CN117887688B - A high-activity, high-stability lipase mutant and its encoding gene and application - Google Patents

Abstract

The invention relates to the field of novel lipase mutants, in particular to a lipase mutant with high activity and high stability, and a coding gene and application thereof, wherein the lipase of thermomyces lanuginosus is taken as a template, and the 113 th amino acid Ala is mutated into Gly, so that the activity of the lipase mutant is improved; then mutating the 74 th amino acid Leu into Asn to form a specific sequence of Asn74-Tyr75-Ser76, and introducing glycosylation to obtain a lipase mutant TLL291/A113G/L74N with enhanced heat stability and organic solvent tolerance. The lipase mutant prepared by the invention can be widely applied to the production process in the fields of paper making, food or medicine, and has high activity, high thermal stability and high methanol/tertiary butanol tolerance.

Description

A high-activity, high-stability lipase mutant and its encoding gene and application

Technical Field

The present invention relates to the field of bioengineering and energy technology, and also to the field of novel lipase mutants, in particular to a lipase mutant with high activity and high stability, a coding gene thereof and an application thereof.

Background technique

Lipase is a triacylglycerol acyl hydrolase, which belongs to the class of carboxyl ester hydrolases. It can act on the ester bonds of triglycerides, catalyze the hydrolysis of natural substrate oils and fats, and generate fatty acids, glycerol, and mono- or di-glycerides. Lipase is widely available, safe and non-toxic, and has a wide range of applications. It has shown important application potential in the fields of food, medicine, papermaking, and energy. However, the preparation or use of many foods, detergents, and biodiesel requires high temperatures, and the low activity, thermal sensitivity, and denaturation of lipase by alcohols are key issues that limit the widespread application of lipase. Therefore, it is of great significance to design a lipase with high activity, thermal stability, and tolerance to organic solvents.

At present, immobilization technology, protein engineering and heterologous expression are common methods to improve the activity, thermal stability and organic solvent tolerance of lipase. Among them, protein engineering methods to improve enzymatic properties include irrational design (directed evolution), semi-rational design and rational design. Semi-rational design mainly uses bioinformatics methods to rationally select multiple amino acid residues as transformation targets based on homologous protein sequence alignment, three-dimensional structure or existing knowledge, and combines the rational selection of effective codons to construct a high-quality mutant library to transform proteins in a targeted manner. Heterologous expression refers to the use of DNA or RNA of another organism as an exogenous gene or expressor in one organism, so that the gene or its product can produce specific functions or traits. In the field of biocatalysis, semi-rational design strategies have occupied a dominant position. With the help of this technology, many enzymes have obtained mutants with enhanced performance.

Studies have shown that two N-acetylglucosamines in Pichia pastoris can link mannose and asparagine to form glycoproteins. During the N-glycosylation process of glycoproteins, pre-synthesized oligosaccharides can be linked to the recognition sequence Asn-Xaa-Ser/Thr (where Xaa is an amino acid other than proline) through the N-glycosidic bond of the asparagine (N) residue. This reaction improves the methanol resistance and thermal stability of proteins by affecting their folding, intracellular localization and structure.

For another example, the invention patent with announcement number CN104762277B discloses a method for improving lipase expression by glycosylation modification, a mutant enzyme and its application. The leading peptide sequence of Rhizopus oryzae lipase is subjected to N-glycosylation mutation, and the SAS and/or NT amino acids are respectively transformed into N-glycosylation sites NGT and/or NLT. The extracellular protein concentrations of the obtained mutant enzymes proROLA, proROLB and proROLAB are increased by 211%, 188% and 233% respectively compared with the non-glycosylated proROL. The enzyme activities during fermentation tank culture reach 8210U·mL ^-1 , 8457U·mL ^-1 and 9366U·mL ^-1 respectively, while the extracellular enzyme activity of the non-mutated proROL is almost zero. For example, the invention patent with announcement number CN115369099B discloses a mutant of Rhizomucor miehei lipase and a method for improving the activity and/or methanol tolerance of Rhizomucor miehei lipase. Glycosylation modification is introduced into the β-sheet secondary structure of the mature peptide of Rhizomucor miehei lipase to obtain a lipase mutant with high activity and high methanol tolerance. Compared with before the modification, the enzyme activities of the mutant enzymes P167N, L242N, R267N and P300N produced by the present invention are increased by 33.90, 17.90, 14.07 and 12.26 times, respectively. After incubation in 60% methanol solution for 1 hour, the residual activity of the mutant enzyme is increased from 19.10% before the modification to more than 60%.

