Alkaline lipase mutant, method for preparing same, use and product thereof

Abstract

The application provides a basic lipase mutant and a preparation method, application and product thereof, and belongs to the field of enzyme engineering. The TDLip sequence shown in the amino acid sequence such as SEQ ID NO. 1 is subjected to G66D, F147L, W226C and G245E mutations to obtain a mutant TDLip25, and the TDLip25 can still maintain more than 60% of enzyme activity at a lower temperature of 20-50 DEG C, so that the technical problems of unstable enzyme property and low enzyme activity of most low-temperature basic lipase products on the market are solved, and the TDLip25 has wide application value in industry, food and medicine and the like.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to an alkaline lipase mutant, its preparation method, application, and product. Background Technology

[0002] Lipase (EC 3.1.1.3), also known as triacylglycerol hydrolase, is an enzyme that breaks down or synthesizes triglyceride ester chains formed by higher fatty acids and glycerol. It is used to catalyze the decomposition, synthesis, and lipid exchange of lipid compounds and has high chemoselectivity and stereoisomerism.

[0003] Lipases are widely found in plants, animals, microorganisms, and the human body. In humans and animals, various lipases control processes such as digestion, absorption, fat reconstruction, and lipoprotein metabolism. In plants, lipases are found in energy storage tissues. Many types of microorganisms can produce a large number of extracellular lipases with different properties. These extracellular lipases are easier to extract and use, and can be produced on a large scale industrially. Therefore, lipases derived from microorganisms have been studied and applied more extensively.

[0004] Due to their excellent activity under low-temperature, alkaline conditions, low-temperature alkaline lipases are widely used in food, animal husbandry, aquaculture, pharmaceuticals, and leather processing. In the pharmaceutical industry, low-temperature alkaline lipases effectively increase the yield of chiral compounds in drug production, reduce the likelihood of side effects to some extent, decrease the energy required for synthesis, and ensure drug quality. In food processing, they prevent the degradation of quality and flavor caused by prolonged high temperatures. In household detergents, they are highly effective at removing lipid contaminants. In lipid processing, they save energy and ensure product quality. When treating lipid-containing waste, the additional heat required is low, safe, efficient, and does not cause secondary pollution, making it of great significance for environmental protection.

[0005] Most commercially available low-temperature alkaline lipase products are currently unstable in their enzymatic properties and have low enzyme activity, which to some extent restricts their industrial application. To obtain industrial-grade alkaline lipases with stable enzyme production characteristics, high enzyme activity, good stability, and high antioxidant properties, cultivating engineered bacteria using genetic engineering technology is an effective way to obtain high-yielding strains of low-temperature alkaline lipase.

[0006] Relevant patent documents retrieved:

[0007] This document, published in China (CN114958807A) on August 30, 2022, discloses an alkaline-resistant lipase mutant and its applications. The alkaline lipase mutant was obtained by changing alanine to valine at position 122 of the amino acid sequence of the lipase from Rhizopus strain ZF071. The optimal pH for this alkaline lipase mutant is 10.5, an increase of 0.5 compared to the original mutant. However, when used at pH less than 9, the relative enzyme activity is less than 75%, limiting its application range.

[0008] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects:

[0009] Most alkaline lipase products are enzymatically unstable. Evidence suggests that existing alkaline lipase mutants have a relative enzyme activity of less than 75% when used at pH less than 9. Summary of the Invention

[0010] The purpose of this invention is to provide:

[0011] An alkaline lipase mutant, its preparation method, application, and product, and related technologies, to solve the technical problems of most currently available low-temperature alkaline lipase products, such as unstable enzymatic properties and low enzyme activity, or combinations thereof.

[0012] Terminology Explanation:

[0013] Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0014] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0015] The definition of standard chemical terminology can be found in the reference "Enzyme Engineering", by Fang Jun, Science Press.

[0016] Unless otherwise specified, conventional methods within the scope of the art, such as plasmid extraction, PCR amplification, double enzyme digestion, vector ligation, and culture medium preparation, shall be used.

