Lipase mutant and application thereof

Abstract

The invention discloses a lipase mutant and application thereof. According to the invention, lipase ROL10x is mutated to obtain a mutant, the residual enzyme activity of which is obviously higher than that of the lipase ROL10x at a high temperature (60 DEG C), the mutant is recorded as lipase mutant G22I, the amino acid sequence of the lipase mutant G22I is shown as SEQ ID NO.3, and the nucleotide sequence of a corresponding coding gene is shown as SEQ ID NO.4. The invention further discloses a preparation method of the lipase mutant G22I. Compared with the lipase ROL10x, the lipase mutant G22I has the advantages that the residual enzyme activity is increased, the half-life period of the lipase mutant G22I at different temperatures (45 DEG C and 65 DEG C) is also remarkably prolonged, the specific activity of the enzyme is also remarkably increased, and the lipase mutant G22I can be used for esterification reaction, ester hydrolysis reaction and the like which need to be carried out at a relatively high temperature and is beneficial to the development of industries such as food and chemical engineering.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to a lipase mutant and application thereof. Background Art

[0002] Lipase (EC 3.1.1.3) is a type of biocatalyst with multiple catalytic functions. It can carry out ester hydrolysis reactions and catalyze the hydrolysis of triglycerides into fatty acids and glycerol. In addition, it can also carry out esterification, acidolysis, alcoholysis and transesterification reactions. It has important application value in the food industry, detergent manufacturing, biodiesel production and medicine.

[0003] In industry, the temperature tolerance of enzymes is a key indicator. At high temperatures, enzymes with good temperature tolerance show higher kinetic stability and operational stability, which not only helps the effective recycling of biocatalysts and reduces the amount of enzyme added during the reaction, but also helps the substrate dissolve, accelerates the reaction rate and reduces the risk of microbial contamination. In addition, enzymes with good temperature tolerance can reduce the loss of enzyme activity during long-term storage. Rhizopus oryzae lipase (ROL) has great potential in specific industrial processes due to its unique substrate specificity and adaptability to reaction conditions. However, ROL has poor temperature tolerance, and its molecular structure is easily destroyed in high temperature environments, resulting in a decrease in enzyme activity or even complete loss, which limits its industrial application range. For example, in the production of medium- and long-chain esters (MLCT) using palmitic acid as a substrate, the melting point of the substrate is about 63°C, and the current ROL is difficult to maintain enzyme stability at this high temperature. Therefore, enhancing the temperature tolerance of the enzyme is of great significance and can lay the foundation for its further industrial application.

[0004] Molecular modification is an important method to improve enzymatic properties, but improving the temperature tolerance of enzymes by this method often results in a decrease in enzyme activity. How to improve the temperature tolerance of enzymes without sacrificing enzyme activity is currently a major technical problem. Although there are reports that lipases with higher temperature tolerance can be obtained by mutating the gene loci of ROL, their half-lives under high temperature (65°C) are still not long enough, and most are less than 2h, while in actual industrial applications, the longer the enzyme half-life, the better. That is, there is an urgent need for a lipase that has relatively higher enzyme activity and a longer half-life at high temperatures. However, the structure of ROL is complex and there are many mutation sites. It is difficult to design and screen a lipase with good temperature tolerance, and it is even more difficult to obtain a lipase with good temperature tolerance and high enzyme activity. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a lipase mutant and application thereof.

[0006] The first object of the present invention is to provide a lipase mutant.

[0007] The second object of the present invention is to provide a gene.

[0008] The third object of the present invention is to provide a recombinant vector.

[0009] The fourth object of the present invention is to provide a genetically engineered host cell.

[0010] A fifth object of the present invention is to provide a composition.

[0011] The sixth object of the present invention is to provide use of the lipase mutant, the host cell or the composition in an esterification reaction.

[0012] The seventh object of the present invention is to provide use of the lipase mutant, the host cell or the composition in the hydrolysis reaction of esters.

[0013] The eighth object of the present invention is to provide use of the lipase mutant, the host cell or the composition in transesterification reaction and / or acidolysis reaction.

[0014] The ninth object of the present invention is to provide the use of the lipase mutant, the gene, the recombinant vector, the host cell or the composition in the preparation of a preparation for esterification reaction, ester hydrolysis reaction, ester exchange reaction and / or acidolysis reaction.

[0015] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0016] The present invention obtains a mutant having a significantly higher residual enzyme activity at high temperature (60°C) than that of lipase ROL_10x by mutating lipase ROL_10x, which is recorded as lipase mutant G22I. In addition to the increased residual enzyme activity, the half-life of the lipase mutant at different temperatures (45°C and 65°C) is also significantly increased, and the specific activity of the enzyme is also significantly increased. Therefore, the present invention requests protection for the lipase mutant.

