A lipase mutant and its encoding gene, product and application

Abstract

The present invention belongs to the field of genetic engineering and enzyme engineering, and specifically relates to a lipase mutant and its encoding gene, product and application. The present invention obtains a lipase mutant LIPA5 by performing D77L, S164C and P184V mutations on the sequence shown in SEQ ID NO.1, and has an amino acid sequence as shown in SEQ ID NO.2. The thermal stability of the lipase mutant LIPA5 is significantly improved, and it can still maintain more than 60% enzyme activity at 75°C for 3min; the pH is treated between 4.0-10.0 for 1 h, and the stability is more than 60%. The lipase mutant LIPA5 of the present invention has the following properties: optimum pH 8.0, optimum temperature 50°C, good stability, and broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and enzyme engineering, and specifically relates to a lipase mutant and its encoding gene, product and application. Background Art

[0002] In the field of biomass conversion, lipase plays a vital role. It is a type of biocatalyst that can decompose triglycerides stored in organisms into fatty acids and glycerol. Traditional lipases, such as pancreatic lipase and gastric lipase, are mainly involved in the fat digestion process in organisms. However, in the field of biomass conversion, the application of lipases goes far beyond this. They can work with other enzymes to act on the complex lipid structures in plant and microbial cells.

[0003] As an important biocatalyst, lipase is gradually showing its potential in industrial applications. Its target is lipids such as triglycerides, which are structurally different from cell wall polysaccharides (such as cellulose and hemicellulose), but also exist in organisms and are important energy storage substances in organisms. Lipase can effectively decompose triglycerides into fatty acids and glycerol through catalysis. The products can not only be directly used as raw materials for biofuels (such as biodiesel), but can also be further converted into other high-value-added chemicals.

[0004] In modern industrial applications, the application scope of lipase is constantly expanding. It not only plays an important role in oil processing and food industry, but also shows its unique value in the fields of bioenergy, biomaterials and environmental remediation. For example, in the production process of biodiesel, lipase can catalyze the transesterification reaction between oil and methanol to produce fatty acid methyl esters (i.e. biodiesel) and glycerol. This process is not only efficient and environmentally friendly, but also can achieve sustainable utilization of oil resources.

[0005] At present, research has found that lipase can catalyze the hydrolysis, acidolysis, alcoholysis, aminolysis, transesterification and esterification synthesis reactions of ester substances, making lipase have a variety of industrial application values ​​in related industries such as food industry, feed industry, paper industry, textile industry and medicine. In the food industry, lipase can change the length of fatty acid chains in oils and fats through transesterification to obtain the required higher-value lipids, thereby improving the economic benefits and development potential of the oil industry. In feed preparation, adding lipase is beneficial to supplement the deficiency of endogenous digestive enzymes in animals at special physiological stages, improve the digestion of feed fat, relieve nutritional stress and promote animal growth. In the papermaking industry, with the assistance of cellulase and lignocellulase, treating pulp with lipase can remove substances such as resin and wax, avoid serious cracking of paper, not only reduce the amount of chemicals needed to treat resin, but also maintain the quality and output of paper. In the textile industry, lipase is mainly used for wool shrinkage prevention, silk degreasing and waxing, and improving the hydrophilicity of polyester, and has initially shown its application prospects. In the medical field, chiral compounds have important application value. Lipase can be used in the synthesis of chiral drugs and the separation of optical isomers.

[0006] With the continuous progress of biotechnology, researchers are improving lipases through various means. Patent CN119193537A provides a high-temperature resistant lipase mutant. Compared with the wild-type lipase RL, the heat resistance of the lipase mutant containing the V274T single-point mutation provided by the invention is significantly improved. Patent CN118956818B discloses a lipase mutant G266C that tolerates organic solvents. After incubation in the final organic solvent for 30 minutes, the residual enzyme activity is significantly improved compared to the wild-type Rhizopus oryzae lipase in terms of organic solvent tolerance, and its enzyme activity is better than that of the wild type. The above improvements not only help to improve the efficiency and product quality of existing industrial processes, but also provide possibilities for the development of new bioconversion technologies and products.