[0017] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0018] The term "alkaline lipase" as used in this article refers to a class of lipases (EC 3.1.1.3, also known as triacylglycerol acyl hydrolase) that can catalyze the decomposition, synthesis, and lipid exchange reactions of lipid compounds under alkaline conditions. It has high chemoselectivity and stereoisomerism and is widely found in animals, plants, and microorganisms.

[0019] The term "expression vector" as used in this article refers to a DNA molecule that can carry and express exogenous genes (such as nucleic acids encoding alkaline lipase mutants) in host cells. It typically contains elements such as promoters, terminators, and resistance genes. The Pichia pastoris expression vector pPIC9K used in this invention can carry the target nucleic acid and express alkaline lipase mutants in Pichia pastoris.

[0020] The term "host cell" as used in this article refers to a cell used to carry and express exogenous genes. In this invention, Pichia pastoris strain GS115 is selected as the host cell. After the recombinant expression vector carrying the target nucleic acid is transferred into the host cell, the alkaline lipase mutant can be synthesized and secreted by culturing the host cell, which facilitates the subsequent recovery and purification of the enzyme.

[0021] The term "enzyme activity" as used in this article refers to an indicator that measures the catalytic ability of an enzyme. In this invention, one unit of enzyme activity (U / g or U / mL) is defined as "the production of 1 μmol of titratable fatty acid by hydrolyzing the substrate within 1 minute under the conditions of 40°C and pH 7.5" to evaluate the catalytic efficiency of alkaline lipase mutants.

[0022] In a first aspect, the present invention provides: an alkaline lipase mutant.

[0023] The amino acid sequence of the alkaline lipase mutant is as follows: at least one mutation is made at positions 66, 147, 226 and 245 of SEQ ID NO.1.

[0024] SEQ ID NO.1:

[0025] SPVRREVSQDLCDQFNLFAQYSAAAYCAKNNDAPAGANVTCSENVCPEVEKADATFLYSFEDSGVGDVTGFLALDNTNRLIVLSFRGSRSLENWIGNINLDLKGIDDICSGCKGHDGFTSSWRSVANTLTQQVQNAV REHPDYRVVFTGHSLGALATVAGASLRGNGYDIDVFSYGAPRVGNRAFAVFLTAQTGGTLYRITHTNDIVPRLPPRELGYSHSSPEYWITSGTLVPVTCNDIVKVEGIDSTDGNNQPNTPDIAAHLWYFGLIGTCL.

[0026] The mutation at position 66 is G66D.

[0027] The mutation at position 147 is F147L.

[0028] The mutation at position 226 is W226C.

[0029] The mutation at position 245 is G245E.

[0030] The preferred amino acid sequence of the alkaline lipase mutant is SEQ ID NO.2.

[0031] SEQ ID NO.2:

[0032] SPVRREVSQDLCDQFNLFAQYSAAAYCAKNNDAPAGANVTCSENVCPEVEKADATFLYSFEDSGVDDVTGFLALDNTNRLIVLSFRGSRSLENWIGNINLDLKGIDDICSGCKGHDGFTSSWRSVANTLTQQVQNAV REHPDYRVVLTGHSLGGALATVAGASLRGNGYDIDVFSYGAPRVGNRAFAVFLTAQTGGTLYRITHTNDIVPRLPPRELGYSHSSPEYCITSGTLVPVTCNDIVKVEEIDSTDGNNQPNTPDIAAHLWYFGLIGTCL.

[0033] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes:

[0034] A low-temperature alkaline lipase mutant, TDLip25, with its amino acid sequence shown in SEQ ID NO.2, exhibits an optimal pH of 9.0 and an optimal temperature of 40°C. It retains over 60% of its enzyme activity at 20-50°C, and after treatment at pH 6.0-11.0 for 60 minutes, the remaining enzyme activity reaches over 70%. The cationic surfactant CTAB significantly activates its enzyme activity, while the anionic surfactant SDS and nonionic surfactant Triton X-100 have no significant effect on enzyme activity. This technical solution solves the technical problems of unstable enzymatic properties and low enzyme activity in currently available low-temperature alkaline lipase products, while also improving its applicability across a wide pH range and in various surfactant environments.

[0035] Secondly, the present invention provides a nucleic acid that encodes any of the aforementioned alkaline lipase mutants.

