High-temperature-resistant high-activity lipase mutant and application thereof

Abstract

The application discloses a high-temperature-resistant high-activity lipase mutant and application thereof, wherein the mutant is obtained by substituting at least one amino acid in three positions of 25th, 242nd and 283rd of the amino acid sequence shown in SEQ ID No. 1; the threonine in the 25th position is mutated into methionine, the serine in the 242nd position is mutated into glycine or alanine, and the aspartic acid in the 283rd position is mutated into proline. The application obtains the mutant by mutating the wild-type lipase, and the mutant can efficiently catalyze the generation of p-nitrophenol from p-nitrophenol palmitate, and the mutant shows significantly enhanced resistance under high-temperature conditions, thereby improving the high-temperature catalytic efficiency and effectively expanding the application range of the lipase under high-temperature conditions.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and genetic engineering, and in particular to a high-temperature resistant, highly active lipase mutant and its applications. Background Technology

[0002] Thermosensitive lipases are a class of biocatalysts that maintain high activity and stability at high temperatures (typically above 60 °C). They are primarily derived from thermophilic microorganisms (such as *Thermomyces lanuginosus* and *Pyrococcus furiosus*) or obtained through protein engineering. Their thermostability stems from optimization of their molecular structure, such as enhancing hydrophobic interactions, increasing salt bridges and disulfide bonds, thereby maintaining conformational stability at high temperatures. These enzymes have wide applications in many fields, including food processing, textile industry, and medical testing. However, industrially produced lipases generally suffer from insufficient tolerance to high temperatures. Currently, their stability is continuously optimized through chemical modification and protein engineering, but bottlenecks such as low heterologous expression efficiency and high purification costs still restrict their industrialization process.

[0003] Directed evolution technology, by simulating natural evolutionary mechanisms, iteratively optimizes enzyme genes and has been successfully applied to modify the thermal stability and pH tolerance of enzymes. However, in terms of improving high-temperature performance, due to the complex flexible regulation mechanism of enzyme conformation, mutants with both high catalytic activity and excellent high-temperature tolerance are still relatively scarce.

[0004] Therefore, developing lipase mutants with high catalytic activity, strong high-temperature stability, and scalable production is of great significance for expanding applications in high-temperature industrial fields and improving process efficiency. Summary of the Invention

[0005] Purpose of the invention: The first purpose of this invention is to provide a lipase mutant with improved catalytic effect and high temperature tolerance; the second purpose of this invention is to provide applications of the high-temperature resistant and highly active lipase mutant.

[0006] Technical solution: The high-temperature resistant, highly active lipase mutant of the present invention is characterized by the substitution of at least one amino acid at positions 25, 242, and 283 of the amino acid sequence shown in SEQ ID No. 1; threonine (T) at position 25 is mutated to methionine (M), serine (S) at position 242 is mutated to glycine (G) or alanine (A), and aspartic acid (D) at position 283 is mutated to proline (P).

[0007] The lipase is derived from Pseudomonas fluorescens, with the amino acid sequence SEQ ID NO.1 and the gene sequence SEQ ID NO.2.

[0008] Preferably, the mutant is T25M, D283P, T25M / D283P, T25M / S242A / D283P, or T25M / S242G / D283P.

[0009] The mutant T25M is formed by the mutation of threonine (T) at position 25 to methionine (M).

[0010] The mutant D283P is formed by the mutation of aspartic acid (D) at position 283 to proline (P).

[0011] The mutant T25M / D283P has a mutation at position 25 (threonine, T) to methionine (M) and position 283 (aspartic acid, D) to proline (P).

[0012] The mutant T25M / S242A / D283P has a mutation at position 25 (threonine (T) to methionine (M), position 242 (serine (S)) to alanine (A), and position 283 (aspartic acid (D)) to proline (P).

[0013] The mutant T25M / S242G / D283P has a mutation at position 25 (threonine (T) to methionine (M), position 242 (serine (S)) to glycine (G), and position 283 (aspartic acid (D)) to proline (P).

[0014] The nucleic acid of this invention encodes the lipase mutant.

[0015] The recombinant vector of the present invention contains the aforementioned nucleic acid.

[0016] Preferably, the original vector of the recombinant vector is a PET series expression vector.

[0017] The recombinant cells of the present invention comprise the recombinant vector described above.

[0018] Preferably, the cells are Escherichia coli.

[0019] The product of the present invention comprises the lipase mutant, the nucleic acid, the recombinant vector, or the recombinant cell.

[0020] Preferably, the product comprises immobilized enzymes or immobilized cells made using immobilization technology.

[0021] The application of the lipase mutant, nucleic acid, recombinant vector, recombinant cell, or product described in this invention in the food industry, feed processing, wastewater treatment, or oil processing.