[0007] Therefore, the current research focus is to further improve lipase through genetic engineering, protein engineering, etc., optimize the structure and performance of lipase, and improve its catalytic efficiency, stability and substrate specificity. Summary of the invention

[0008] In view of the above-mentioned deficiencies, the present invention provides a lipase mutant and its encoding gene, product and application. The present invention obtains the lipase mutant LIPA5 by performing D77L, S164C and P184V mutations on the sequence shown in SEQ ID NO.1, and has an amino acid sequence as shown in SEQ ID NO.2. The thermal stability of the lipase mutant LIPA5 is significantly improved, and it can still maintain more than 60% enzyme activity at 75°C for 3 minutes; the stability is more than 60% after being treated at a pH between 4.0-10.0 for 1 hour. The lipase mutant LIPA5 of the present invention has the following properties: optimal pH 8.0, optimal temperature 50°C, good stability and broad application prospects.

[0009] The technical solution of the present invention includes:

[0010] In a first aspect, the present invention provides a lipase mutant, wherein the lipase mutant is the lipase mutant LIPA5, having an amino acid sequence as shown in SEQ ID NO.2.

[0011] SEQ ID NO.2:

[0012] KRVYTSTETSHIDQESYNFFEKYARLANIGYCVGPGTKIFKPFNCGLQCAHFPNVELIEEFHDPRLIFDVSGYLAVLHASKQIYLVIRGTHSLEDVITDIRIMQAPLTNFDLAANISSTATCDDCLVHNGFIQSYNNTYNQIGPKLDSVIEQ YPDYQIAVTGHCLGGAAALLFGINLKVNGHDVLVVTLGQPIVGNAGFANWVDKLFFGQENPDVSKVSKDRKLYRITHRGDIVPQVPFWDGYQHCSGEVFIDWPLIHPPLSNVVMCQGQSNKQCSAGNTLLQQVNVIGNHLQYFVTEGVCGI.

[0013] Specifically, the lipase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1.

[0014] SEQ ID NO.1:

[0015] KRVYTSTETSHIDQESYNFFEKYARLANIGYCVGPGTKIFKPFNCGLQCAHFPNVELIEEFHDPRLIFDVSGYLAVDHASKQIYLVIRGTHSLEDVITDIRIMQAPLTNFDLAANISSTATCDDCLVHNGFIQSYNNTYNQIGPKLDSVIEQ YPDYQIAVTGHSLGGAAALLFGINLKVNGHDPLVVTLGQPIVGNAGFANWVDKLFFGQENPDVSKVSKDRKLYRITHRGDIVPQVPFWDGYQHCSGEVFIDWPLIHPPLSNVVMCQGQSNKQCSAGNTLLQQVNVIGNHLQYFVTEGVCGI.

[0016] Preferably, the mutation is to mutate the aspartic acid at position 77 of SEQ ID NO.1 to leucine, the serine at position 164 to cysteine, and the proline at position 184 to valine.

[0017] In a second aspect, the present invention provides a coding gene, wherein the coding gene encodes the above-mentioned lipase mutant.

[0018] Specifically, the coding gene is lipa5 Gene.

[0019] Preferably, the encoding gene has a nucleotide sequence as shown in SEQ ID NO.3.