In summary, how to obtain new and improved high-performance tolerant lipase mutants through protein engineering is a technical problem that needs to be solved urgently.

Summary of the invention

In view of this, the purpose of the present invention is to propose a novel lipase mutant and its encoding gene and application, and to obtain a lipase mutant with high activity, high thermal stability and enhanced alcohol tolerance through site-directed mutagenesis and introduction of glycosylation.

Based on the above purpose, the present invention provides a high-activity and high-stability lipase mutant, which is obtained by mutating the amino acid Ala at position 113 of the lipase with an amino acid sequence of SEQ ID NO:1 to Gly, and mutating the amino acid Leu at position 74 to Asn, forming a specific sequence of Asn74-Tyr75-Ser76, and its amino acid sequence is SEQ ID NO:4.

Preferably, the amino acid sequence of the Thermomyces lanuginosus lipase is as shown in SEQ ID NO:2, wherein the amino acid sequence of the intermediate mutant formed by the mutation of the 113th amino acid Ala to Gly in SEQ ID NO:1.

Preferably, the nucleotide sequence encoding the intermediate mutant is shown in SEQ ID NO:3.

The present invention also provides a coding gene for the lipase mutant with high activity and high stability.

Preferably, the nucleotide sequence of the encoding gene is as shown in SEQ ID NO:5.

The invention also provides a recombinant plasmid into which the coding gene of the lipase mutant is inserted.

The present invention further provides a recombinant Pichia pastoris genetically engineered bacterium: the genetically engineered bacterium comprises the recombinant plasmid, is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, is classified and named Pichia sp ., has a deposit date of 2023.12.21, and a deposit number of CGMCC No.29367.

The present invention further provides the application of the lipase mutant in food processing, drug synthesis, leather production or papermaking industry.

The present invention further provides a method for preparing a lipase mutant, comprising the following steps:

(1) A wild-type lipase was synthesized using the lipase gene from Thermomyces lanuginosus as a template, and the enzyme was named TLL291. The amino acid sequence of the enzyme is shown in SEQ ID NO: 1;

(2) Obtain the crystal structure of lipase, search for lipase genes with high homology in the NCBI database, and determine the amino acid sequence of the "lid" structure of lipase by analyzing the sequence consistency; using TLL291 as a template, select the amino acid residue Ala113 in the "lid" structure that is inconsistent with the homologous sequence and mutate it to Gly. The amino acid sequence of the mutant is shown in SEQ ID NO:2, and the nucleotide sequence is shown in SEQ ID NO:3;

The Ala113Gly mutation primer is:

A113G For:

5’-GTTGAAAATTGGATTGGCAATTTGGCTGCTGAT-3’

A113G Rev:

5′-GCCAATCCAATTTTCAACAGATCTTGAACCTCT-3′;

(3) analyzing the full-length amino acid sequence of the lipase, and based on step (2), mutating the amino acid residue Leu74 to Asn to form a glycosylation sequence of Asn74-Tyr75-Ser76. The amino acid sequence of the mutant is shown in SEQ ID NO:4, and the nucleotide sequence is shown in SEQ ID NO:5;

The Leu74Asn mutation primer is:

L74N For:

5’-GCTGATGCTACTTTTAACTATTCTTTTGAAGATTCT-3’

L74N Rev:

5′-GTTAAAAGTAGCATCAGCAGCATCAACTTCAGGACA-3′;

(4) The plasmid vector used to express the wild-type lipase and its mutants in the above step is pPICZαA;

(5) The host cell of the recombinant expression plasmid in step (4) is Pichia pastoris GS115 ;

In the above steps, the original lipase and its mutants were inserted into the pPICZαA vector using Kpn I- Xba I as cloning sites, and linearized using endonuclease Kpn Ⅰ or Xba Ⅰ.

Beneficial effects of the present invention:

The present invention firstly improves the activity of lipase by site-directed mutation of amino acids on the "lid" structure; and introduces glycosylation site mutation by analyzing the amino acid sequence. After glycosylation, the thermal stability of the lipase mutant is significantly improved, and the tolerance to methanol and tert-butanol is significantly enhanced. It can be applied to the fields of energy, chemical industry, and pharmaceuticals, and has greater industrial application value.

