CN111117981B

CN111117981B - Lipase mutant and application thereof in decontamination

Info

Publication number: CN111117981B
Application number: CN201911353179.8A
Authority: CN
Inventors: 王睿; 喻晓蔚; 徐岩
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-05-31
Filing date: 2019-05-31
Publication date: 2021-07-13
Anticipated expiration: 2039-05-31
Also published as: CN110172455B; CN110951712B; CN110172455A; CN110904074B; CN110904073B; CN111117981A; WO2020238339A1; CN110904073A; CN110904074A; CN110951712A

Abstract

The invention discloses a lipase mutant and application thereof in decontamination, belonging to the technical field of enzyme engineering. The lipase mutant has good thermal stability, good pH stability and good washing performance, so the lipase mutant has important application in the aspects of decontamination, grease processing, dairy processing, wheaten food processing, meat processing, drug synthesis, diesel oil synthesis, polymer synthesis, chiral compound synthesis, leather production, detergent preparation, paper making and the like.

Description

Lipase mutant and application thereof in decontamination

Technical Field

The invention relates to a lipase mutant and application thereof in decontamination, belonging to the technical field of enzyme engineering.

Background

Lipases (Lipase, EC 3.1.1.3, glyceride hydrolases) are a generic term for a class of enzymes that hydrolyze fats. In some reaction systems, lipases can catalyze hydrolysis, alcoholysis, transesterification, hydrogenolysis, indirect redox transesterification, and in addition, lipases have the ability to hydrolyze racemic mixtures and ester synthesis and synthesis of peptide bonds, and thus, lipases have important applications in many fields, such as:

1. the lipase can be applied to grease processing, and the lipase hydrolysis of the grease can be carried out at normal temperature and normal pressure, so that biological substances such as highly unsaturated fatty acid, tocopherol and the like in the grease can not be denatured;

2. the lipase can be applied to dairy product processing, and the lipase is applied to milk fat hydrolysis in the dairy product, so that the flavors of cheese, milk powder and cream can be enhanced, the maturity of the cheese is promoted, and the quality of the dairy product is improved;

3. the lipase can be applied to the processing of wheaten food, the lipase is added into the wheaten food, the elasticity of the product can be improved to different degrees, the wrappers are thin and transparent but are not easy to break, the mouthfeel is improved, in addition, the lipase is added into the wheaten food, the monoglyceride can be released, the decay is delayed, and the fresh-keeping capacity of the wheaten food is improved;

4. the lipase can be applied to meat processing, and the lipase is added into meat, so that lipids in the meat can be effectively degraded, and fat-free meat is prepared;

5. the lipase can be applied to the synthesis of medicines, the microbial lipase is used for enriching polyunsaturated fatty acids from animals and plants, free polyunsaturated fatty acids and mono-diglyceride thereof are used for producing various medicines, and the lipase is used in the synthesis of medicines such as sulcotrione, naproxen, captopril and the like;

6. the lipase can be applied to diesel oil synthesis, the diesel oil synthesized by an enzymatic method is that ester exchange reaction between animal and vegetable oil and low-carbon alcohol is catalyzed by the lipase to generate corresponding fatty acid ester, and compared with the traditional diesel oil synthesized by chemical catalysis, the method has the advantages of simple purification process, less investment equipment, low energy consumption, low pollution, low requirement on oil raw materials and the like, and has increasingly attracted general attention of people;

7. lipase can be applied to polymer synthesis, and currently, a polymer material artificially synthesized causes serious environmental pollution, so that the molecular material which can be degraded by microorganisms is more and more valued by people when being synthesized by an enzyme catalysis method;

8. the lipase can be applied to the synthesis of chiral compounds, can play a role in non-hydrolyzates, and can synthesize pharmaceutical and pesticide intermediates with optical activity, such as alcohol, fatty acid, lactone and the like through the enzyme catalytic reaction in the non-hydrolyzates.

9. The lipase can be applied to leather production, and in the leather processing process, an important step is to remove residual fat and protein which are tightly connected with leather or fur, wherein intradermal fat is protected by a fat membrane and is difficult to remove by chemical treatment methods such as lime treatment and the like, and the intradermal fat can easily enter the fat to be emulsified by utilizing the decomposition effect of the lipase or mixing with protease, so that the leather quality is obviously improved;

10. the lipase can be applied to the preparation of detergents, and the development of the lipase for detergents by applying genetic engineering is one of the most successful applications of the large-scale industrialization of modern biotechnology;

11. lipase can be used in papermaking synthesis, and is a general term for all hydrophobic components in wood, including triglyceride and wax lipid, which can be deposited on a drying column to affect the quality and productivity of paper, and lipase can remove the "lipid" from pulp to improve the quality of paper.

However, most of the applications of lipase have certain requirements on the stability of lipase, for example, higher temperature is often used in the process of drug synthesis, which has higher requirements on the thermal stability of lipase; the leather processing and paper making process needs strong alkali, which has higher requirements on the pH stability of lipase under alkaline conditions, but most of the existing lipases have poor stability, for example, the lipase RAL derived from Rhizopus arrhizus (Rhizopus arrhizus) has the optimum temperature of only 35 ℃, only 40% of enzyme activity can be remained after heat treatment at 50 ℃ for 10min, the enzyme activity can be completely lost after the heat treatment for 40min, and the enzyme activity can be lost by more than 70% under the condition that the pH is more than 9.5, which undoubtedly greatly limits the further development of the lipase in the application.

Therefore, there is an urgent need to provide lipases which are stable, in particular thermostable and strongly pH-stable under alkaline conditions.

Disclosure of Invention

[ problem ] to

The technical problem to be solved by the invention is to provide Lipase (Lipase, EC 3.1.1.3) with high stability, especially thermal stability and strong pH stability under alkaline conditions.

