mg of lysozyme and 0.25 mg of RNase A were added, and the cells were lysed by gently shaking at 37 ° C for 30 minutes. Thereafter, the mixture was heated at 60 ° C. for 10 minutes to completely solubilize. To this solution, add an equal volume of phenol saturated with TE buffer (10 mM Tris-HCl buffer (pH 8) containing 1 mM EDTA), mix gently by inversion, and collect the upper aqueous layer by centrifugation. Was repeated three times. To the third aqueous layer collected, twice the amount of ethanol cooled to 120 ° C was added, and the precipitate was wound up with a plastic rod. The precipitate was rinsed with ethanol, dried under reduced pressure, and reconstituted in 0.5 ml of TE buffer. Dissolved.

Example 2: Preparation of a chromosome DNA library

After partially degrading the chromosomal DNA with the restriction enzyme Ec0RI, a DNA fragment was recovered by extraction with phenol / chloroform and ethanol precipitation. On the other hand, cosmid pWE15 was similarly degraded with EcoRI and treated with alkaline phosphatase. Both were ligated using T4DNA ligase. This was packaged into phage particles. Add the phage to 600 μl of E. coli culture cultured in L medium containing maltose and magnesium sulfate, incubate at 37 ° C for 15 minutes, and add L medium to lml. Incubated at 37 ° C for 1 hour. This was applied to an L plate medium (a L medium solidified with 1.5% agarose) containing 50 ppm of ampicillin, and cultured at 37 ° C for 1 晚. As a result, about 1000 transformants were obtained.

Example 3: Preparation of oligonucleotide probe

The N-terminal amino acid sequence of the purified lipase was analyzed using an amino acid sequencer, and the result shown in SEQ ID NO: 3 was obtained. Based on this, an oligonucleotide probe represented by SEQ ID NO: 4 was synthesized using a DNA synthesizer. _Thereto, Ί - ³² Ρ- A Τ Ρ and T 4 Porinuku Reochi Dokinaze was added, reacted at at 37, was radiolabeled.

Example 4: Isolation of a gene encoding lipase

About 1000 colonies of the transformant were cultured at 37 ° C for 1 晚 on a two-cellulose filter placed on an L plate medium. Remove the filter, lyse for 10 minutes on filter paper soaked in 0.5M sodium hydroxide Z 1.5M sodium chloride solution, and add 1.5M sodium chloride, 1M Tris-HCl buffer (pH 7). Neutralized twice on soaked filter paper for 7 minutes. The filter is calcined at 80 ° C for 2 hours, and 0.5% sodium dodecyl sulfate (SDS) / 6 XSSC (1 XS SC is 150 mM sodium chloride Zl 5 mM sodium citrate solution. n XSSC means sodium chloride / sodium citrate solution with a concentration of n times that of 1 XSSC. ) The bacterial residue was washed in. Prehybridization of the filter in 0.1% sodium dodecyl sulfate (SDS) / 5X Denhardts solution (0.1% icoico11, 0.1% polyvinylpyrrolidone, 0.1% BSA) / 5xSSC One shot was performed at 37 ° C for 1 hour. The above-mentioned radiolabeled oligonucleotide probe was added to the same solution, and the filter was subjected to hybridization for 1 hour at 37 ° C. Thereafter, the filter was washed with 4 XSSC for 10 minutes, 2 XSSC twice for 20 minutes, and 1 XSSC twice for 20 minutes.

As a result of such colony hybridization, several positive colonies were obtained. Cosmid was recovered from one of the obtained colonies, the fragment digested with Ec0RI was separated by agarose electrophoresis, and adsorbed to a Nitrocellulose filter. Southern hybridization was performed in the same procedure as for colony hybridization. As a result, it hybridized to an 18 kbp fragment.

This fragment was recovered, ligated to the EcoRI site of plasmid PUC119, and transformed into E. coli JMl01. This plasmid was recovered, digested with the restriction enzyme SacI, and subjected to Southern hybridization in the same manner. As a result, a 2.3 kbp fragment was obtained.