Compared with the unmodified wild-type lipase, the lipase mutant constructed by the present invention has an activity increased by 98.1% at a substrate concentration of 1.5 mM, and still has 72% of residual activity after being incubated in a 50°C water bath for 24 hours. The inventors also found that the lipase mutant TLL291/A113G/L74N still has 33.6% of residual activity after being incubated in 60% (v/v) methanol for 24 hours, and is particularly easy to maintain high activity in 60% (v/v) tert-butanol. After incubation in tert-butanol for 12 hours, the activity is still 85.8%, and after incubation for 24 hours, the activity remains 38.6%, showing excellent organic solvent tolerance.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

FIG1 is a color reaction diagram of lipase hydrolysis of pNPP in Example 2 of the present invention;

FIG2 is a standard curve diagram of lipase activity measured by the pNPP method in Example 2 of the present invention;

FIG3 is a comparison diagram of the activity detection of the wild-type lipase and its mutant TLL291/A113G at different pNPP substrate concentrations in Example 2 of the present invention; wherein WT represents the activity curve of the wild-type lipase, and A113G represents the activity curve of the lipase mutant TLL291/A113G;

FIG4 is a comparison diagram of the half-lives of the wild-type lipase and its mutant TLL291/A113G/L74N in Example 3 of the present invention;

FIG5 is a comparison of the residual activities of the wild-type lipase and its mutant TLL291/A113G/L74N in Example 4 of the present invention when incubated in a methanol environment;

FIG6 is a comparison of the residual activities of the wild-type lipase and its mutant TLL291/A113G/L74N in Example 4 of the present invention when incubated in a tert-butanol environment.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

Example 1 Construction and acquisition of lipase and its mutants

(1) An artificial gene was synthesized using the lipase gene from Thermomyces lanuginosus as a template. The enzyme was named TLL291, and its amino acid sequence is shown in SEQ ID NO: 1. The enzyme was inserted into the pPICZαA vector using Kpn I- Xba I as the cloning site, and the wild recombinant expression plasmid pPICZαA-TLL291 was obtained by PCR amplification technology;

Amino acid sequence of TLL291 lipase (SEQ ID NO: 1):

MRSSLVLFFLSAWTALARPVRRAVPQDLLDQFELFSQYSAAAYCAANNHAPVGSDVTCSENVCPEVDAADATFLYSFEDSGLGDVTGLLALDNTNKLIVLSFRGSRSVENWIANLAADLTEISDICSGCEGHVGFVTSWRSVADTIREQVQNAVNEHPDYRVVFTGHSLGGALATIAAAALRGNGYNIDVFSYGAPRVGNRAFAEFLTAQTGGTLYRITHTNDIVPRLPPRDWGYSHSSPEYWVTSGNDVPVTANDITVVEGIDSTDGNNQGNIPDIPSHLWYFGPISECD;

(2) Obtaining the crystal structure of lipase, searching for highly homologous fat sequences in the NCBI database, and determining that the "lid" structure of TLL291 is the Ser105-Asp118 region by analyzing the sequence consistency, using the wild recombinant expression plasmid described in step (1) as a template, designing primers, and mutating the amino acid residue at position Ala113 to Gly by whole plasmid PCR to obtain the lipase mutant TLL291/A113G, whose amino acid sequence is shown in SEQ ID NO:2, and whose nucleotide sequence is shown in SEQ ID NO:3;

The Ala113Gly mutation primer is:

A113G For:

5’-GTTGAAAATTGGATTGGCAATTTGGCTGCTGAT-3’

A113G Rev:

5′-GCCAATCCAATTTTCAACAGATCTTGAACCTCT-3′;

Amino acid sequence of lipase mutant TLL291/A113G (SEQ ID NO: 2):

MRSSLVLFFLSAWTALARPVRRAVPQDLLDQFELFSQYSAAAYCAANNHAPVGSDVTCSENVCPEVDAADATFLYSFEDSGLGDVTGLLALDNTNKLIVLSFRGSRSVENWIGNLAADLTEISDICSGCEGHVGFVTSWRSVADTIREQVQNAVNEHPDYRVVFTGHSLGGALATIAAAALRGNGYNIDVFSYGAPRVGNRAFAEFLTAQTGGTLYRITHTNDIVPRLPPRDWGYSHSSPEYWVTSGNDVPVTANDITVVEGIDSTDGNNQGNIPDIPSHLWYFGPISECD;