[ solution ]

In order to solve the problems, the invention provides a lipase mutant, which is obtained by mutating the 151 th, 235 th, 171 th, 343 th, 275 th, 332 th, 310 th or 180 th amino acid of lipase with the starting amino acid sequence shown as SEQ ID NO. 1;

or the enzyme mutant is obtained by simultaneously mutating 316 th and 340 th amino acids of the lipase with the starting amino acid sequence shown as SEQ ID NO. 1;

or the enzyme mutant is obtained by simultaneously mutating 178 th and 238 th amino acids of the lipase with the starting amino acid sequence shown as SEQ ID NO. 1;

or the enzyme mutant is obtained by simultaneously mutating 316 th, 340 th, 178 th and 238 th amino acids of the lipase with the starting amino acid sequence shown as SEQ ID NO. 1;

or the enzyme mutant is obtained by simultaneously mutating 235 th, 343 th, 332 th and 310 th amino acids of the lipase with the starting amino acid sequence shown as SEQ ID NO. 1;

or the enzyme mutant is obtained by simultaneously mutating the 151 th, 171 th, 275 th and 180 th amino acids of the lipase with the starting amino acid sequence shown as SEQ ID NO. 1;

or the enzyme mutant is obtained by simultaneously mutating 235 th, 343 th, 332 th, 310 th, 151 th, 171 th, 275 th and 180 th amino acids of the lipase with the starting amino acid sequence shown in SEQ ID NO. 1;

or the enzyme mutant is obtained by simultaneously mutating 235 th, 343 th, 332 th, 310 th, 151 th, 171 th, 275 th, 180 th, 316 th, 340 th, 178 th and 238 th amino acids of the lipase with the starting amino acid sequence shown in SEQ ID NO. 1.

In one embodiment of the present invention, the lipase mutant is obtained by mutating serine 151 to asparagine of a lipase having an original amino acid sequence shown in SEQ ID No.1 (S151N), designated as L1;

or the lipase mutant is obtained by mutating serine at position 235 of the lipase with an original amino acid sequence shown as SEQ ID NO.1 into alanine (S235A) and is named as L2;

or the lipase mutant is obtained by mutating lysine 171 of the lipase with the starting amino acid sequence shown as SEQ ID NO.1 into arginine (K171R), and is named as L3;

or the lipase mutant is obtained by mutating 343 rd site serine of the lipase with an original amino acid sequence shown as SEQ ID NO.1 into tyrosine (S343Y) and is named as L4;

or the lipase mutant is obtained by mutating glycine 275 position of the lipase with an original amino acid sequence shown as SEQ ID NO.1 into alanine (G275A), and is named as L5;

or the lipase mutant is obtained by mutating glutamine 332 of the lipase with the starting amino acid sequence shown as SEQ ID NO.1 into phenylalanine (Q332F) and is named as L6;

or the lipase mutant is obtained by mutating aspartic acid at the 310 th site of the lipase with the starting amino acid sequence shown as SEQ ID NO.1 into valine (D310V) and is named as L7;

or the lipase mutant is obtained by mutating leucine at position 180 of the lipase with an original amino acid sequence shown as SEQ ID NO.1 into histidine (L180H), and is named as L8;

or the lipase mutant is obtained by mutating phenylalanine 316 to cysteine and glycine 340 to cysteine of the lipase with the starting amino acid sequence shown as SEQ ID NO.1 (F316C \ G340C), and is named as L9;

or the lipase mutant is obtained by mutating serine at position 178 to cysteine and glutamine at position 238 to cysteine of the lipase with the starting amino acid sequence shown as SEQ ID NO.1 (S178C \ Q238C), and is named as L10;

or the lipase mutant is obtained by mutating phenylalanine at position 316 to cysteine, glycine at position 340 to cysteine, serine at position 178 to cysteine and glutamine at position 238 to cysteine of the lipase with an original amino acid sequence shown as SEQ ID No.1 (F316C \ G340C \ S178C \ Q238C), and is named as L11;

or the lipase mutant is obtained by mutating serine at position 235 to alanine, serine at position 343 to tyrosine, glutamine at position 332 to phenylalanine, and aspartic acid at position 310 to valine (S235A \ S343Y \ Q332F \ D310V) of the lipase with an original amino acid sequence shown as SEQ ID NO.1 (named as L12);

or the lipase mutant is obtained by mutating serine at position 151 to asparagine, lysine at position 171 to arginine, glycine at position 275 to alanine, and leucine at position 180 to histidine of the lipase with an original amino acid sequence shown as SEQ ID NO.1 (S151N \ K171R \ G275A \ L180H), and is named as L13;

or the lipase mutant is obtained by mutating serine at position 235 to alanine, serine at position 343 to tyrosine, glutamine at position 332 to phenylalanine, aspartic acid at position 310 to valine, serine at position 151 to asparagine, lysine at position 171 to arginine, glycine at position 275 to alanine and leucine at position 180 to histidine of the lipase with an original amino acid sequence shown in SEQ ID NO.1 (S235A \ S343Y \ Q332F \ D310V, S151N \ K171R \ G275A \ L180H) and is named as L14;

or the lipase mutant is obtained by mutating serine at position 235 to alanine, serine at position 343 to tyrosine, glutamine at position 332 to phenylalanine, aspartic acid at position 310 to valine, serine at position 151 to asparagine, lysine at position 171 to arginine, glycine at position 275 to alanine, leucine at position 180 to histidine, phenylalanine at position 316 to cysteine, glycine at position 340 to cysteine, serine at position 178 to cysteine and glutamine at position 238 to cysteine of the lipase with the starting amino acid sequence shown in SEQ ID No.1 (S235A \ S343Y \ Q332F D310V \ S151 \ K171 \ 5 \ G275A \ L180 \ F H \ F58316 \ G C \ 178S 178 \ Q46 \ Q23), and the name of the lipase mutant is L38724.