[0036] The preferred nucleotide sequence of the alkaline lipase mutant is SEQ ID NO.3.

[0037] SEQ ID NO.3:

[0038] .

[0039] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the second aspect of the present invention includes:

[0040] A gene encoding a low-temperature alkaline lipase mutant, TDLip25. tdlip25Its nucleic acid sequence is shown in SEQ ID NO. 3. This technical solution addresses the problems of low enzyme activity and poor stability of existing low-temperature alkaline lipase gene expression products, further achieving compatibility between the gene sequence and the Pichia pastoris expression system. This ensures that the recombinant strain can efficiently synthesize lipases with the target activity, thereby improving industrial production efficiency.

[0041] Thirdly, the present invention provides an expression vector that expresses any of the above-mentioned alkaline lipase mutants and / or carries any of the above-mentioned nucleic acids.

[0042] The expression vector is either a eukaryotic expression vector or a prokaryotic expression vector.

[0043] The eukaryotic expression vectors include, but are not limited to, any one or more of the following: pPIC3, pPIC9, pYES2 / NT C, pFastBac, pEGFP-N1, pcDNA3.1, and pLVX.

[0044] The eukaryotic expression vector is preferably pPIC3 or pPIC9.

[0045] The eukaryotic expression vector is further preferably pPIC9K.

[0046] The prokaryotic expression vectors include, but are not limited to, any one or more of pMD19, pET28a, pET32a, pGEX-4T, pMAL-p2x, and pMAL-c2X.

[0047] The prokaryotic expression vector is preferably pMD19.

[0048] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the third aspect of the present invention includes:

[0049] A gene containing low-temperature alkaline lipase tdlip25 The recombinant expression vector has a backbone of Pichia pastoris expression vector pPIC9K, and the inserted gene is as described in the second aspect. tdlip25 (SEQ ID NO.3), via EcoRI and NotI double restriction sites tdlip25 Targeted insertion into the pPIC9K vector. This technical solution addresses the challenge of constructing vectors containing... tdlip25 Building upon existing gene recombination vector technology, we further utilized the strong promoter and secretion signal of the pPIC9K vector to achieve efficient secretory expression of TDLip25 in Pichia pastoris, facilitating subsequent enzyme recovery and purification.

[0050] Fourthly, the present invention provides a cell comprising any of the alkaline lipase mutants, nucleic acids, or expression vectors described above.

[0051] The cells in question are eukaryotic or prokaryotic cells.

[0052] The eukaryotic cells include, but are not limited to, any one or more of yeast, filamentous fungi, insect cells, mammalian cells, or plant cells.

[0053] The eukaryotic cells are preferably yeast.

[0054] The yeast is further preferably one or more of Pichia pastoris, brewer's yeast, or polymorphonuclear yeast.

[0055] The prokaryotic cells include, but are not limited to, any one or more of the following: Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, Corynebacterium glutamicum, thermophilic bacteria, cyanobacteria, or halophilic bacteria.

[0056] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the fourth aspect of the present invention includes:

[0057] A gene containing low-temperature alkaline lipase tdlip25 The host cell, the host strain is Pichia pastoris GS115, and the vector introduced is the recombinant expression vector described in the third aspect ( pPIC9K - tdlip25 This technical solution addresses the technical challenges of constructing recombinant strains capable of expressing TDLip25. Furthermore, by utilizing the eukaryotic expression system of Pichia pastoris GS115, it reduces protein misfolding, ensuring that the expressed TDLip25 possesses complete enzymatic activity. Simultaneously, the strain can efficiently produce enzymes after culturing at 28-32℃ and 180-220rpm for 110-130h, making it suitable for industrial-scale cultivation.

[0058] Fifthly, the present invention provides a method for preparing any of the above-mentioned alkaline lipase mutants, comprising the following steps:

[0059] (1) Transform any of the above expression vectors into host cells to obtain recombinant strains;

[0060] (2) Cultivate recombinant strains;

[0061] (3) Recover and purify alkaline lipase mutants.

[0062] In step (1), the host cell includes, but is not limited to, any one or more of Pichia pastoris, brewer's yeast, or polymorphonuclear yeast.