[0020] SEQ ID NO.3:

[0021] AAACGTGTATATACTTCCACCGAAACTTCACATATAGATCAAGAGAGTTACAACTTTTTCGAGAAGTATGCCAGGCTAGCAAACATTGGCTATTGTGTCGGACCAGGTACAAAGATTTTTAAGCCATTCAACTGTGGCTTGCAATGTGCTCATTTTCCAAACGTAGAACTAATTGAAGAGTTTCACGATCCCAGATTGATCTTTGATGTGTCAGGATATCTTGCTGTCTTGCATGCTAGTAAACAGATATATTTAGTGATACGTGGCACCCATTCACTGGAAGATGTGATCACTGACATTCGAATTATGCAGGCTCCCTTGACGAATTTTGATCTAGCTGCCAATATTAGTTCCACCGCTACTTGTGATGATTGCTTAGTGCATAACGGATTCATCCAATCATACAACAATACGTACAATCAAATCGGCCCTAAGTTGGACTCCGTGATTGAACAGTACCCAGATTACCAAATTGCAGTCACTGGACATTGTCTGGGAGGTGCTGCAGCTCTACTTTTCGGCATTAATTTGAAAGTCAACGGTCATGATGTGTTGGTCGTTACACTGGGCCAACCTATTGTTGGTAACGCCGGTTTCGCAAATTGGGTTGATAAGCTTTTTTTCGGCCAAGAAAACCCTGATGTGAGTAAAGTTTCAAAGGATAGAAAGCTTTACAGGATAACTCACAGAGGTGATATAGTGCCACAAGTACCTTTCTGGGATGGTTATCAACATTGTTCCGGTGAGGTTTTTATTGACTGGCCTCTAATTCACCCTCCTCTTTCCAACGTCGTTATGTGTCAAGGTCAATCTAACAAACAGTGTTCCGCCGGCAACACACTGTTGCAGCAAGTTAACGTGATCGGAAACCATCTGCAATATTTTGTCACAGAAGGTGTGTGTGGAATT。

[0022] In a third aspect, the present invention provides a genetic engineering vector containing the above-mentioned coding gene.

[0023] Specifically, the genetic engineering vector is constructed by fusing the above coding gene with an expression vector.

[0024] Preferably, the expression vector is selected from one or more of pPIC9K, pET-28a, pEZZ18, pTA1529, pINIII-ompA, pUB110, pE194, pUCX05-bgaB, pHT304, pMK3, pPIC9, pHIL-S1, pPICZα, pYAM75P, and PNZ8149-usp45.

[0025] More preferably, the expression vector is pPIC9K.

[0026] In a fourth aspect, the present invention provides a genetically engineered cell containing the above-mentioned genetically engineered vector.

[0027] Specifically, the genetically engineered cells are obtained by transforming genetically engineered vectors into host cells.

[0028] Preferably, the host cell comprises a Pichia pastoris cell, a Saccharomyces cerevisiae cell or a Pseudomonas polymorpha cell.

[0029] More preferably, the host cell is Pichia pastoris.

[0030] More preferably, the host cell is Pichia pastoris GS115.

[0031] In a fifth aspect, the present invention provides a genetically engineered preparation, which comprises one or more of the above-mentioned genetically engineered cell culture, culture extract, cell disruptor, bacterial body, fermentation broth, fermentation broth precipitate, and lyophilized powder.

[0032] In a sixth aspect, the present invention provides the use of the above-mentioned lipase mutant, encoding gene, genetic engineering vector, genetic engineering cell or genetic engineering preparation in food production, feed production, pharmaceutical production, oil processing, pulp and paper making or textile.

[0033] The beneficial effects of the present invention are:

[0034] The thermal stability of the lipase mutant LIPA5 provided by the present invention is significantly improved. At 75°C for 3 minutes, it can still maintain more than 60% enzyme activity; after treatment for 1 hour at a pH between 4.0-10.0, the stability is more than 60%. The lipase mutant LIPA5 of the present invention has the following properties: optimal pH 8.0, optimal temperature 50°C, good stability, and broad application prospects.

[0035] The lipase mutant LIPA5 provided by the present invention can significantly reduce or eliminate the anti-nutritional effects caused by fat deposition in the feed industry by effectively decomposing fat in the feed, thereby significantly improving the growth efficiency and production capacity of animals. In the process of brewing beverages and alcoholic beverages, lipase, with its unique catalytic ability, effectively reduces the fat content in the wort, significantly improves the filtration efficiency of the fermentation liquid, and ensures the clarity and pure flavor of the product. In the field of biomass conversion, lipase, with its excellent catalytic performance, significantly improves the efficiency and stability of the cellulose decomposition process, injecting new vitality into the production of biofuels and the development of biochemical products. It not only promotes the optimization and upgrading of existing industrial processes, but also opens up a broad road for exploring new bioconversion technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the optimal pH of the recombinant lipase mutant.