The invention also provides a gene, and the gene codes the lipase mutant.

The invention also provides a recombinant plasmid which carries the gene.

In one embodiment of the present invention, the expression vector of the recombinant plasmid is a pPIC9K vector, a ppic3.5k vector, a pPICZ α vector, or a pPICZ vector.

The invention also provides a host cell, which carries the gene or the recombinant plasmid.

In one embodiment of the invention, the host cell is a fungus or a bacterium.

In one embodiment of the invention, the host cell is pichia pastoris.

The invention also provides a detergent containing the lipase mutant.

In one embodiment of the present invention, the detergent contains the lipase mutant, a surfactant, and a buffer.

In one embodiment of the present invention, the surfactant may be Sodium Dodecyl Sulfate (SDS), sodium ethoxylated alkyl sulfate (AES), fatty alcohol polyoxyethylene ether (AEO-9), and/or disodium lauryl sulfosuccinate monoester (DLS).

In one embodiment of the present invention, the buffer may be a phosphate buffer, a Tris buffer, a citrate buffer, or a sodium carbonate buffer.

In one embodiment of the invention, the concentration of the lipase mutant in the detergent is 10-400U/L.

The invention also provides a decontamination method, which is characterized in that the lipase mutant is added into an article to be decontaminated; or the method is to add the detergent into the articles to be decontaminated.

The invention also provides the use of the lipase mutant or the gene or the recombinant plasmid or the host cell in oil processing, dairy processing, pasta processing, meat processing, drug synthesis, diesel synthesis, polymer synthesis, chiral compound synthesis, leather production, detergent preparation or paper making.

The invention also provides the application of the lipase mutant or the detergent or the decontamination method in decontamination.

[ advantageous effects ]

(1) The heat stability of the lipase mutant is obviously improved compared with the wild type, wherein the optimum temperature of L11 can reach 45 ℃, the T of L11 is improved by 5 ℃ compared with the wild type_mThe value can reach 49.2 ℃, the temperature is improved by 4.2 ℃ compared with the wild type, and the half-life period of L11 at 65 ℃ is 3.9min, which is 4.59 times of that of the wild type; the optimum temperature of L12 is 45 deg.C, which is increased by 5 deg.C compared with wild type, and T_mThe value can reach 50.3 ℃, is increased by 5.3 ℃ compared with the wild type, and the half-life period of L12 at 65 ℃ is 4.9min, which is 5.76 times of that of the wild type; the optimum temperature of L13 is 43 deg.C, which is increased by 3 deg.C compared with wild type, and T is higher than wild type_mThe value can reach 53.8 ℃, the temperature is improved by 8.8 ℃ compared with the wild type, and the half-life period of L13 at 65 ℃ is 7.6min, which is 8.94 times of that of the wild type; the optimum temperature of L14 is 48 deg.C, which is 8 deg.C higher than wild type, and T is_mThe value can reach 60.8 ℃, the temperature is increased by 15.8 ℃ compared with the wild type, and the half-life period of L14 at 65 ℃ is 19.8min, which is 23.29 times of that of the wild type; the optimum temperature of L15 can reach 50 deg.C, increased by 10 deg.C compared with wild type, and T_mThe value may be 68.The temperature is increased by 23.8 ℃ compared with the wild type, and the half-life period of L15 at 65 ℃ is 56.2min, which is 66.11 times of that of the wild type; compared with the wild type, the thermal stability of L1-L10 is also improved to a certain extent;

(2) the pH stability of the lipase mutant is obviously improved compared with that of a wild type, wherein the optimum pH of L9 is increased to 8.5 from 8.0 of the wild type; the optimum pH of the L11 is increased to 9.0 from 8.0 of the wild type, the enzyme activity of the L11 under the condition that the pH is 10 can reach 80.5 percent of the enzyme activity under the condition that the pH is 10, the enzyme activity is improved by 10.73 times compared with the wild type, the residual enzyme activity of the L11 after being treated for 30min under the condition that the pH is 10 can reach 90.8 percent, and the enzyme activity is improved by 2.20 times compared with the wild type; the optimum pH of L15 was raised from 8.0 of wild type to 9.0; the enzyme activity of the L15 under the condition that the pH value is 10 can reach 75.2 percent of the enzyme activity under the condition of the optimum pH value, the enzyme activity is improved by 10.02 times compared with the wild type, the residual enzyme activity of the L15 after being treated for 30min under the condition that the pH value is 10 can reach 100 percent, and the residual enzyme activity is improved by 2.43 times compared with the wild type;

(3) the lipase mutant has good washing performance, wherein the oil removal rate of the oil stain cloth washed by using L9 can reach 92.8%; the oil removal rate of the oil stain cloth washed by the L11 can reach 93.5%; the oil removal rate of the oil stain cloth washed by the L15 can reach 93.6 percent;

(4) the lipase mutant has good thermal stability, good pH stability and good washing performance, so the lipase mutant has important application in the aspects of decontamination, grease processing, dairy processing, wheaten food processing, meat processing, drug synthesis, diesel oil synthesis, polymer synthesis, chiral compound synthesis, leather production, detergent preparation, paper making and the like.

Drawings

FIG. 1: a whole plasmid PCR amplification electrophoretogram containing recombinant plasmids encoding different mutant genes;

wherein, M is DL10000 DNA molecular weight standard, 1 is the whole plasmid PCR amplification electrophoresis result of the recombinant plasmid containing the gene coding mutant S151N, 2 is the whole plasmid PCR amplification electrophoresis result of the recombinant plasmid containing the gene coding mutant S235A, and 3 is the whole plasmid PCR amplification electrophoresis result of the recombinant plasmid containing the gene coding mutant K171R.