[0063] In step (2), the conditions for culturing the recombinant strain are 28-32℃ and 180-220rpm shaking culture for 110-130h.

[0064] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the fifth aspect of the present invention includes:

[0065] A method for preparing low-temperature alkaline lipase TDLip25 includes the following steps: (1) using the recombinant expression vector described in the third aspect ( pPIC9K-tdlip25 The recombinant strain was transferred into Pichia pastoris GS115 host cells and screened to obtain the recombinant strain. GS115 / tdlip25 (2) The recombinant strain was inoculated into BMGY medium and cultured at 30℃ and 200 rpm for 48 h with shaking. Then it was transferred to BMMY medium and induced to culture at 30℃ and 200 rpm for 72 h with shaking. (3) The culture supernatant was collected by centrifugation and TDLip25 was recovered and purified by salting out and ion exchange chromatography. This technical solution solved the technical problem of preparing TDLip25.

[0066] In a sixth aspect, the present invention provides the application of any of the above-mentioned alkaline lipase mutants, nucleic acids, or expression vectors in food, animal husbandry, aquatic products, detergents, pharmaceuticals, and / or leather processing.

[0067] In a seventh aspect, the present invention provides a product comprising any of the above-described alkaline lipase mutants, any of the above-described nucleic acids, or any of the above-described expression vectors.

[0068] The products include food, feed additives, detergents, or pharmaceutical compositions.

[0069] Compared with the prior art, the present invention has the following advantages:

[0070] 1. The alkaline lipase mutant provided by this invention has stable properties, high activity, good stability, and high antioxidant properties. It maintains more than 60% of enzyme activity at 20-50℃. The cationic surfactant CTAB can significantly activate enzyme activity, while the anionic surfactant SDS and the nonionic surfactant Triton X-100 have no significant effect on enzyme activity. Attached Figure Description

[0071] Figure 1 The optimal pH for the low-temperature alkaline lipase mutant.

[0072] Figure 2 pH stability of the low-temperature alkaline lipase mutant.

[0073] Figure 3This is the optimal temperature for the low-temperature alkaline lipase mutant.

[0074] Figure 4 The effect of surfactants on the activity of low-temperature alkaline lipase. Detailed Implementation

[0075] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0076] 1. Strains and vectors: The low-temperature alkaline lipase mutant gene of this invention tdlip25 The Pichia pastoris expression vector was synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd. pPIC9K and strains GS115 Purchased from Invitrogen.

[0077] 2. Enzymes and other biochemical reagents: Endonucleases were purchased from TaKaRa, ligases from Invitrogen, and polyvinyl alcohol from Sigma. All other reagents were domestically produced (available from general biochemical reagent companies).

[0078] 3. Culture medium:

[0079] (1) Yeast culture medium YPD: 1% peptone, 0.5% yeast extract, 1% glucose, 2% agar, pH 7.0.

[0080] (2) Escherichia coli culture medium LB: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0.

[0081] (3) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (V / V).

[0082] (4) BMMY medium: except that 0.5% methanol is used instead of glycerol, the other components are the same as those of BMGY.

[0083] Note: Molecular biology experimental methods not specifically described in the following examples were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd Edition), or according to the kit and product instructions.

[0084] Example 1: DuPont thermophile ( Thermomyces dupontii The low-temperature alkaline lipase mutant encoding gene tdlip25 Synthesis

[0085] This invention utilizes alkaline lipase derived from DuPont thermophile. tdlip25 Using the gene as a reference, its sequence was mutated as follows (G66D, F147L, W226C, G245E), and insertions were made at the 5' and 3' ends of the mutated sequence, respectively. EcoR I and Not The sequence was sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for artificial gene synthesis after restriction enzyme sites were identified. The amino acid sequence of the artificially synthesized low-temperature alkaline lipase mutant is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.3.

[0086] Example 2: Low-Temperature Alkaline Lipase Gene tdlip25 Cloning

[0087] The synthesized gene vector was preserved as a puncture culture. The puncture culture was picked up with a sterile toothpick in a clean bench and placed in an LB shaker containing antibiotic Amp (working concentration: 100 μg / ml). It was cultured overnight at 37°C and 220 rpm. The next day, the gene-containing vector was extracted according to the instructions of the Kangwei Century Plasmid Extraction Kit PurePlasmid Mini Kit (CW0500).