[0037] Figure 2 pH stability of the recombinant lipase mutants.

[0038] Figure 3 is the optimal temperature of the recombinant lipase mutant.

[0039] Figure 4 The thermal stability of recombinant lipase mutants. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0041] 1. Strains and vectors: The lipase genes lip5, lipa5, and lipa5l of the present invention were synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd., and the Pichia pastoris expression vector pPIC9K and strain GS115 were purchased from Invitrogen.

[0042] 2. Enzymes and other biochemical reagents: Endonucleases were purchased from TaKaRa, ligases were purchased from Invitrogen, and the others were domestic reagents that can be purchased from common biochemical reagent companies.

[0043] 3. Culture medium:

[0044] (1) Yeast medium YPD: 1% peptone, 0.5% yeast extract, 1% glucose, 2% agar, pH 7.0.

[0045] (2) Escherichia coli culture medium LB: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0.

[0046] (3) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, and 1% glycerol.

[0047] (4) BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, and 0.5% methanol.

[0048] Note: The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods listed in the book Molecular Cloning Experiment Guide (3rd Edition) by J. Sambrook, or according to the kits and product instructions.

[0049] Example 1 Yarrowia sp. Yarrowia ) Lipase encoding gene lipa5 Synthesis

[0050] The present invention uses lipase from Yarrowia yeast lip5 The gene was used as a reference, and its sequence (SEQ ID NO.1) was mutated to D77L, S164C, and P184V, and the 5' and 3' ends of the mutated sequence were added respectively. EcoR I and Not I The restriction enzyme cutting site was removed and the sequence was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for artificial gene synthesis. The amino acid sequence of the artificially synthesized lipase mutant LIPA5 is shown in SEQ ID NO.2. lipa5 The gene nucleotide sequence is shown as SEQ ID NO.3.

[0051] Example 2 Lipase gene lipa5 Clone

[0052] The synthesized target gene lipa5 The vector was stored in the form of puncture bacteria. The puncture bacteria were picked up with a sterile toothpick in the clean bench and placed in an LB shaking tube containing Kan (working concentration: 100 μg / ml) antibiotics. The tubes were cultured overnight at 37°C and 220 rpm. The next day, the vector containing the gene was extracted according to the instructions of the PurePlasmid Mini Kit (CW0500).

[0053] According to the lipase gene sequence, the following primers were designed and synthesized:

[0054] P1 (SEQ ID NO.4):

[0055] CCGGAATTCAAGCGTGTCTACACCTCTACCGA;

[0056] P2 (SEQ ID NO.5):

[0057] TAAAGCGGCCGCAAATTTAAATACCACAGACACCCTCGG.

[0058] The extracted vector was used as a template for PCR amplification. The PCR reaction parameters were: denaturation at 94°C for 5 min; then denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1 min, and 32 cycles followed by incubation at 72°C for 10 min. A fragment of about 912 bp was obtained, which was recovered and connected to the pMD19 vector and sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The predicted protein molecular weight was 33.7 kDa.

[0059] According to the nucleotide sequence obtained by sequencing, the obtained nucleotide sequence was compared with lipa5 The sequences were compared and confirmed to be correct.

[0060] Example 3 Preparation of recombinant lipase LIPA5

[0061] The expression vector pPIC9K Double enzyme digestion ( EcoR I+Not I ), while the gene encoding the lipase mutant LIPA5 lipa5 (SEQ ID NO.3) double enzyme digestion ( EcoR I+Not I ), enzyme cut out the gene fragment encoding lipase and the expression vector pPIC9K Ligation to obtain lipase gene lipa5 Recombinant plasmid pPIC9K-lipa5 Pichia pastoris GS115 , obtain recombinant Pichia pastoris strains GS115 / LIPA5 .