Nucleotide sequence of lipase mutant TLL291/A113G (SEQ ID NO: 3):

ATGAGATCTTCTTTGGTTTTGTTTTTTTTGTCTGCTTGGACTGCTTTGGCTAGACCTGTTAGAAGAGCTGTTCCACAAGATTTGTTGGATCAATTTGAATTGTTTTCTCAATATTCTGCTGCAGCTTATTGTGCTGCTAATAATCATGCTCCTGTTGGTTCTGATGTTACTTGTTCTGAAAATGTTTGTCCTGAAGTTGATGCTGCTGATGCTACTTTTTTGTATTCTTTTGAAGATTCTGGTTTGGGAGATGTTACTGGTTTGTTAGCATTAGATAACACTAATAAATTGATTGTTTTGTCTTTTAGAGGTTCAAGATCTGTTGAAAATTGGATTGGCAATTTGGCTGCTGATTTGACTGAAATTTCTGATATTTGTTCTGGTTGTGAAGGTCATGTTGGTTTTGTTACTTCTTGGAGATCTGTTGCTGATACTAT TAGAGAACAAGTTCAAAATGCTGTTAATGAACATCCTGATTATAGAGTTGTTTTTACTGGTCATTCTTTGGGTGGTGCTTTGGCTACTATTGCTGCAGCTGCATTAAGGGGTAATGGTTATAATATTGATGTTTTTTCTTATGGTGCTCCAAGAGTTGGTAATAGAGCTTTTGCTGAATTTTTGACTGCTCAAACTGGTGGTACTTTGTATAGAATTACTCATACTAATGATATTGTTCCAAGATTGCCACCAAGAGATTGGGGTTATTCTCATTCTTCTCCTGAATATTGGGTTACTTCTGGTAATGATGTTCCTGTTACTGCTAATGATATTACTGTTGTTGAAGGTATTGATTCTACTGATGGTAATAATCAAGGTAATATTCCTGATATTCCATCTCATTTGTGGTATTTTGGTCCAATTTCTGAATGTGAT;

(3) Analyze the full-length amino acid sequence of the lipase. Based on step (2), design primers and mutate the amino acid residue Leu74 to Asn by whole-plasmid PCR to form a glycosylation sequence of Asn74-Tyr75-Ser76, thereby obtaining the lipase mutant TLL291/A113G/L74N, whose amino acid sequence is shown in SEQ ID NO:4 and nucleotide sequence is shown in SEQ ID NO:5;

The Leu74Asn mutation primer is:

L74N For:

5’-GCTGATGCTACTTTTAACTATTCTTTTGAAGATTCT-3’

L74N Rev:

5′-GTTAAAAGTAGCATCAGCAGCATCAACTTCAGGACA-3′;

Amino acid sequence of lipase mutant TLL291/A113G/L74N (SEQ ID NO: 4):

MRSSLVLFFLSAWTALARPVRRAVPQDLLDQFELFSQYSAAAYCAANNHAPVGSDVTCSENVCPEVDAADATFNYSFEDSGLGDVTGLLALDNTNKLIVLSFRGSRSVENWIGNLAADLTEISDICSGCEGHVGFVTSWRSVADTIREQVQNAVNEHPDYRVVFTGHSLGGALATIAAAALRGNGYNIDVFSYGAPRVGNRAFAEFLTAQTGGTLYRITHTNDIVPRLPPRDWGYSHSSPEYWVTSGNDVPVTANDITVVEGIDSTDGNNQGNIPDIPSHLWYFGPISECD;

Nucleotide sequence of lipase mutant TLL291/A113G/L74N (SEQ ID NO: 5):