[0088] Based on the low-temperature alkaline lipase gene sequence, the following primers were designed and synthesized:

[0089] P1 (SRQ ID NO.4):5'-CCGGAATTCCGGTCTCCAGTCAGACGTG-3';

[0090] P2 (SRQ ID NO.5): 5'-TTGCGGCCGCAATTACAAACAAGTACCA-3'.

[0091] PCR amplification was performed using the extracted vector as a template. The PCR reaction parameters were: denaturation at 94℃ for 5 min; followed by denaturation at 94℃ for 30 sec, annealing at 55℃ for 30 sec, extension at 72℃ for 2 min, and 30 cycles, followed by incubation at 72℃ for 10 min. A fragment of approximately 825 bp was obtained. This fragment was recovered, ligated into the pMD19 vector, and sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. The predicted protein molecular weight was 29.6 kDa.

[0092] Based on the nucleotide sequences obtained from sequencing, the DNAMan software was used to compare the obtained nucleotide sequences with... tdlip25 The sequences were compared and confirmed to be correct.

[0093] Example 3 Preparation of recombinant low-temperature alkaline lipase TDLip25

[0094] expression carrier pPIC9K Double enzyme digestion ( Eco R I+ Not I), and simultaneously the gene encoding low-temperature alkaline lipase. tdlip25 Double enzyme digestion ( Eco R I+ Not I) The gene fragment encoding mature low-temperature alkaline lipase and its expression vector were digested with enzymes. pPIC9K Linkage was performed to obtain a gene containing a low-temperature alkaline lipase. tdlip25 recombinant plasmid pPIC9K- TDLip25 And transform Pichia pastoris GS115 Recombinant Pichia pastoris strain was obtained GS115 / TDLip25 .

[0095] Take the sample containing the recombinant plasmid. GS115 Strains and control strains (i.e., unmutated strains) GS115 / TDLip25 The cells were inoculated into 300 mL of BMGY medium and cultured at 30°C with shaking at 200 rpm for 48 h. The cells were then collected by centrifugation. The cells were then resuspended in 150 mL of BMGY medium and cultured at 30°C with shaking at 200 rpm. After induction for 72 h, the supernatant was collected by centrifugation, and the activity of the low-temperature alkaline lipase was determined.

[0096] Example 4 Activity analysis of low-temperature alkaline lipase TDLip25

[0097] Lipase activity assay: The specific method is as follows: Take two 100 mL Erlenmeyer flasks, add 4 mL of substrate solution and 5 mL of phosphate buffer (pH 7.5) to the blank flask (A) and the sample flask (B) respectively, and then add 15 mL of 95% ethanol to flask A. Preheat in a water bath at 40℃±0.2℃ for 5 min. Then add 1 mL of the enzyme solution to be tested (appropriately diluted enzyme solution) to each of flasks A and B, mix well immediately, and count. After reacting accurately for 15 min, immediately add 15 mL of 95% ethanol to flask B to terminate the reaction and remove the flask. Add two drops of phenolphthalein indicator to flasks A and B, and titrate with sodium hydroxide standard solution until a faint red color remains for 30 s as the titration endpoint. Record the volume of sodium hydroxide standard solution consumed, and calculate the enzyme activity using the lipase preparation according to the formula. At 40°C and pH 7.5, 1 g of solid enzyme powder or 1 mL of liquid enzyme hydrolyzes the substrate for 1 min to produce 1 μmol of titratable fatty acid, which is defined as 1 unit of enzyme activity, expressed as U / g (U / ml).

[0098] Example 5: Determination of the properties of the low-temperature alkaline lipase TDLip25

[0099] The enzymatic properties of recombinant alkaline lipase TDLip25 and unmutated alkaline lipase TDLip were determined and compared. Simultaneously, the mutant low-temperature alkaline lipase TDLip32 was added for enzymatic property comparison. The TDLip32 sequence was modified from SEQ ID NO.1 by mutations of D13V, E193G, and G245E. The specific preparation methods for the TDLip25 mutant and the TDLip32 recombinant enzyme are as described in the above examples.