FIG. 2: protein purification SDS-PAGE electrophoresis picture of fermentation supernatant of pichia pastoris engineering bacteria containing genes coding different mutants;

wherein, M is DL10000 DNA molecular weight standard, 1 is protein purification SDS-PAGE electrophoresis result of fermentation supernatant of pichia pastoris engineering bacteria containing genes for coding mutant S151N, 2 is protein purification SDS-PAGE electrophoresis result of fermentation supernatant of pichia pastoris engineering bacteria containing genes for coding mutant S235A, and 3 is protein purification SDS-PAGE electrophoresis result of fermentation supernatant of pichia pastoris engineering bacteria containing genes for coding mutant K171R.

FIG. 3: relative enzyme activities of wild type, L1, L2, L3, L4, L5, L6, L7, L8, L9 and L10 at different temperatures.

FIG. 4: relative enzyme activities of wild type, L11, L12, L13, L14, L15, L16 and L17 at different temperatures.

FIG. 5: relative enzyme activities of wild type, L1, L2, L3, L4, L5, L6, L7, L8, L9 and L10 under different pH values.

FIG. 6: relative enzyme activities of wild type, L11, L12, L13, L14, L15, L16 and L17 at different pH.

Detailed Description

The invention is further illustrated with reference to specific examples.

Escherichia coli JM109 to which the following examples refer was purchased from Biotechnology engineering (Shanghai) Co., Ltd; the pichia pastoris GS115 referred to in the examples below was purchased from Invitrogen; the pPIC9K vector referred to in the examples below was purchased from Invitrogen.

The detection methods referred to in the following examples are as follows:

the detection method of lipase activity comprises the following steps:

pNPP method, specific references: pencreach G et al, enzyme and Microbial Technol.1996,18: 417-.

Wherein, the lipase activity is defined as: the reaction was carried out at 40 ℃ at pH 8.0, and the amount of the enzyme producing 1. mu. mol of p-nitrophenol per minute was one enzyme activity unit (1U).

The media involved in the following examples are as follows:

LB liquid medium (m/m): peptone 1%, yeast extract 0.5%, NaCl 1%, pH7.0.

YPD liquid medium (m/m): 1% of yeast extract, 2% of tryptone and 2% of glucose, and autoclaving at 121 ℃ for 20 min.

YPD plates (m/m): 1% of yeast extract, 2% of tryptone, 2% of glucose and 2% of agar, and autoclaving at 121 ℃ for 20 min.

MD plate (m/m): nitrogen source of non-amino yeast 1.34%, biotin 4X 10^-5% glucose, 2% agar and 2%.

MM plate (m/m): nitrogen source of non-amino yeast 1.34%, biotin 4X 10^-5Percent, methanol 0.5 percent and agar 2 percent.

BMGY medium (m/m): 1% of yeast extract and 2% of tryptone.

Example 1: expression of wild-type Lipase

The method comprises the following specific steps:

amplifying a gene of lipase with an amino acid sequence shown as SEQ ID NO.1 from Rhizopus chinensis (Rhizopus chinensis); connecting the obtained gene proRCL with a pPIC9K vector through Not I and EcoR I double enzyme digestion to obtain a recombinant plasmid pPIC 9K-proRCL; transforming the recombinant plasmid pPIC9K-proRCL into Escherichia coli JM109 to obtain Escherichia coli engineering bacteria E.coli JM109/pPIC 9K-proRCL; e.coli JM109/pPIC9K-proRCL was spread on an LB plate (containing 100mg/L kanamycin), cultured at 37 ℃ for 10 hours, and then positive clones were selected and sequenced to confirm, thereby obtaining a correctly verified recombinant plasmid pPIC 9K-proRCL; after the recombinant plasmid pPIC9K-proRCL is linearized by Sal I, the recombinant plasmid is electrically transformed into Pichia pastoris GS115 to obtain Pichia pastoris engineering bacteria Pichia pastoris GS115/pPIC 9K-proRCL; coating Pichia pastoris GS115/pPIC9K-proRCL on an MD (MD) plate (containing 100mg/L kanamycin), culturing at 37 ℃ for 10 hours, extracting a genome, and identifying by PCR (polymerase chain reaction) to determine a positive clone to obtain a positive transformant; the positive transformants were inoculated into 250mL shake flasks containing 25mLBMGY medium and cultured at 30 ℃ and 250r/min to OD₆₀₀Obtaining a bacterial liquid when the concentration is 4.0; centrifuging the bacterial liquid at 8000r/min for 5min, collecting thallus, and resuspending the thallus to OD with 100mL BMMY culture medium₆₀₀1.0, obtaining a resuspension; continuously culturing the heavy suspension at 30 ℃ and 250r/min, supplementing 0.5% (volume ratio) methanol every 24h for induction expression, collecting bacterial liquid after 96h, and centrifuging the bacterial liquid at 8000r/min for 5min to obtain supernatant containing wild enzyme; performing affinity chromatography on the supernatant by using a nickel column, eluting the target protein at the concentration of 250mmol/L imidazole to obtain purified wild enzyme, and naming the wild enzyme as wild;

wherein, the amplification primers are as follows:

Fm：5’-TCAAGATCCCTAGGGTTCCTGTTGCTGGTCATAAAGGTTC-3’(SEQ ID NO.2)；

Rm：5’-AATTCCAGTGCGGCCGCTTAATGATGATGATGATGATGAGAAGAACCCAAACAGCTTCCTTCGTTAATACC-3’(SEQ ID NO.3)。

example 2: preparation and expression of lipase mutant

The method comprises the following specific steps:

carrying out site-directed mutagenesis by using a whole plasmid PCR technology and taking the recombinant plasmid pPIC9K-proRCL obtained in example 1 as a template to obtain mutants S151N, S235A, K171R, S343Y, G275A, Q332A, D310A, L180A, F316A \ G340A, S178A \ Q238A, F316A \ G340 \ S178A \ Q238A, S235A \ S343A \ Q332A \ D310A, S151A \ K171A, G275A L A, S235A \ S343 \ Q332 \ D310 \ S151A \ S151 \ A \ K171 \ A \ G A \ D A \ S151 \ S275 \ S72 \ S A \ S275 \ S310 \ A \ S310 \ A \ D A \ S151 \ A \ S275 \ A \ S310 \ A \ F A \;

wherein, the mutation primer is:

S151N：

S151N-Fm：5’-CTGCTTACTGTCGTAACGTCGTTCCAGGTACC-3’(SEQ ID NO.4)；

S151N-Rm：5’-GGTACCTGGAACGACGTTACGACAGTAAGCAG-3’(SEQ ID NO.5)；

S235A：

S235A-Fm：5’-GTTTCCTTTCCGCATACAACCAAGTTGTCAAAG-3’(SEQ ID NO.6)；

S235A-Rm：5’-CTTTGACAACTTGGTTGTATGCGGAAAGGAAAC-3’(SEQ ID NO.7)；

K171R：

K171R-Fm：5’-GTCTCAAGTATGTTCCTGATGGTAGGCTTATCAAGAC-3’(SEQ ID NO.8)；

K171R-Rm：5’-GTCTTGATAAGCCTACCATCAGGAACATACTTGAGAC-3’(SEQ ID NO.9)；

S343Y：

S343Y-Fm：5’-CCCGGTGTCGAATATTGGATCAAGGAAGAC-3’(SEQ ID NO.10)；

S343Y-Rm：5’-GGGGTGAAGATAACCGGCGGCTTGAGGAGG-3’(SEQ ID NO.11)；

G275A：

G275A-Fm：5’-GCCTTGCTCGCTGCCATGGATCTCTACCAACGTG-3’(SEQ ID NO.12)；

G275A-Rm：5’-CACGTTGGTAGAGATCCATGGCAGCGAGCAAGGC-3’(SEQ ID NO.13)；

Q332F：

Q332F-Fm：5’-CCCTCATGTTCCTCCTTTCGCCTTCGGTTATCTTC-3’(SEQ ID NO.14)；

Q332F-Rm：5’-GAAGATAACCGAAGGCGAAAGGAGGAACATGAGGG-3’(SEQ ID NO.15)；

D310V：

D310V-Fm：5’-CATTCGCTTACTACGTCGTCAGCACCGGAATTCCC-3’(SEQ ID NO.16)；

D310V-Rm：5’-GGGAATTCCGGTGCTGACGACGTAGTAAGCGAATG-3’(SEQ ID NO.17)；

L180H：

L180H-Fm：5’-GACCTTCACTTCTCTTCACACTGATACCAATGGC-3’(SEQ ID NO.18)；

L180H-Rm：5’-GCCATTGGTATCAGTGTGAAGGCAAGTGAAGGTC-3’(SEQ ID NO.19)；

F316C\G340C：

F316C-Fm：5’-CACCGGAATTCCCTGCCACCGTACCGTTCAC-3’(SEQ ID NO.20)；

F316C-Rm：5’-GTGAACGGTACGGTGGCAGGGAATTCCGGTG-3’(SEQ ID NO.21)；

G340C-Fm：5’-GTTATCTTCACCCCTGCGTCGAATCTTGGATC-3’(SEQ ID NO.22)；

G340C-Rm：5’-GATCCAAGATTCGACGCAGGGGTGAAGATAAC-3’(SEQ ID NO.23)；

S178C\Q238C：

S178C-Fm：5’-GACCTTCACTTGCCTTCTCACTGATACCAATG-3’(SEQ ID NO.24)；

S178C-Rm：5’-CATTGGTATCAGTGAGAAGGCAAGTGAAGGTC-3’(SEQ ID NO.25)；

Q238C-Fm：5’-CCTTTCCTCATACAACTGCGTTGTCAAAGACTACTTCCCCG-3’(SEQ ID NO.26)；

Q238C-Rm：5’-CGGGGAAGTAGTCTTTGACAACGCAGTTGTATGAGGAAAGG-3’(SEQ ID NO.27)；

F316C \ G340C \ S178C \ Q238C, S235C \ S343C \ Q332C \ D310C, S151C \ K171 \ G275C \ L180C, S235C \ S343C \ Q332C \ D310C \ S151C \ K171 \ G275C \ L180C and S235C \ S343C \ Q332C \ D310C \ S151C \ K171 \ G275C \ L180 \ F316C \ G340 \ S C \ S332C \ Q332C \ D310C \ S151C \ G275C \ L180C \ F316C \ F340 \ G340 \ S C \ Q36238: the same as above;

D159R；

D159R-Fm：5’-GGTACCAAGTGGCGATGTAAGCAATGTCTC-3’(SEQ ID NO.28)；

D159R-Rm：5’-GAGACATTGCTTACATCGCCACTTGGTACC-3’(SEQ ID NO.29)；

T319V：

T319V-Fm：5’-CCCTTCCACCGTGTCGTTCACAAGCGTG-3’(SEQ ID NO.30)；

T319V-Rm：5’-CACGCTTGTGAACGACACGGTGGAAGGG-3’(SEQ ID NO.31)；

the PCR system is as follows: mu.L each of 10. mu.M forward primer and reverse primer, 1. mu.L of dNTPmix 2. mu.L, 5 XPS Buffer 2.5. mu.L, PrimeStar GXLPMERASE 0.5. mu.L, template 0.5. mu.L, and 25. mu.L of double distilled water;

the PCR conditions were: 2min at 95 ℃; at the temperature of 95 ℃ for 20s, at the temperature of 55-63 ℃ for 1min and at the temperature of 68 ℃ for 11min, and performing 18 cycles; 5min at 68 ℃.