Example 5: Nucleotide sequencing of lipase gene

The 2.3 kbp fragment obtained in Example 4 was ligated to the SacI site of pUC119 to transform Escherichia coli JM101. This plasmid was named pSDL23. FIG. 1 shows a restriction map of plasmid PSDL23. Using this plasmid, the Sanger dideoxy method (Sanger, F., Nicklen, S., Coulson, AR (1977) Pro Natl. Acad. Sci. USA., 74, 5463) Determined the nucleotide sequence of the lipase gene DNA. That is, the nucleotide sequence was analyzed by a DNA sequencer using ADI's didoxytermine overnight-sequencing kit. As a result, a nucleotide sequence encoding lipase such as SEQ ID NO: 2 was obtained. The amino acid sequence deduced from this sequence is shown in SEQ ID NO: 1.

Example 6: Construction of mutant lipase gene by site-directed mutation

Mutants in which the amino acid residue of lipase was site-specifically substituted (A1a60

The V a1 or Le u 223 Phe) gene was constructed as follows. In order to convert amino acids in a partially specific manner, the oligonucleotides shown in SEQ ID NO: 5 and SEQ ID NO: 6 were chemically synthesized and phosphorylated at the 5 'end using T4 polynucleotide kinase before use. On the other hand, for the type I mutation, a plasmid pSDL23 (FIG. 1) was used. E. coli BW31 3 strain (H fr KL16 POZ45 [lysA (61-62)], dut1, nu1, thy-1 and re1A1) was transformed with pSDL23, Single-stranded DNA was prepared from the transformants in accordance with the method of Meshing et al. (Viera. J. and Messing, J. (1987) Method in Enzymology, 153, 3-11), and a part of the deoxythymine in the DNA was prepared. A single-stranded DNA was obtained, which was replaced with deoxyduracil, and this was used as type I.

Annealing buffer (2 OmM Tris-HCl buffer (pH 8), 10 mM magnesium chloride, 5 OmM sodium chloride, ImM DTT) was prepared by annealing the mutagenic oligonucleotide and type I DNA prepared as described above. The mixture was allowed to stand at 65 ° C for 15 minutes and then at 37 ° C for 15 minutes to allow the mutagenic oligonucleotide to anneal to the target site of the mutation.

Next, the DN A mixture of 2.5 volumes of Extension buffer (50 mM T ris HC l, pH8.0, 6 OmM CHgCOONH ,. 5 mM M g C l 2, 5 m MD TT, I mM NAD, 0.5 m M dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP), and E. coli DNA ligase and T4 DNA polymerase. Was done. Thereafter, Escherichia coli BMH71-18 mut S was transformed with the DNA, and ampicillin-resistant colonies were selected. Plasmid DNA was prepared from several of the above transformants, the nucleotide sequence was determined by the dideoxy method as in Example 5, and converted to codons corresponding to the target amino acid residues. It was confirmed.

After the plasmid prepared as described above was completely digested with SacI, the DNA fragment containing the mutant lipase gene portion was fractionated by agarose gel electrophoresis, and extracted and purified from agarose gel. After pM FY42, which is a broad host range vector, is completely digested with SacI, it is mixed with the DNA fragment containing the mutant lipase gene portion purified as described above, and mixed with T4 DNA ligase. A ligation reaction was performed to transform Escherichia coli JMl01 strain, and a kanamycin-resistant colony was selected. Plasmid DNA was extracted, purified, and analyzed from these transformants to obtain a plasmid in which a SacI DNA fragment containing a mutant lipase gene was inserted into the SacI cleavage site of PMFY42. Example 7: Preparation of lipase