[0100] 1. The optimal pH and pH stability of recombinant alkaline lipase TDLip25 were determined as follows:

[0101] Purified lipases TDLip, TDLip25, and TDLip32 were subjected to enzymatic reactions at different pH levels to determine their optimal pH. Appropriately diluted enzyme solutions were subjected to enzymatic hydrolysis reactions at different pH conditions (2.0-12.0) to determine their optimal reaction pH. The buffers used were as follows: pH 2.0-3.0: 0.1 mol / L glycine-hydrochloric acid buffer; pH 4.0-8.0: 0.1 mol / L phosphate buffer; pH 9.0-12.0: 0.1 mol / L Tris-HCl buffer. The pH suitability of lipases in different pH buffer systems was measured at 40℃. Figure 1 The results show that the optimal pH for all three is 9.0, and the enzyme can maintain more than 80% of its activity within the pH range of 8.0-10.0.

[0102] Purified alkaline lipases TDLip, TDLip25, and TDLip32 were treated at 40°C for 60 min in various buffer solutions with different pH values, and then their activity was measured at 40°C in a pH 7.5 buffer system to study the pH tolerance of the enzymes. Results ( Figure 2 The results showed that the recombinant alkaline lipase TDLip25 was very stable between pH 6.0 and 11.0. After treatment within this pH range for 60 min, the remaining enzyme activity was more than 70%, which was significantly higher than that of TDLip and TDLip32. This indicates that the recombinant enzyme has good pH stability over a wide pH range.

[0103] 2. The optimal temperature for recombinant alkaline lipase TDLip25 is determined as follows:

[0104] The optimal temperature for alkaline lipase was determined by conducting enzymatic reactions in a pH 7.5 buffer system at different temperatures (10-70℃) for 30 min. Results of the optimal temperature determination for recombinant alkaline lipase TDLip25 were presented. Figure 3 This indicates that its optimal temperature is 40℃, and it maintains more than 60% enzyme activity at 20-50℃.

[0105] 3. The method for determining the effect of surfactant on enzyme activity of recombinant alkaline lipase TDLip25 is as follows:

[0106] The purified alkaline lipases TDLip, TDLip25, and TDLip32 were added to the above reaction system, followed by the addition of solutions of anionic surfactant (SDS), cationic surfactant (CTAB), and nonionic surfactant (Triton X-100), respectively, to a final concentration of 0.10%. The results were then measured. Figure 4 The cationic surfactant CTAB significantly activated enzyme activity, while the anionic surfactant SDS and the nonionic surfactant Triton X-100 had no significant effect on enzyme activity. This indicates that lipases have good tolerance to the tested surfactants and have potential for application in detergent formulations.

[0107] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A basic lipase mutant, characterized in that, The amino acid sequence of the alkaline lipase mutant is shown as SEQ ID NO.

2.

2. A nucleic acid, characterized in that, The nucleic acid encodes the alkaline lipase mutant of claim 1.

3. The nucleic acid of claim 2, wherein, The nucleotide sequence of the nucleic acid is shown as SEQ ID NO.

3.

4. An expression vector, characterized by, The expression vector expresses the alkaline lipase mutant of claim 1 and / or carries the nucleic acid of any one of claims 2-3.

5. A cell, comprising: The cell comprises the alkaline lipase mutant of claim 1 or the nucleic acid of any one of claims 2-3 or the expression vector of claim 4.

6. The method of producing the basic lipase mutant according to claim 1, characterized by, The method comprises the following steps: (1) transforming the expression vector into host cells to obtain recombinant strains; (2) culturing the recombinant strains; (3) recovering and purifying the alkaline lipase mutant.

7. The alkaline lipase mutant of claim 1 or the nucleic acid of any one of claims 2-3 or the expression vector of claim 4 or the cell of claim 5 is used in food, livestock, aquaculture, detergent and / or leather processing.

8. A product characterized by, The product comprises the alkaline lipase mutant of claim 1 or the nucleic acid of any one of claims 2-3 or the expression vector of claim 4 or the cell of claim 5.

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