[0062] Recombinant Pichia pastoris strain GS115 / LIPA5 Compared with the control strain (i.e., the non-mutated strain GS115 / LIP5 , the preparation method is the same as GS115 / LIPA5 ), inoculated in 300 mL BMGY culture medium, cultured at 30°C 200 rpm for 48 h, and then centrifuged to collect the cells. Then resuspended in 150 mL BMMY medium, cultured at 30°C 200 rpm. After 72 h of induction, the supernatant was collected by centrifugation to obtain the recombinant lipase LIPA5 and the non-mutated lipase LIP5, and the lipase activity was measured.

[0063] Example 4 Activity analysis of recombinant lipase LIPA5

[0064] The method for determining lipase activity is as follows: 4 mL of olive oil emulsion is used as substrate, 5 mL of Tris-HCl (pH = 7.0, 50 mM) is used as buffer, 1 mL of appropriately diluted fermentation supernatant is added, and the reaction is carried out at 40 ° C for 15 minutes, then taken out and placed on ice and 15 mL of 95% ethanol is added to terminate the reaction. 30 μl of phenolphthalein is added to each bottle of the sample group and the control group as an indicator, and titrated with 50 mmol / L NaOH standard solution. When the reaction solution turns red, it is judged to have reached the titration endpoint; the lipase activity is calculated by the volume difference of the NaOH standard solution consumed by the sample group and the control group. 1 mL of liquid enzyme hydrolyzes the substrate under the above temperature and pH conditions for 1 minute to produce 1 μmol of titratable fatty acid, which is one unit of enzyme activity.

[0065] The measurement results showed that the expression level of the recombinant lipase LIPA5 was 673U / mL; the expression level of the unmutated lipase LIP5 was 580U / mL.

[0066] Example 5 Determination of the optimal pH and pH stability of recombinant lipase LIPA5

[0067] The enzymatic properties of the recombinant lipase LIPA5 and the non-mutated lipase LIP5 were measured and compared. At the same time, the mutant recombinant lipase LIPA5L was added for enzymatic property comparison. The recombinant lipase LIPA5L was mutated with H229L on the basis of SEQ ID NO.1 and had an amino acid sequence as shown in SEQ ID NO.6. The specific preparation methods of the LIPA5 mutant and the recombinant enzyme LIPA5L are as described in the above examples.

[0068] SEQ ID NO.6:

[0069] KRVYTSTETSHIDQESYNFFEKYARLANIGYCVGPGTKIFKPFNCGLQCAHFPNVELIEEFHDPRLIFDVSGYLAVDHASKQIYLVIRGTHSLEDVITDIRIMQAPLTNFDLAANISSTATCDDCLVHNGFIQSYNNTYNQIGPKLDSVIEQ YPDYQIAVTGHSLGGAAALLFGINLKVNGHDPLVVTLGQPIVGNAGFANWVDKLFFGQENPDVSKVSKDRKLYRITLRGDIVPQVPFWDGYQHCSGEVFIDWPLIHPPLSNVVMCQGQSNKQCSAGNTLLQQVNVIGNHLQYFVTEGVCGI.

[0070] 1. Optimal pH determination

[0071] Purified non-mutated lipase LIP5, recombinant lipase LIPA5, and recombinant lipase LIPA5L were subjected to enzymatic reaction at 37°C for 15 min at different pH values ​​to determine their optimal pH. The enzyme activity of lipase at pH = 6.5-8.0 (phosphate buffer), pH = 8.0-9.5 (Tris-Hcl buffer), and pH = 9.5-11.0 (glycine-sodium hydroxide buffer) was determined by alkaline titration to explore its optimal reaction pH.