[0017] Specifically, the amino acid sequence of the mutant is shown in SEQ ID NO.3.

[0018] The present invention also claims protection for the gene encoding the lipase mutant.

[0019] As one of the options, the nucleotide sequence of the gene is shown as SEQ ID NO.4.

[0020] The present invention also claims to protect a recombinant vector.

[0021] Specifically, the recombinant vector contains the gene encoding the lipase mutant.

[0022] Specifically, the recombinant vector includes a recombinant expression vector.

[0023] Optionally, the vector used to construct the recombinant expression vector is pPICZaA.

[0024] The present invention also claims a genetically engineered host cell.

[0025] Specifically, the host cell expresses the lipase mutant, or contains the gene or the recombinant vector.

[0026] Optionally, the host cell is Pichia pastoris.

[0027] The present invention also claims a composition.

[0028] Specifically, it contains the lipase mutant or the host cell and auxiliary materials.

[0029] The present invention also claims to protect the use of the lipase mutant, the host cell or the composition in an esterification reaction.

[0030] The present invention also claims to protect the use of the lipase mutant, the host cell or the composition in the hydrolysis reaction of esters.

[0031] The present invention also claims to protect the use of the lipase mutant, the host cell or the composition in transesterification reaction and / or acidolysis reaction.

[0032] The present invention also claims to protect the use of the lipase mutant, the gene, the recombinant vector, the host cell or the composition in preparing a preparation for esterification reaction, ester hydrolysis reaction, transesterification reaction and / or acidolysis reaction.

[0033] The present invention has the following beneficial effects:

[0034] The present invention obtains a mutant having a significantly higher residual enzyme activity at high temperature (60°C) than that of lipase ROL_10x by mutating lipase ROL_10x, which is recorded as lipase mutant G22I, and its amino acid sequence is shown in SEQ ID NO.3, and the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO.4. Compared with lipase ROL_10x, in addition to the increased residual enzyme activity, the half-life of the lipase mutant G22I at different temperatures (45°C and 65°C) is also significantly increased, and the specific activity of the enzyme is also significantly increased, and it can be used for esterification reactions and ester hydrolysis reactions that need to be carried out at higher temperatures, which is beneficial to the development of industries such as food and chemical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1It is an agarose gel electrophoresis diagram of the overexpression vectors of the six mutants obtained by PCR amplification, and lanes 1 to 6 in the figure correspond to the overexpression vectors of lipase mutant T18I, lipase mutant G22I, lipase mutant S61T, lipase mutant G105S, lipase mutant S144T and lipase mutant T229I, respectively.

[0036] Figure 2 This is the SDS-PAGE test result of the recombinant proteins of the expressed lipase ROL_10x and the six mutants. In the figure, M is a molecular weight marker, and lanes 1 to 7 correspond to the recombinant proteins of lipase ROL_10x, lipase mutant T18I, lipase mutant G22I, lipase mutant S61T, lipase mutant G105S, lipase mutant S144T and lipase mutant T229I, respectively.

[0037] Figure 3 The residual enzyme activity test results of lipase ROL_10x and the six mutants after incubation at 60°C for 90 minutes. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0040] Example 1 Obtaining lipase ROL_10x and its mutants

[0041] The present invention uses lipase ROL_10x as a template, combines the analysis and comparison results and the inventor's experience, and designs 6 different lipase mutants, which are sequentially recorded as lipase mutant T18I, lipase mutant G22I, lipase mutant S61T, lipase mutant G105S, lipase mutant S144T and lipase mutant T229I. The amino acid sequence of the lipase ROL_10x is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2. The lipase mutant T18I refers to mutating the 18th amino acid T of the lipase ROL_10x to I, and so on.

[0042] In order to perform recombinant expression of lipase ROL_10x and the mutant, the present invention respectively constructed overexpression vectors of lipase ROL_10x and the mutant, and the process is as follows:

[0043] Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize the ROL_10x gene, and EcoRⅠ and SalⅠ restriction sites were introduced at both ends of the gene, which was then inserted into the vector pPICZaA through enzyme digestion to construct the ROL_10x overexpression vector pPICZaA-ROL_10x.

[0044] Based on the overexpression vector pPICZaA-ROL_10x, six pairs of primers, T18I-F / R, G22I-F / R, S61T-F / R, G105S-F / R, S144T-F / R, and T229I-F / R, were designed (the nucleotide sequences of the primers are shown in Table 1) to introduce mutation sites, thereby using pPICZaA-ROL_10x as a template and obtaining an overexpression vector of the lipase mutant T18I, the lipase mutant G22I, the lipase mutant S61T, the lipase mutant G105S, the lipase mutant S144T, or the lipase mutant T229I by PCR amplification.