ATGAGATCTTCTTTGGTTTTGTTTTTTTTGTCTGCTTGGACTGCTTTGGCTAGACCTGTTAGAAGAGCTGTTCCACAAGATTTGTTGGATCAATTTGAATTGTTTTCTCAATATTCTGCTGCAGCTTATTGTGCTGCTAATAATCATGCTCCTGTTGGTTCTGATGTTACTTGTTCTGAAAATGTTTGTCCTGAAGTTGATGCTGCTGATGCTACTTTTAACTATTCTTTTGAAGATTCTGGTTTGGGAGATGTTACTGGTTTGTTAGCATTAGATAACACTAATAAATTGATTGTTTTGTCTTTTAGAGGTTCAAGATCTGTTGAAAATTGGATTGGCAATTTGGCTGCTGATTTGACTGAAATTTCTGATATTTGTTCTGGTTGTGAAGGTCATGTTGGTTTTGTTACTTCTTGGAGATCTGTTGCTGATACTAT TAGAGAACAAGTTCAAAATGCTGTTAATGAACATCCTGATTATAGAGTTGTTTTTACTGGTCATTCTTTGGGTGGTGCTTTGGCTACTATTGCTGCAGCTGCATTAAGGGGTAATGGTTATAATATTGATGTTTTTTCTTATGGTGCTCCAAGAGTTGGTAATAGAGCTTTTGCTGAATTTTTGACTGCTCAAACTGGTGGTACTTTGTATAGAATTACTCATACTAATGATATTGTTCCAAGATTGCCACCAAGAGATTGGGGTTATTCTCATTCTTCTCCTGAATATTGGGTTACTTCTGGTAATGATGTTCCTGTTACTGCTAATGATATTACTGTTGTTGAAGGTATTGATTCTACTGATGGTAATAATCAAGGTAATATTCCTGATATTCCATCTCATTTGTGGTATTTTGGTCCAATTTCTGAATGTGAT;

The recombinant expression plasmid described in the above steps is linearized by endonuclease Kpn Ⅰ or Xba Ⅰ, and the linearized plasmid is transformed into Pichia pastoris GS115 competent cells by electroporation, and a genetically engineered bacterium capable of stable expression is obtained after bleomycin resistance screening, enzyme digestion, electroporation and SDS-PAGE verification; wherein, the Pichia pastoris GS115 /TLL291/A113G/L74N lipase genetically engineered bacterium is currently deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration (No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing), and is classified and named Pichia sp ., with a deposit date of 2023.12.21 and a deposit number of CGMCC No.29367;

(4) The genetically engineered bacteria were inoculated into a YPD liquid medium containing 100 μg/mL bleomycin, and cultured in a shake flask at 28°C and 220 r/min for 24 h; wherein the YPD liquid medium formula is: 1 wt% yeast powder + 2 wt% tryptone + 2 wt% glucose;

Then, the cells were transferred to BMGY medium at a volume inoculation rate of 1%, and cultured in a shaking flask at 28°C and 220 r/min for 24 h; the BMGY medium formula was: 100 mM sodium phosphate buffer (pH 6.0) + 1 wt% yeast powder + 2 wt% tryptone + 0.34 wt% amino acid-free yeast nitrogen source (YNB) + 4×10 ^-5 wt% biotin + 1 wt% glycerol + 100 μg/mL bleomycin;

The cells were collected and redispersed in BMMY medium, and 1% (v/v) methanol was added every 24 h for induction culture. The induction culture temperature was 28 °C and the rotation speed was 180 r/min. The BMMY medium formula was: 100 mM sodium phosphate buffer (pH 6.0) + 1 wt% yeast powder + 2 wt% peptone + 0.34 wt% amino acid-free yeast nitrogen source (YNB) + 4×10 ^-5 wt% biotin, 1% (v/v) methanol + 100 μg/mL bleomycin.

After 120 hours of induction culture, the supernatant was removed by centrifugation to obtain the crude enzyme solution of the lipase mutant, and the enzyme solution was concentrated by centrifugation and ultrafiltration for later use.

The method for preparing the crude enzyme solution of wild-type lipase is the same as above, except that the wild recombinant expression plasmid pPICZαA-TLL291 is used to replace the above recombinant expression plasmid, which is linearized and directly transformed into Pichia pastoris GS115 competent cells for expression.

Example 2 Determination of lipase activity

Under heating conditions at 50° C. in a water bath, 10 mL of isopropanol was used to dissolve 30 mg of p-nitrophenol palmitate pNPP, and then 90 mL of 0.01 M sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution at pH = 8 was added to prepare a substrate solution. 1 mL of the substrate solution was added to 1 mL of the crude enzyme solution of the lipase mutant or the crude enzyme solution of the wild-type lipase obtained in Example 1, and 1 mL of the substrate solution was added to 1 mL of blank culture medium as a control. The reaction was kept in a water bath at 50° C. for 10 min, and 1 mL of acetone was added to terminate the reaction.