Using the plasmid containing the mutant lipase gene or the plasmid containing the wild-type lipase gene obtained in Example 6, the lipase-deficient strain LD9 (Pseudomonas mendocina) SD702 was used. A mutant strain obtained by reacting with N-methyl-N, mono-nitro-N-nitrosoguanidine, culturing on a plate medium containing a lipase substrate, and selecting a strain that does not form a clear zone) was obtained. Transformation was carried out by the kanamycin method, and kanamycin-resistant colonies were selected. That is, first, 9 strains of LD were grown in 5 ml of L medium until OD = 0.7. Thereafter, the cells were collected by centrifugation. The cells were suspended in sterile water, collected again, and then resuspended in 5001 sterile water. After adding plasmid DNA to the bacterial suspension and transferring it to a 4 mm cell with electrodes, high voltage was applied using Gene Pulser electropolation system (BI0RAD). electrical ⁰ Noresu (¾ pressure 2.5k V, capacitance 2 5 F, resistor 1000 [Omega]) were given. Then, add 1 lm of L medium to the cell suspension, and culture with shaking at 37 ° C for 1 hour. Was applied. Cultures grown at 37 ° C for 1 晚 were selected from those that formed a clear zone around the colonies among the grown colonies to obtain transformed strains.

This transformant was shaken at 35 ° C for 14 hours in 30 Om1 of a lipase production medium containing 1% Tween 80 and adjusted to PH9 with sodium carbonate. After culturing, the mutant lipase was secreted and produced in the medium. The culture was centrifuged, and the supernatant was taken to prepare a crude enzyme solution. This was subjected to ammonium sulfate fractionation, and after removing ammonium sulfate by dialysis, treated with a CM cellulose column and purified by SDS polyacrylamide gel electrophoresis until a single band was obtained.

Example 8: Specific activity of mutant lipase

The number of molecules of the mutant lipase due to the site-specific mutation purified in Example 7 was determined.

Activity was measured at a constant absorbance of 280 nm and compared to wild-type lipase. The activity was measured by the following method.

Dissolve p-nitrophenyl palmitate (hereinafter abbreviated as p NPP) in isopropyl alcohol to a concentration of 2 mgZm1. This pNPP solution and 100 mM Bicine buffer (pH 8.0) are mixed at a ratio of 1:10 to prepare a substrate solution. Enzyme solution 201 in 500 μl substrate solution After reacting at room temperature for 1 to 10 minutes, add 1N hydrochloric acid (200 1) to stop the reaction, and measure the absorbance at 405 nm using a spectrophotometer.

Table 1 shows specific activity values when the activity of the wild-type enzyme is 100. Example 9: Thermostability of mutant lipase

The thermostability of the wild-type lipase and the mutants (Ala60Val and Leu223Phe) produced by the site-specific mutation prepared above were measured as follows. The mutant lipase solution and the wild-type lipase solution having the same activity value were incubated in a 5 OmM borate buffer (pHIO) at 60 ° C for 10 minutes, and then the lipase activity was measured. Table 1 shows the results when the activity of the wild-type enzyme before heat treatment was set to 100.

Enzyme specific activity Thermal stability

Wild type 1 00 51

Ala 60 Val 98 59

Leu 223 Phe 64 84 Effect of the Invention

The gene of the present invention facilitates the production and modification of the lipase according to the present invention, that is, the wild-type lipase LP or its mutant lipase.

In addition, the modified lipase LP according to the present invention has a novel amino acid sequence, and its physical properties or chemical properties, such as an increase in heat stability or specific activity, are different from those of lipase LP depending on the field of use. It is possible to obtain a mutant lipase which has been changed so as to be compatible, and it is possible to provide a lipase useful in industrial fields such as detergents, food processing, and papermaking. Sequence Listing SEQ ID NO: 1

