A lipase mutant and its application

Abstract

The present invention discloses a lipase mutant and its application. By mutating lipase ROL_10x, the present invention obtains a mutant having significantly higher residual enzyme activity at high temperature (60°C) than lipase ROL_10x. This mutant is designated 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 lipase mutant G22I also exhibits a significantly increased half-life at different temperatures (45°C and 65°C) and a significantly increased specific enzyme activity. This mutant can be used in esterification reactions and ester hydrolysis reactions that require higher temperatures, thus benefiting 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, enzyme temperature tolerance is a key performance indicator. Enzymes with good temperature tolerance exhibit enhanced kinetic and operational stability at high temperatures. This not only facilitates the efficient recycling of biocatalysts and reduces the amount of enzyme required during the reaction, but also aids substrate dissolution, accelerates reactions, and reduces the risk of microbial contamination. Furthermore, enzymes with good temperature tolerance can reduce the loss of enzyme activity during long-term storage. Rhizopus oryzae lipase (ROL) exhibits 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 at high temperatures, resulting in decreased or even complete loss of enzyme activity, limiting its industrial application. For example, in the production of medium- and long-chain esters (MLCTs) using palmitic acid as a substrate, the melting point of this substrate is approximately 63°C, and current ROLs struggle to maintain enzyme stability at these elevated temperatures. Therefore, enhancing the temperature tolerance of this enzyme is of great significance and could pave the way for its further industrial application.

[0004] Molecular modification is an important method to improve enzymatic properties, but increasing 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 challenge. Although there are reports that lipases with higher temperature tolerance can be obtained by mutating the ROL gene site, their half-lives under high temperature (65°C) conditions are still not long enough, and most are less than 2 hours. In actual industrial applications, the longer the enzyme half-life, the better. In other words, 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 relatively 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 response to 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, transesterification reaction and / or acidolysis reaction.

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

[0016] By mutating lipase ROL_10x, the present invention obtained a mutant, designated lipase mutant G22I, that exhibits significantly higher residual enzyme activity at high temperature (60°C) than lipase ROL_10x. In addition to the increased residual enzyme activity, the mutant also exhibits significantly increased half-life at different temperatures (45°C and 65°C) and a significantly higher specific enzyme activity. Therefore, the present invention claims protection for this mutant lipase.

[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 option, the nucleotide sequence of the gene is shown in SEQ ID NO.4.

[0020] The present invention also claims protection for 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 protection for 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 protection for 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 protection for 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 protection for 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] By mutating lipase ROL_10x, the present invention obtained a mutant with significantly higher residual enzyme activity at high temperature (60°C) than ROL_10x. This mutant is designated as lipase mutant G22I, and its amino acid sequence is shown in SEQ ID NO. 3, and the nucleotide sequence of the corresponding encoding gene is shown in SEQ ID NO. 4. Compared to ROL_10x, in addition to increased residual enzyme activity, lipase mutant G22I also exhibits significantly increased half-life at different temperatures (45°C and 65°C) and higher specific enzyme activity. This mutant can be used in esterification and ester hydrolysis reactions that require higher temperatures, thus benefiting the development of industries such as food and chemical engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1This is an agarose gel electrophoresis diagram of the overexpression vectors of the six mutants obtained by PCR amplification. 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 These are the SDS-PAGE test results of the expressed recombinant proteins of 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 will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. 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 were commercially available.

[0040] Example 1 Obtaining lipase ROL_10x and its mutants

[0041] The present invention uses lipase ROL_10x as a template, and combining analysis and comparison results with the inventors' experience, we designed six different lipase mutants, designated 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 the gene encoding it is shown in SEQ ID NO. 2. The lipase mutant T18I refers to a mutation in which the amino acid T at position 18 of the lipase ROL_10x is mutated to I, and so on.

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

[0043] Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize the ROL_10x gene. EcoRⅠ and SalⅠ restriction sites were introduced at both ends of the gene, respectively. The gene was 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. Thus, using pPICZaA-ROL_10x as a template, an overexpression vector of the lipase mutant T18I, lipase mutant G22I, lipase mutant S61T, lipase mutant G105S, lipase mutant S144T, or lipase mutant T229I was obtained 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 each of upstream and downstream primers, and ddH2O added to 25 μL.

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

[0050] The overexpression vectors of the six mutants amplified were detected by agarose gel electrophoresis. Figure 1 As shown. Figure 1 It can be seen that 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 all have amplified bands, and the sizes are consistent with expectations, indicating that the amplification is successful.