Lipase hydrolyzes pNPP to release p-nitrophenol pNP, and the p-nitrophenol solution is yellow-green under alkaline conditions, as shown in Figure 1. Use 0.01M, pH=8 PBS sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution to dissolve p-nitrophenol pNP, prepare solutions of different concentrations, test the absorbance to draw a standard curve, and calculate the enzyme activity based on the standard curve, as shown in Figure 2. Enzyme activity is defined as: 1 μmol of p-nitrophenol is released by hydrolyzing p-nitrophenol palmitate for every 1 mg of lipase in 1 minute, which is 1 enzyme activity unit U, μmol/mg·min.

The results are shown in Figure 3. The activity of the TLL291/A113G lipase mutant was significantly improved compared to the original wild-type lipase at different substrate concentrations. At a substrate concentration of 1.5 mM, the activity of the mutant lipase was increased by 98.1%.

Example 3 Determination of thermal stability of lipase

The enzyme solution was incubated in a 50°C water bath, and the time t ₅₀ ^1/2 when the lipase lost 50% of its activity was determined. The specific determination method is as follows: the wild-type lipase and the mutant lipase obtained in Example 1 were incubated at 50°C, and samples were taken at different treatment times. The activity at 0 h of incubation was taken as 100%, and the pNPP method in Example 2 was used to determine and calculate the residual activity of the lipase.

The results are shown in FIG4 . The half-life t ₅₀ ^1/2 of the wild-type lipase is less than 0.5 h, while the lipase mutant TLL291/A113G/L74N still has 72% residual activity after 24 h of incubation.

Example 4 Determination of methanol and tert-butanol tolerance of lipase

It should be noted that when lipase catalyzes the preparation of biodiesel from oil, methanol is usually used as an acyl donor, and the molar ratio of methanol to oil in the reaction is often more than 4:1. Dimethyl carbonate can also be used as an acyl donor to undergo an ester exchange reaction with oil. At this time, some lipases need to add other organic solvents as reaction media to play a better catalytic effect. At the same time, organic solvents also play a solubilizing effect. Tert-butyl alcohol is a commonly used organic solubilizing agent.

60% methanol or 60% tert-butanol was added to the crude enzyme solution according to the volume ratio (i.e., 60 mL of methanol or tert-butanol was used for every 40 mL of crude enzyme solution), and then the solution was kept warm in a 50°C water bath. Samples were taken at different treatment times, and the activity at treatment time 0 h was taken as 100%. The residual activity of the lipase was determined by the pNPP method in Example 2.

The results are shown in Figures 5 and 6. The wild-type lipase completely lost its activity after being incubated in methanol or tert-butanol for 1 hour; while the lipase mutant TLL291/A113G/L74N still had 33.6% residual activity after being incubated in methanol for 24 hours, and was more likely to maintain stable activity in tert-butanol, with 85.8% of activity still detected after 12 hours of incubation and 38.6% remaining activity after 24 hours.

The above-mentioned determination experiments show that the lipase mutant constructed in Example 1 has higher activity and more superior thermal stability and organic solvent tolerance, and can be widely used in food processing, drug synthesis, leather production or papermaking industry, and has high commercial value.

It should be understood by those skilled in the art that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims (5)

1. A lipase mutant with high activity and high stability is characterized in that: the lipase mutant is obtained by mutating 113 th amino acid Ala in an amino acid sequence of thermomyces lanuginosus lipase into Gly and mutating 74 th amino acid Leu into Asn to form a specific sequence of Asn74-Tyr75-Ser76, and the amino acid sequence is SEQ ID NO. 4.

2. A lipase mutant with high activity and high stability is characterized in that: the amino acid sequence of the intermediate mutant formed by mutating the 113 rd amino acid Ala in the amino acid sequence of the thermomyces lanuginosus lipase into Gly is shown as SEQ ID NO. 2.

3. A nucleic acid molecule characterized in that: the sequence of which is shown in SEQ ID NO. 3, and which encodes an intermediate mutant according to claim 2.

4. A recombinant plasmid, characterized in that: comprising the nucleotide sequence shown as SEQ ID NO. 5.

5. A recombinant pichia pastoris genetic engineering bacterium is characterized in that: the genetically engineered bacterium comprises the recombinant plasmid disclosed in claim 4, and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 29367.