[0072] The results of the test are as follows Figure 1 As shown in the figure, the optimum pH of the unmutated lipase LIP5, recombinant lipase LIPA5, and recombinant lipase LIPA5L is 8.0. Among them, the recombinant enzyme LIPA5 has more than 50% relative enzyme activity at pH 4.0-9.0, while the unmutated lipase LIP5 and recombinant lipase LIPA5L have less than 50% enzyme activity at pH 4.0, indicating that the pH application range of the recombinant enzyme LIPA5 has been significantly improved.

[0073] 2. Determination of pH stability

[0074] Purified non-mutated lipase LIP5, recombinant lipase LIPA5, and recombinant lipase LIPA5L were treated at pH = 6.5-8.0 (phosphate buffer), pH = 8.0-9.5 (Tris-Hcl buffer), or pH = 9.5-11.0 (glycine-sodium hydroxide buffer) at 37°C for 1 h, and then the enzyme activity was measured in a pH 8.0 buffer system at 37°C to study the pH tolerance of the enzyme.

[0075] The results are as follows Figure 2 As shown in the figure, the recombinant lipase LIPA5 is very stable between pH 4.0-10.0. After treatment for 1 hour in this pH range, the remaining enzyme activity is above 60%, and the relative enzyme activity is significantly improved compared with LIP5 and LIPA5, which indicates that the recombinant enzyme has good pH stability in a wider pH range.

[0076] Example 6 Determination of the optimum temperature and thermal stability of recombinant lipase LIPA5

[0077] 1. Determination of optimum temperature

[0078] Purified non-mutated lipase LIP5, recombinant lipase LIPA5, and recombinant lipase LIPA5L were reacted at different temperatures for 15 min in a phosphate buffer system at pH = 8.0, and the enzyme activity was determined.

[0079] The results of the optimum temperature determination are as follows Figure 3As shown, the results showed that the optimum temperature of the unmutated lipase LIP5, recombinant lipase LIPA5, and recombinant lipase LIPA5L was 50°C, and the recombinant lipase LIPA5 maintained a high enzyme activity at 20-60°C.

[0080] 2. Thermal stability determination

[0081] The temperature resistance test was as follows: purified non-mutated lipase LIP5, recombinant lipase LIPA5, and recombinant lipase LIPA5L were treated at 75°C for 3 min in a phosphate buffer solution pH=8.0 system, and then the enzyme activity was tested at 50°C.

[0082] The results of the enzyme thermal stability test are as follows Figure 4 As shown in the figure, the recombinant lipase LIPA5 has good thermal stability and can maintain more than 60% of the enzyme activity after incubation at 75°C for 3 minutes. The remaining enzyme activity of the unmutated LIP5 is less than 10%, and the recombinant lipase LIPA5L only has 30%. It can be seen that the thermal stability of LIPA5 has been significantly improved and it has good heat resistance.

[0083] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A lipase mutant, characterized in that: The lipase mutant is the lipase mutant LIPA5, and the amino acid sequence is shown in SEQ ID NO.

2.

2. A coding gene, characterized in that The coding gene encodes the lipase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The coding gene is lipa5 The gene, the nucleotide sequence is shown in SEQ ID NO.

3.

4. A genetic engineering vector containing the coding gene according to any one of claims 2 to 3.

5. A genetically engineered cell containing the genetically engineered vector according to claim 4, characterized in that: The genetically engineered cells are obtained by transforming the genetically engineered vector into a host cell; the host cell is selected from Pichia pastoris cells, Saccharomyces cerevisiae cells or Pseudomonas polymorpha cells.

6. A genetic engineering preparation, characterized in that: The genetically engineered preparation comprises the fermentation broth of the genetically engineered cells according to claim 5.

7. A method for preparing a lipase mutant, characterized in that: The preparation method comprises: culturing the genetically engineered cells according to claim 5.

8. Use of the lipase mutant according to claim 1, the encoding gene according to any one of claims 2-3, the genetic engineering vector according to claim 4, the genetic engineering cell according to claim 5 or the genetic engineering preparation according to claim 6 in food production, feed production, oil processing, papermaking or textiles.

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