Array length: 2 9 3

Sequence type: amino acid

Topology: linear

Sequence type: protein

Origin

Organism name: Pseudomonas mendocina; strain name: SD 7 0 2

Array

Ala Trp Phe Gly Ser Ser Gly Tyr Thr Gin Thr Lys Tyr Pro lie Val 1 5 10 15

Leu Gly His Gly Met Leu Gly Phe Asp Ser He Leu Gly Val Asn Tyr

20 25 30

Trp Tyr Gly lie Pro Ala Ala Leu Arg Arg Asp Gly Ala Ser Val Tyr

35 40 45

Val Thr Glu Val Ser Gin Leu Asp Thr Ser Glu Ala Arg Gly Glu Gin 50 55 60

Leu Leu Gin Gin Val Glu Asp lie Val Ala lie Ser Gly Lys Gly Lys

65 70 75 80

Val Asn Leu He Gly His Ser His Gly Gly Pro Thr Thr Arg Tyr Val

85 90 95

Ala Ala Val Arg Pro Asp Leu Val Ala Ser Val Thr Ser Val Gly Ala

100 105 110

Pro His Lys Gly Ser Ala Ala Ala Asp Phe Leu Lys Gly lie Ser Asp

115 120 125

Gly Pro Ala Gly Pro Val Ala Thr Pro Leu Leu Ala Gly lie Val Asn 130 135 140 Gly Leu Gly Thr Leu lie Asn Phe Leu Ser Gly Ser Ser Ser Thr Thr 145 150 155 160

Pro Gin Asn Ala Leu Gly Ser Leu Glu Ser Leu Asn Ser Glu Gly Ala

165 170 175 Ala Arg Phe Asn Ala Lys Phe Pro Gin Glylie Pro Thr Ser Ala Cys

180 185 190

Gly Glu Gly Ala Tyr Ser Val Asn Gly Val Arg Tyr Tyr Ser Trp Ser

195 200 205

Gly Thr Ser Pro Leu Thr Asn Val Leu Asp Pro Ser Asp Leu Leu Met 210 215 220

Gly Ala Ser Ser Leu Thr Phe Gly Ser Glu Ala Asn Asp Gly Leu Val 225 230 235 240

Gly Arg Cys Ser Ser Arg Met Gly Gin Val lie Arg Asp Asn Tyr Arg

245 250 255 Met Asn His Leu Asp Glu Val Asn Gin Thr Leu Gly Leu Thr Ser Leu

260 265 270

Phe Glu Thr Asp Pro Val Thr Val Tyr Arg Gin His Ala Asn Arg Leu

275 280 285

Lys Asn Ala Gly Leu

290 SEQ ID NO: 2

Array length: 8 7 9

Sequence type: nucleic acid

Number of chains: double strand

Topology: linear

Sequence type: Genomic DNA

Origin

Organism name: Pseudomonas mendocina Stock Name: SD 7 0 2

Array features

Characteristic symbol: mat peptide

Location: 1. .879

How the features were determined: S

Array

GCC TGG TTC GGC TCC TCC GGC TAC ACC CAG ACC AAG TAC CCC ATC GTC 48 Ala Trp Phe Gly Ser Ser Gly Tyr Thr Gin Thr Lys Tyr Pro lie Val 1 5 10 15

CTC GGC CAC GGC ATG CTC GGC TTC GAC AGC ATC CTC GGC GTC AAT TAC 96

Leu Gly His Gly Met Leu Gly Phe Asp Ser lie Leu Gly Val Asn Tyr

20 25 30

TGG TAT GGC ATC CCG GCC GCC CTG CGC CGC GAC GGT GCC AGC GTC TAC 144 Trp Tyr Glylie Pro Ala Ala Leu Arg Arg Asp Gly Ala Ser Val Tyr

35 40 45

GTC ACC GAG GTC AGT CAG CTC GAT ACC TCC GAA GCG CGC GGC GAG CAG 192 Val Thr Glu Val Ser Gin Leu Asp Thr Ser Glu Ala Arg Gly Glu Gin

50 55 60

TTG CTG CAG CAA GTC GAG GAT ATC GTC GCC ATC AGC GGC AAA GGC AAG 240 Leu Leu Gin Gin Val Glu Asp lie Val Ala lie Ser Gly Lys Gly Lys