[0051] The PCR amplification product was then digested with DnpI to obtain a digestion product. The digestion product was then transformed into E. coli TOP10 competent cells. The digestion product and E. coli TOP10 were gently mixed, incubated on ice for 30 minutes, heat-shocked in a 42°C water bath for 90 seconds, and immediately cooled on ice for 2 minutes. The cells were then added with 500 μL of LLB liquid medium (1% peptone, 0.5% yeast extract, 0.5% NaCl) and revived at 37°C at 200 rpm for 45 minutes. The bacterial suspension was spread onto LLB solid plates (1% peptone, 0.5% yeast extract, 0.5% NaCl, 1% agar powder) containing 25 μg / mL zeocin and incubated at 37°C for 12 hours. Single colonies were then 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 cultured 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] Mix 5 μg of the linearized recombinant plasmid with a Pichia pastoris X-33 competent cell, transfer to a pre-chilled electroporation cuvette, and then perform electroporation. Refer to the Invitrogen manual for Pichia pastoris competent cell preparation and plasmid electroporation.

[0055] After the electroporation is completed, immediately add 1 mL of 1 mol / L sorbitol solution, gently pipette to suspend the bacteria, transfer the bacterial solution from the electroporation cup to an EP tube, and incubate in a 30°C incubator for 60 minutes. Then centrifuge the bacterial solution, remove 800 μL of the supernatant, resuspend the bacteria, and draw 100 μL of the bacterial solution to spread on a YPD plate (2% peptone, 1% yeast extract, 2% glucose, 1% agar powder) containing 100 μg / mL Zeocin. Incubate at 30°C for 72 hours, then pick single colonies and identify them. 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 the pH value reached 0.8-1.0, it was inoculated (inoculation volume was 10%) into 400 mL BMMY medium (1% yeast extract, 2% peptone, 1.34% YNB, 1% methanol, 10% 1 M pH 6.0 phosphate buffer), and cultured in a shaker at 30°C and 250 rpm for 96 h, and 1% methanol was added every 24 h for induction.

[0057] After the culture is completed, the culture medium is centrifuged at 4°C and 7000 rpm / min for 40 minutes, and the supernatant is collected. It is then purified using a nickel column affinity chromatography gravity column with a nickel filler volume of 2 mL. The chromatography column is first equilibrated with 20 mL of buffer A (20 mM phosphate buffer containing 20 mM imidazole, pH 7.4), and then the supernatant is combined with the chromatography column. Impurities are eluted with 20 mL of buffer A, and the target protein is eluted with 15 mL of buffer B (20 mM phosphate buffer containing 300 mM imidazole, pH 7.4). An ultrafiltration tube is then used to further remove imidazole from the sample, and the recombinant protein sample is finally dissolved in 20 mM phosphate buffer (pH 7.4).

[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] 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 incubated in a 60°C water bath for 90 minutes, and 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 containing 10 μL of p-Nitrophenylcaprylate (4-nitrophenyl octanoate) (10 mM, dissolved in anhydrous ethanol) solution and 80 μL of 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 The residual enzyme activities of mutants G22I and ROL_10x were 83.2% and 32.5%, respectively. The residual enzyme activity of mutant G22I was 2.6 times that of ROL_10x. With the exception of mutant G22I, the residual enzyme activities of the remaining mutants were lower than or comparable to those of lipase ROL_10x. Therefore, only the temperature tolerance of mutant G22I was further tested. The amino acid sequence of mutant G22I is shown in SEQ ID NO. 3, and the nucleotide sequence of the gene encoding this 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 measured. Enzyme samples were incubated in water baths at 45°C and 65°C, respectively. 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 511.4 hours and 4.9 hours, respectively, representing 1.5 and 2.2 times the ROL_10x.

[0064] Table 2 Half-life at different temperatures

[0065]

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

[0067] Enzyme activity was determined using the olive oil titration method: First, an olive oil emulsion was prepared by mixing a 4% PVA (Chinese name) solution and olive oil (mass ratio 4:3). The mixture was then homogenized for 6 minutes using a high-speed homogenizer. 4 g of the olive oil emulsion and 5 mL of 20 mM phosphate buffer (pH 7.4) were added to a 100 mL Erlenmeyer flask and preheated at 30°C for 5 minutes. The enzyme sample was appropriately diluted with 20 mM phosphate buffer (pH 7.4). Then, 1 mL of the enzyme sample (experimental group) or 1 mL of 20 mM phosphate buffer (pH 7.4, control group) was added to the Erlenmeyer flask and incubated at 30°C for 10 minutes. The reaction was terminated by adding 15 mL of 95% ethanol. Free fatty acid release was determined by titration using 0.05 M sodium hydroxide solution using phenolphthalein solution as an indicator. The specific enzyme activity (U) was defined as the amount of enzyme required to generate 1 μmol of fatty acid per minute under these reaction conditions. The specific activity determination results are shown in Table 3. The specific activities of ROL 10x and G22I were 1328.2 U / mg and 1939.1 U / mg, respectively. Compared with ROL_10x, the specific activity of mutant G22I 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 considered as equivalent replacement methods and are included in the scope of protection 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 Containing 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 ester hydrolysis reaction.

8. 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 ester hydrolysis reaction.

Images