[0045] Table 1 Primer sequences

[0046]

[0047]

[0048] The PCR amplification reaction system was as follows: 1 μL DNA template, 12.5 μL PrimeSTAR Max Premix (2×), 1 μL upstream and downstream primers, and 1 μL ddH2O added to 25 μL.

[0049] The reaction program was: 98°C for 3 min; 98°C for 10 s, 55°C for 30 s, 72°C for 5 min, 30 cycles; and extension at 72°C for 5 min.

[0050] Agarose gel electrophoresis was used to detect the overexpression vectors of the six mutants amplified. The results are as follows: Figure 1 As shown. Figure 1 It can be seen that there are amplified bands in the PCR amplification products of the overexpression vectors of lipase mutant T18I, lipase mutant G22I, lipase mutant S61T, lipase mutant G105S, lipase mutant S144T and lipase mutant T229I, and the sizes are consistent with expectations, indicating that the amplification is successful.

[0051] Then, the PCR amplification product was digested with DnpⅠ enzyme to obtain the digestion product. Subsequently, the digestion product was transferred into E. coli TOP10 competent cells, the digestion product and E. coli TOP10 were gently mixed, incubated on ice for 30 minutes, placed in a 42°C water bath for heat shock for 90 seconds, and then immediately placed on ice for 2 minutes, and then 500 μL LLB liquid medium (1% peptone, 0.5% yeast extract, 0.5% NaCl) was added, and the cells were revived at 37°C shaker at 200 rpm for 45 minutes. The bacterial liquid was spread on LLB solid plates (1% peptone, 0.5% yeast extract, 0.5% NaCl, 1% agar powder) containing 25 μg / mL Zeocin, and after culturing at 37°C for 12 hours, single colonies were selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0052] The correctly sequenced single colony was inoculated into LLB liquid culture medium containing 25 μg / mL Zeocin, and after culturing at 37°C, 200 rpm for 16 h, the recombinant plasmid was extracted to obtain a recombinant plasmid (overexpression vector) overexpressing lipase mutant T18I, lipase mutant G22I, lipase mutant S61T, lipase mutant G105S, lipase mutant S144T and lipase mutant T229I.

[0053] The recombinant plasmids of lipase ROL_10x and the obtained 6 mutants were linearized using PmeⅠ restriction endonuclease and then purified and recovered.

[0054] 5 μg of linearized recombinant plasmid was evenly mixed with Pichia pastoris X-33 competent cells, transferred to a pre-cooled electroporation cup, and then electroporated. The preparation of Pichia pastoris competent cells and electroporation of plasmids were referred to the Invitrogen manual.

[0055] After the electroporation, immediately add 1mL of 1mol / L sorbitol solution, gently blow and suspend the bacteria, transfer the bacterial solution from the electroporation cup to the EP tube, and incubate in a 30°C incubator for 60min. Then centrifuge the bacterial solution, remove 800μL of supernatant, resuspend the bacteria, and apply 100μL of bacterial solution on a YPD plate (2% peptone, 1% yeast extract, 2% glucose, 1% agar powder) containing 100μg / mL Zeocin, and culture at 30°C for 72h, then select single colonies and identify them, and select single colonies that have been identified as positive transformants for subsequent experiments.

[0056] The obtained single colony was inoculated into 50 mL of BMGY medium (1% yeast extract, 2% peptone, 1.34% YNB, 1% glycerol, 10% 1M pH 6.0 phosphate buffer) and cultured in a shaker at 30°C and 250 rpm. 600 When it reaches 0.8-1.0, inoculate it (inoculation volume is 10%) into 400 mL BMMY medium (1% yeast extract, 2% peptone, 1.34% YNB, 1% methanol, 10% 1M pH 6.0 phosphate buffer), and culture it in a shaker at 30°C and 250 rpm for 96 h, adding 1% methanol every 24 h for induction.

[0057] After the culture is completed, the culture solution is centrifuged at 4°C, 7000rpm / min for 40min, and the supernatant is collected. Then, a nickel column affinity chromatography gravity column is used for purification, and the volume of the nickel filler is 2mL. The chromatography column is first balanced with 20mL of buffer A (20mM phosphate buffer, containing 20mM imidazole, pH 7.4), and then the supernatant is combined with the chromatography column. Use 20mL buffer A to elute the impurities, and 15mL buffer B (20mM phosphate buffer, containing 300mM imidazole, pH 7.4) to elute the target protein. Then use an ultrafiltration tube to further remove the imidazole in the sample, and finally dissolve the recombinant protein sample in 20mM phosphate buffer (pH7.4).

[0058] The recombinant proteins of the expressed lipase ROL_10x and the six mutants were detected by 12% protein gel electrophoresis (SDS-PAGE). Figure 2 As shown, the recombinant proteins of lipase ROL_10x and the six mutants were successfully expressed, and the purity of the seven recombinant proteins met the requirements for subsequent determination of enzymatic properties.