65 70 75 80

GTC AAT CTG ATC GGC CAC AGC CAC GGC GGC CCC ACC ACG CGC TAC GTT 288 Val Asn Leulie Gly His Ser His Gly Gly Pro Thr Thr Arg Tyr Val

85 90 95

GCC GCC GTG CGG CCG GAC CTG GTC GCC TCG GTC ACC AGC GTC GGT GCA 336

Ala Ala Val Arg Pro Asp Leu Val Ala Ser Val Thr Ser Val Gly Ala

100 105 110

CCG CAC AAG GGC TCG GCC GCT GCC GAC TTC CTC AAG GGC ATC AGC GAT 384 Pro His Lys Gly Ser Ala Ala Ala Asp Phe Leu Lys Gly He Ser Asp -

m 313 393 QVV 339 V0 9V3 100 3VX 3X3 33V 0X9 933 XV9 33V 9V0 OIL

m 092

J "31 Π31 iqi ui3 usy ΐ¾ nig dsy ng ^ si {{usy H

918 3X3 30V 33V 9X3 330 9X3 33V 9V3 3VV 3X3 3V3 3V0 3X3 0Ώ 3VV 9XV 丄 usy dsy Sjy UI9; 3M S-iV s-iy

89L 093 3VI 3VV 3VD 0D3 9V3 309 9XV 333 03X 10V 30X 393 330

Λ dsy usy u io sqd -iqi naiI3S s BTV

310 3X3 300 3V0 OVV 339 3V0 031 3D9 3X1 30V 013 931 33X 339 300 02 n9q dsy JGS OJJ dsy usv I nsq 0Jd S

2Z9 DIV 013 9X3 OVO 33V 333 OVO 313 919 3VV 03V 013 933 39V 33V 300

SOS 002 96T

^J Interview ΑΐΟ usy m Interview ^ TV niO 5ΐ ^ 9 3DV 901 0.丄 3VI IVl 393 310 390 3VV 013 39V 3VI V3G 990 WO 399

06T 081

JRI OJJ 3TI 9¾i sAq BIV usy sqd

X3X 330 33V 33V 333 OXV 300 9V3 333 3XX 3VV ODD XVV 3XX 303 333

S9I eieiV ηΐθ usv J3S Aio Π9 ^ IV usy u [9 OJJ

330 333 VVG 33V 3VV 313 D3I 9V0 913 03X 100 0X0 339 3VV 9V3 933

09T SSI OS!

■ I9S J3S usy an nsq • iqi naq Λο

08 ^ 33V 33V 30V 331 XOV 309 331 j OkLtL 3VV OXV 0X3 03V 390 013 390

O

usy A 3Π Βΐν BIV m BTV ojj Ai

Z 3VV 310 DIV 309 D 010 0X3 333 VOV 339 310 933 900 330 333 390

021

S96lO / t? 6df / XDd £ 8 I / S6 OAV Phe Glu Thr Asp Pro Val Thr Val Tyr Arg Gin His Ala Asn Arg Leu

275 280 285

AAG AAC GCC GGG CTA 879 Lys Asn Ala Gly Leu

290 SEQ ID NO: 3

Array length: 30

Organism: Shootmonas mendocina (Pseudomonas mendocina

Stock Name: SD 7 0 2

Array

Ala Trp Phe Gly Ser Ser Gly Tyr Thr Gin Thr Lys Tyr Pro lie Val 1 5 10 15

Leu Gly His Gly Met Leu Gly Phe Asp Ser lie Leu Gly Val

20 25 30 SEQ ID NO: 4

Array length: 20

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: Other nucleic acids Synthetic DNA

Array

CACGGCATGC TCGGCTTCGA 20 SEQ ID NO: 5

Array length: 2 2

Sequence type: nucleic acid

Number of chains: single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence

ACCTCCGAAG TCCGCGGCGA GC 22 SEQ ID NO: 6

Array length: 29

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: Other nucleic acid Synthetic DNA sequence

TCGACCCCAG CGATCTGTTC ATGGGCGCC 29

Claims

The scope of the claims

1) A gene that encodes the lipase described in SEQ ID NO: 1 and includes all or a part of the nucleotide sequence described in SEQ ID NO: 2.