Detecting the PCR product by 1% agarose gel electrophoresis, digesting the detected correct PCR product by Dpn I, and transforming the product into Escherichia coli JM109 to obtain Escherichia coli engineering bacteria containing genes encoding the mutant; the engineered Escherichia coli was spread on LB plate (containing 100mg/L kanamycin), cultured at 37 ℃ for 10 hours, screened for positive clones and confirmed by sequencing to obtain correctly verified recombinant plasmids (containing all recombinant plasmids encoding the genes of mutants S151N, S235A, K171R)The plasmid PCR amplification electrophoretogram is shown in FIG. 1); after the recombinant plasmid is linearized by Sal I, the recombinant plasmid is electrically transformed into Pichia pastoris GS115 to obtain Pichia pastoris engineering bacteria containing genes of the coding mutant; coating pichia pastoris engineering bacteria on an MD (MD) plate (containing 100mg/L kanamycin), culturing for 10 hours at 37 ℃, extracting a genome, and identifying by PCR (polymerase chain reaction) to determine positive clone to obtain a positive transformant; the positive transformants were inoculated into 250mL shake flasks containing 25mLBMGY medium and cultured at 30 ℃ and 250r/min to OD₆₀₀Obtaining a bacterial liquid when the concentration is 4.0; centrifuging the bacterial liquid at 8000r/min for 5min, collecting thallus, and resuspending the thallus to OD with 100mL BMMY culture medium₆₀₀1.0, obtaining a resuspension; continuously culturing the heavy suspension at 30 ℃ and 250r/min, supplementing 0.5 percent (volume ratio) of methanol for induction expression every 24h, collecting bacterial liquid after 96h, and centrifuging the bacterial liquid at 8000r/min for 5min to obtain supernatants respectively containing different mutants (the protein purification SDS-PAGE electrophoresis chart of the fermentation supernatant of the pichia pastoris engineering bacteria containing the genes for coding the mutants S151N, S235A and K171R is shown in figure 2); and (2) carrying out affinity chromatography on the supernatant by using a nickel column, eluting the target protein at the concentration of 250mmol/L imidazole to obtain purified mutants S151, S235, K171, S343, G275, Q332, D310, L180, F316 \ G340, S178 \ Q238, F316 \ G340 \ Q238, S235 \ S343 \ Q332 \ D310, S151 \ K171 \ G275 \ L180, S235 \ S343 \ Q332 \ D310 \ S151 \ K171 \ G275 \ L180, S235 \ S275 \ D310S 151 \ K171 \ K275 \ L275 \ F316 \ G340 \ S178 \ Q238, D159 or T319 which are respectively named as L, L275 \ L180, L and L319.

Example 3: study of the enzymatic Properties of different lipases

The method comprises the following specific steps:

1. optimum temperature (T)_opt)

The lipase enzyme activities of the wild type obtained in example 1 and L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16 and L17 obtained in example 2 were measured at 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ and 65 ℃ respectively, and the relative enzyme activities were calculated based on the highest enzyme activity as 100% and the other enzyme activities compared thereto, to examine the optimum action temperature of the enzyme (see FIGS. 3-4 for the results of the detection).

As can be seen from FIGS. 3 to 4, the optimum temperatures of the wild type were all 40 ℃; the optimal temperatures of L1, L2, L3, L4, L5, L6, L8, L9, L10, L16 and L17 are all 40 ℃, and are not improved compared with the wild type; the optimal temperature of the L7 is raised to 45 ℃; the optimal temperature of the L13 is raised to 43 ℃; the optimal temperature of L11 and L12 is increased to 45 ℃; the optimal temperature of L14 is raised to 48 ℃; the optimum temperature of L15 was increased by 50 ℃ to the maximum, which is 10 ℃ higher than that of wild type.

2. Denaturation temperature (T)_m)

The wild type obtained in example 1 and L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16 and L17 obtained in example 2 are respectively treated in a constant-temperature water bath at 40-70 ℃ for 30min, then are respectively subjected to ice bath for 20min, finally, the lipase enzyme activity is measured at 40 ℃, and when the enzyme activity is 50% of the initial enzyme activity, the heat treatment temperature is the enzyme denaturation temperature (the detection result is shown in Table 1).

As can be seen from Table 2, the denaturation temperatures of L1-L15 were increased to different degrees compared with the wild type, wherein the increased degrees were L11-L15, the denaturation temperatures were respectively 49.2 deg.C, 50.3 deg.C, 53.8 deg.C, 60.8 deg.C and 68.8 deg.C, and the denaturation temperatures were respectively increased by 4.2 deg.C, 5.3 deg.C, 8.8 deg.C, 15.8 deg.C and 23.8 deg.C compared with the wild type; the denaturation temperature of L16 was 40.1 deg.C and that of L17 was 38.5 deg.C, which were reduced by 4.9 deg.C and 6.5 deg.C, respectively, compared to wild type.

TABLE 1 denaturation temperatures (T) of different lipases_m)

3. Half life (t)_1/2)

The wild type obtained in example 1 and L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16, and L17 obtained in example 2 were placed in a constant-temperature water bath at 65 ℃ respectively, and samples were taken at intervals to measure the residual enzyme activity and compare the thermal stability (see Table 2 for the results of the measurement).