[0059] Example 2 Determination of temperature tolerance of lipase mutants

[0060] The recombinant protein samples of lipase ROL_10x and six lipase mutants obtained in Example 1 were diluted with 20 mM phosphate buffer (pH 7.4) to a concentration of 0.2 mg / mL. The recombinant protein samples were placed in a 60° C. water bath and incubated for 90 min, and the residual enzyme activity was determined using the pNP colorimetric method.

[0061] The specific method of the pNP colorimetric method is as follows: first prepare a reaction mixture, the reaction mixture contains 10 μL p-Nitrophenylcaprylate (4-nitrophenyl octanoate) (10 mM, dissolved in anhydrous ethanol) solution and 80 μL reaction buffer (20 mM phosphate buffer, pH 7.4), then add 10 μL of recombinant protein sample to the reaction mixture and react at 30°C for 5 minutes; then add 100 μL of isopropanol to terminate the reaction, and measure its absorbance at a wavelength of 405 nm.

[0062] The residual enzyme activity test results of lipase ROL_10x and the six mutants are as follows Figure 3 As shown. Figure 3 It can be seen that the residual enzyme activities of mutants G22I and ROL_10x are 83.2% and 32.5% respectively, and the residual enzyme activity of mutant G22I is 2.6 times that of ROL_10x. Except for mutant G22I, the residual enzyme activities of the other mutations are lower than or not much different from lipase ROL_10x, so only the temperature tolerance of mutant G22I is further determined in the future. The amino acid sequence of the mutant G22I is shown in SEQ ID NO.3, and the nucleotide sequence of the gene encoding the mutant is shown in SEQ ID NO.4.

[0063] To further evaluate the temperature tolerance of the lipase mutant G22I, its half-life at 45°C and 65°C was determined. The enzyme samples were incubated in a water bath at 45°C and 65°C, respectively, and then the residual enzyme activity at different time points was measured, and the half-life was calculated. The results are shown in Table 2. The half-life of the lipase mutant G22I at 45°C and 65°C was 511.4h and 4.9h, respectively, which were 1.5 and 2.2 times of ROL_10x, respectively.

[0064] Table 2 Half-life at different temperatures

[0065]

[0066] Example 3 Determination of specific activity of lipase mutants

[0067] The enzyme activity was determined by olive oil titration method: first, olive oil emulsion was prepared, that is, 4% PVA (Chinese name) solution and olive oil (mass ratio of 4:3) were weighed and mixed, and then processed with a high-speed homogenizer for 6 minutes to make olive oil emulsion. 4g olive oil emulsion and 5mL 20mM phosphate buffer (pH 7.4) were added to a 100mL conical flask, and preheated at 30℃ for 5min; the enzyme sample was appropriately diluted with 20mM phosphate buffer (pH 7.4), and then 1mL enzyme sample (experimental group) or 1mL 20mM phosphate buffer (pH 7.4, control group) was added to the conical flask, and reacted at 30℃ for 10min; then 15mL 95% ethanol solution was added to terminate the reaction; phenolphthalein solution was used as an indicator, and 0.05M sodium hydroxide solution was used for titration to determine the release of free fatty acids. The enzyme specific activity U is defined as the amount of enzyme required to generate 1μmol fatty acid per minute under the reaction conditions. The results of specific activity determination are shown in Table 3. The specific activities of ROL 10x and G22I are 1328.2 U / mg and 1939.1 U / mg, respectively. Compared with ROL_10x, the specific activity of mutant G22I is increased by 46%.

[0068] Table 3 Specific activity determination results

[0069]

[0070] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

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

3.

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

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

4.

4. A recombinant vector, characterized in that: Contains the gene according to claim 2 or 3.

5. A genetically engineered host cell, characterized in that The host cell expresses the lipase mutant according to claim 1, or contains the gene according to claim 2 or 3, or the recombinant vector according to claim 4.

6. A composition, characterized in that Contains the lipase mutant according to claim 1 or the host cell according to claim 5 and auxiliary materials.

7. Use of the lipase mutant according to claim 1, the host cell according to claim 5 or the composition according to claim 6 in an esterification reaction.

8. Use of the lipase mutant according to claim 1, the host cell according to claim 5 or the composition according to claim 6 in the hydrolysis reaction of esters.

9. Use of the lipase mutant according to claim 1, the host cell according to claim 5 or the composition according to claim 6 in transesterification reaction and / or acidolysis reaction.

10. Use of the lipase mutant according to claim 1, the gene according to claim 2 or 3, the recombinant vector according to claim 4, the host cell according to claim 5 or the composition according to claim 6 in the preparation of a preparation for esterification reaction, ester hydrolysis reaction, transesterification reaction and / or acidolysis reaction.

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