2) The gene according to claim 1, comprising a part of the nucleotide sequence set forth in SEQ ID NO: 2.

3) The gene according to claim 1, wherein the gene is a lipase gene containing the entire nucleotide sequence of SEQ ID NO: 2.

4) A mutant lipase in which at least one amino acid residue in the amino acid sequence described in SEQ ID NO: 1 has been substituted with another amino acid residue.

5) The amino acid sequence described in SEQ ID NO: 1 or the following position in the amino acid sequence having at least 50% homology with the amino acid sequence: 18, 21, 25, 31 , 43, 60, 75, 79, 93, 104, 107, 1 25, 142, 1 45, 1 48, 1 55, 1 56, 1 59,

1 64, 1 8 3, 1 9 8, 2 0 3 2 1 0, 2 1 6, 22 2, 22 3, 2 2 4, 242, 2 46, 2 4 7 2 5 7, 2 6 6, 2 6 A mutant lipase in which at least one amino acid residue among 7, 276, and 292 is substituted with another amino acid residue.

6) The following substitutions: G1y18A1aMet21Leu; Asp25Leu; Asp25Arg; Asn31Asp; Asp43Asn; A 1 a 60 V a 1; I le 75 Leu; Gly 79 Pro; T hr 93 V a 1; V a 1104 I le; V all 07 A la; G lyl 25 G ln; I lel 42 Leu; G lyl 45 Ser; T hrl 48 A la; Gly 15 55 Ser; Ser 15 56 G 1 y; T hr 15 G 1 y; A 1 a 1

64 S er; P hel 83 Tyr; S er 198 Lys; Arg 203

Ser; Thr 210 Ser; Val 2 16 P he; Leu 222 A 1 A; Leu223Phe; Met224Leu; Arg242Thr; Arg246His; Met247Leu; Met257Leu; Thr266Va1; Leueu267Phe A mutant lipase according to claim 5, comprising at least one of: Asp276Ser; and Gy292Ser.

7) A gene comprising all or a part of the nucleotide sequence encoding the mutant lipase according to any one of claims 4 to 6.

8) The following positions in the amino acid sequence described in SEQ ID NO: 1, 18, 21, 25, 31, 43, 60, 75, 79, 93, 104, 107, 125, 142, 145, 148, 155, 156, 159, 164, 183, 198, 203, 210, 216, 222, 223, 224, 242, 246, 247, 257, 266, 267, 276, or 292 at least one amino acid residue A gene in which the corresponding site in the nucleotide sequence set forth in SEQ ID NO: 2 has been changed so that it encodes a lipase whose group has been replaced with another amino acid residue.

9) Substitution of the following amino acid residue in the amino acid sequence described in SEQ ID NO: 1: Glyl8Ala; Met21Leu; Asp25Leu; Asp25Arg; Asn31Asp A sp60 A sn; A la 60 V a 1; I le 75 Leu; G ly 79 Pro; Th r 93 V al; V a 1104 1 le; V all 07A la; G lyl 25 G ln; I lei 42 Leu; G lyl 45 Ser; Thrl 48 A la; Glyl 55 Ser; Serl 56 Gly; Thrl 59 Gly; Alal 64 Ser; Phel 83 Tyr; Serl 98 Lys; Arg 203 Ser; Thr 210 Ser; Val 216 Phe; Leu 222 A la; Leu 223 Phe; Met 224 Leu; Arg242 Thr; Arg2 46 H is; Me t 247 L eu; Me t 257 L eu; T hr 266 SEQ ID NO: 2 to encode a lipase in which at least one of V a1; Leu267Phe; Asp276Ser; and Gly2992Ser is encoded. 9. The gene according to claim 8, wherein a site corresponding to the described nucleotide sequence is changed.