As can be seen from table 3, compared with the wild type, the half-lives of L1 to L15 are extended to different degrees at 65 ℃, wherein the extended degrees are L11 to L15, and the half-lives are respectively 3.9min, 4.9min, 7.6min, 19.8min and 56.2min, which are respectively 4.59 times, 5.76 times, 8.94 times, 23.29 times and 66.11 times longer than the wild type; and the half-life period of L16 is 0.75min, and the half-life period of L17 is 0.5min, which are respectively reduced by 11.76% and 41.18% compared with the wild type.

TABLE 2 half-lives of different lipases after treatment at 65 ℃

4. Optimum pH

A citric acid buffer solution (pH 6) with the concentration of 0.05mol/L, a phosphate buffer solution (pH 6-8) with the concentration of 0.05mol/L, a Tris-HCl buffer solution (pH 8-9) with the concentration of 0.05mol/L and a carbonate buffer solution (pH 9-10) with the concentration of 0.05mol/L are respectively prepared to replace buffer solutions in the lipase activity measuring method, lipase enzyme activities of the wild type obtained in example 1 and the L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16 and L17 obtained in example 2 are measured at 40 ℃, the highest enzyme activity is 100 percent, and relative enzyme activities are calculated compared with the rest enzyme activities, so as to investigate the optimal action pH of the enzyme (the detection result is shown in a figure 5-6).

As can be seen from FIGS. 5 to 6, the optimum pH of the wild type was 8.0; the optimum pH of L1, L2, L3, L4, L5, L6, L7, L8, L10, L12, L13, L14, L16 and L17 was also 8.0, and was unchanged from the wild type; the optimum pH of L9 is 8.5, which is 0.5 higher than that of wild type; the optimum pH of both L11 and L15 increased to 9.0, which was 1 higher than that of wild type, and more favorable to alkaline conditions.

5. Alkali resistance

From the results in table 4, the ratio of the enzyme activities of the wild type obtained in example 1 and L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16, and L17 obtained in example 2 under the condition of pH 10 to the enzyme activity under the optimum pH condition was calculated, and the calculation results are shown in table 3.

As can be seen from table 3, the enzyme activities of L9, L11, and L15 under the condition of pH 10 account for 90.6%, 80.5%, and 75.2% of the enzyme activities under the optimum pH condition, and are respectively increased by 12.1 times, 10.7 times, and 10.0 times as compared with the wild type; the enzyme activities of L16 and L17 under the condition that the pH is 10 account for 7.2 percent and 6.9 percent of the enzyme activities under the condition of the optimal pH, and are slightly reduced compared with the wild type; the ratio of the enzyme activity of other mutants under the condition that the pH is 10 to the enzyme activity under the condition of the optimal pH is not much different from that of the wild type or slightly increased.

TABLE 3 ratio of enzyme activity of different lipases under pH 10 to enzyme activity under optimum pH

6. Stability of pH

Carbonate buffer with the concentration of 0.05mol/L, pH of 10 is prepared to replace buffer in the method for measuring the lipase activity, the wild type obtained in example 1 and the L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16 and L17 obtained in example 2 are respectively stored in the buffer system at 25 ℃ for 30min, the lipase activity is measured at 40 ℃, the initial enzyme activity is 100%, and the residual enzyme activity is calculated by comparing the enzyme activity after storage, so as to examine the pH stability (the detection result is shown in Table 4).

As can be seen from Table 4, the residual enzyme activity of L5 after being treated for 30min under the condition that the pH value is 10 is 65.0 percent, which is increased by 57.3 percent compared with the wild type; the residual enzyme activity of the L7 treated for 30min under the condition that the pH value is 10 is 55.3 percent, which is improved by 34.2 percent compared with the wild type; the residual enzyme activity of the L9 treated for 30min under the condition that the pH value is 10 is 87.4 percent, which is improved by 112 percent compared with the wild type; the residual enzyme activity of the L11 treated for 30min under the condition that the pH value is 10 is 98.8 percent, which is improved by 139.8 percent compared with the wild type; the residual enzyme activity of the L12 treated for 30min under the condition that the pH value is 10 is 55.8 percent, which is improved by 35.4 percent compared with the wild type; after the L13 and the L14 are treated for 30min under the condition that the pH value is 10, the residual enzyme activity is 65.2 percent and 65.8 percent, which are respectively improved by 58.3 percent and 59.7 percent compared with the wild type; the enzyme activity of the L15 treated for 30min under the condition that the pH value is 10 is not lost, the enzyme activity is still maintained at 100 percent, and the enzyme activity is improved by 142.7 percent compared with that of a wild type; the residual enzyme activities of L16 and L17 after being treated for 30min under the condition that the pH value is 10 are 39.4 percent and 38.5 percent, which are slightly reduced compared with the wild type; the residual enzyme activity of other mutants treated for 30min under the condition of pH 10 is not much or slightly increased compared with that of the wild type.

TABLE 4 residual enzyme activity after 30min of treatment of different lipases at pH 10

Numbering	Residual enzyme activity
		Wild type	41.2％
L1	40.6％
		L2	48.3％
L3	44.4％
		L4	47.2％
L5	65.0％
		L6	37.8％
L7	55.3％
		L8	42.1％
L9	87.4％
		L10	40.5％
L11	90.8％
		L12	55.8％
L13	65.2％
		L14	65.8％
L15
		100％
L16			39.4％
	L17	38.5％

Example 4: evaluation of the Wash Performance of different lipases

The method comprises the following specific steps:

1. preparation of greasy dirt cloth

Cutting cotton cloth into square cotton cloth blocks of 6 × 6cm, boiling in boiling chloroform for degreasing for 4 hr to obtain degreased cotton cloth; adding olive oil into toluene to obtain an oil stain solution with the concentration of 200 mg/mL; dropwise adding the greasy dirt liquid to two sides of the absorbent cotton cloth twice by 0.1mL each time to obtain the absorbent cotton cloth dropwise added with the greasy dirt liquid; and (3) drying the degreased cotton cloth dropwise added with the greasy dirt liquid in a fume hood, and then drying the degreased cotton cloth in an oven at the temperature of 60 ℃ for 1 hour to obtain the greasy dirt cloth.

2. Preparation of detergents

Preparing 0.1mol/L phosphate buffer solution (pH 9.0), 0.1mol/L Tris buffer solution (pH 10.0, 10.5 and 11.0) and 0.5% (m/m) SDS solution, preparing washing solution with the wild type obtained in example 1 and the L9, L11, L15, L16 and L17 obtained in example 2, wherein the final volume of the washing solution is 100mL, and the formula is as follows:

detergent 1: 50mL of buffer solution and 50mL of deionized water;

2-7 parts of a detergent: 50mL of buffer, L9, L11, L15, L16 or L17100U obtained in example 1 and wild type obtained in example 2, was added deionized water to a total volume of 100 mL;

and (4) a detergent 8: 50mL of buffer solution and 25mL of SDS solution are added with deionized water to the total volume of 100 mL;

9-14 parts of detergent: 50mL of buffer, L9, L11, L15, L16 or L17100U obtained in example 1 and L9, L11, L15, L16 or L17100U obtained in example 2, 25mL of SDS solution, and deionized water was added to a total volume of 100 mL.

3. Washing of soiled cloths

And respectively putting the oil stain cloth into the laundry detergent 1-14, washing for 30min at 50 ℃ and 200rpm, taking out the oil stain cloth after washing, respectively washing for 3 times at 40 ℃ and 200rpm for 2min each time by using 100mL of purified water, airing, and then continuously drying the oil stain cloth in an oven at 60 ℃ for 1 h.

4. Evaluation of washing Performance

Weighing the mass of the greasy dirt cloth before and after washing by using a precision balance, wherein the oil removal rate takes the weight of oil before and after washing as a measurement standard:

oil removal rate (%) [ (pre-wash oil heavy)/pre-wash oil heavy ] × 100;

15 pieces of the soiled cloths were repeatedly made under each condition, and the washing effect of different lipases on the soiled cloths is shown in table 5.

As can be seen from Table 5, the oil removal rate was not improved and slightly decreased compared to the case where the wild type was added under four conditions of pH 9.0 and 10.0, pH 10.5 and pH 11.0, indicating that the wild type had no washing performance;

after L9 is added under four conditions of pH 9.0 and 10.0, 10.5 and 11.0, the oil removal rate is improved by nearly one time compared with that of the washing only by buffer solution, and when SDS is contained in the detergent, L9 is added, the oil removal rate can be respectively increased to 88.1%, 90.5%, 92.8% and 85.5% from 79.9%, 78.6%, 75.8% and 76.1%, and the oil removal rate can be respectively increased to 10.3%, 15.3%, 22.4% and 12.4%; after L11 is added under four conditions of pH 9.0 and 10.0, 10.5 and 11.0, the oil removal rate is doubled compared with that of the washing only by buffer solution, when SDS is contained in the detergent, the oil removal rate can be respectively increased to 87.5%, 90.4%, 93.5% and 86.3% from 79.9%, 78.6%, 75.8% and 76.1% by adding L11, and the oil removal rate can be respectively increased to 9.9%, 15.0%, 23.4% and 13.4%; after L15 is added under four conditions of pH 9.0 and 10.0, 10.5 and 11.0, the oil removal rate is doubled compared with that of the washing only by buffer solution, when the detergent contains SDS, L15 is added, the oil removal rate can be respectively increased from 79.9%, 78.6%, 75.8% and 76.1% to 87.1%, 89.1%, 93.6% and 86.5%, the increase range is 9.0%, 13.4%, 23.4% and 13.7%, and the results show that L9, L11 and L15 have very good washing effect and wide application prospect;

after addition of L16 or L17 at pH 9.0 and 10.0, 10.5 and 11.0, the oil removal rate was not improved compared to that without the addition, indicating that L16 and L17 also had no cleaning performance.

TABLE 5 Wash Performance of detergents containing different lipases at different pH

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

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<120> lipase mutant and application thereof in decontamination

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Claims

1. A lipase mutant is characterized in that the lipase mutant is obtained by mutating glycine 275 position of lipase with an original amino acid sequence shown as SEQ ID NO.1 into alanine;

or the lipase mutant is obtained by mutating serine at position 151 to asparagine, lysine at position 171 to arginine, glycine at position 275 to alanine, and leucine at position 180 to histidine of the lipase with an original amino acid sequence shown as SEQ ID No. 1.

2. A gene encoding the lipase mutant of claim 1.

3. A recombinant plasmid carrying the gene of claim 2.

4. A host cell carrying the gene of claim 2 or the recombinant plasmid of claim 3.

5. A detergent comprising the lipase mutant according to claim 1.

6. The detergent according to claim 5, wherein the detergent comprises the lipase mutant according to claim 1, a surfactant and a buffer.

7. A method for degreasing, comprising adding the lipase mutant of claim 1 to an article to be decontaminated; alternatively, the method is to add the detergent of claim 5 or 6 to an article to be decontaminated.

8. Use of the lipase mutant according to claim 1 or the gene according to claim 2 or the recombinant plasmid according to claim 3 or the host cell according to claim 4 in oil and fat processing, dairy processing, pasta processing, meat processing, pharmaceutical synthesis, diesel synthesis, polymer synthesis, synthesis of chiral compounds, leather production, detergent preparation or paper production.

9. Use of the lipase mutant of claim 1 or the detergent of claim 5 or 6 or the degreasing method of claim 7 for degreasing.