[0022] Preferably, the application is in the catalytic production of p-nitrophenol palmitate from p-nitrophenol.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0024] (1) The lipase mutant of the present invention is a lipase mutant that can efficiently catalyze the formation of p-nitrophenol palmitate from p-nitrophenol compared with wild-type lipase. It exhibits significantly enhanced tolerance under high temperature conditions, thereby improving high temperature catalytic efficiency and effectively expanding the application range of lipase in high temperature environment.

[0025] (2) The catalytic efficiency of the mutant T25M / S242G / D283P of the present invention is 1.5 times that of the wild-type lipase;

[0026] (3) The mutant T25M / S242G / D283P of the present invention, after high temperature tolerance treatment at 95°C for 30 min, has a residual enzyme activity of 30%, and its high temperature tolerance is 1.6 times that of wild-type lipase. Attached Figure Description

[0027] Figure 1 This is a standard curve for lipase activity detection;

[0028] Figure 2 The enzyme activity effect of wild-type lipase and lipase mutant catalyzing the production of p-nitrophenol from p-nitrophenol palmitate is shown in the embodiments of this application.

[0029] Figure 3 The residual enzyme activity of wild-type lipase and lipase mutant in catalyzing the production of p-nitrophenol from p-nitrophenol palmitate after high-temperature tolerance treatment at 95℃ for 30 min is shown in the figure. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the embodiments.

[0031] Example 1: Construction and preparation of wild-type lipase

[0032] The examples used lipase from *Pseudomonas fluorescens* as the original enzyme (wild type), and the gene for this enzyme was synthesized by Sangon Biotech (Shanghai) Co., Ltd. Its amino acid sequence is shown in SEQ ID NO: 1, and its gene sequence is shown in SEQ ID NO: 2.

[0033] The recombinant plasmid pET28b-Kan-PfLipase was constructed, and the wild-type lipase gene Lipase was ligated with the enzyme-digested plasmid pET28b (provided by Sangon Biotech (Shanghai) Co., Ltd.) to obtain the recombinant expression vector pET28b-Kan-PfLipase. The constructed recombinant expression vector pET28b-Kan-PfLipase was first transformed into E. coli DH5α competent cells [hereinafter referred to as E. coli DH5α, purchased from Shanghai Titan Technology Co., Ltd.] for amplification. The resulting recombinant bacteria were inoculated onto the surface of LB solid medium containing 30 mg / mL kanamycin sulfate and cultured at 37 ℃ for 12 h. Single colonies were then picked and cultured in LB liquid medium at 37 ℃ and 220 rpm for 12 h with shaking. After culture, the cells were centrifuged at 4,000 rpm for 10 min and collected. Plasmids were extracted from E. coli DH5α / pET28b-Kan-PfLipase using a high-purity plasmid miniprep kit and used as templates for subsequent mutant construction.

[0034] The plasmids were further transformed into competent Escherichia coli BL21(DE3) cells [hereinafter referred to as E. coli BL21(DE3), purchased from Shanghai Titan Technology Co., Ltd.], plated onto agar plates containing kanamycin sulfate at a final concentration of 30 mg / mL, and then single colonies were picked and inoculated into culture tubes containing 5 mL of LB liquid medium containing 30 mg / mL kanamycin sulfate. After incubation at 37 ℃ and 220 rpm for 12 h with shaking, 3 mL of the inoculum was transferred to 500 mL of resistant LB medium containing 30 mg / mL kanamycin sulfate. 600 When the concentration reaches approximately 0.6, add 0.5 mL of 0.5 M IPTG (i.e., isopropyl-β-D-thiopyranoside) to make the final IPTG concentration 0.5 mM, and induce at 20℃ for about 16 h.

[0035] After induction, the bacterial suspension was centrifuged at 4 °C and 4000 rpm for 10 min to collect the bacterial cells. The cells were resuspended in an appropriate amount of 100 mM pH 7.5 phosphate buffer and the bacterial suspension was adjusted to OD. 600 =3, which can be used for subsequent enzyme activity assays. Alternatively, the bacterial cells can be further sonicated under ice bath conditions (3 seconds working, 6 seconds interval, total working time 5 minutes), and the lysate can be centrifuged at 4 ℃ and 4000 rpm for 10 minutes. The resulting supernatant is the crude enzyme solution, which can be aliquoted and stored at -20 ℃ for later use.

[0036] Example 2: Lipase mutant T25M and its preparation method

[0037] This embodiment provides a lipase mutant T25M, the preparation method of which is as follows:

[0038] Step 1: Construct the nucleic acid encoding the lipase mutant and the expression vector encoding the nucleic acid.

[0039] The wild-type lipase gene of Pseudomonas fluorescens was mutated using whole plasmid PCR to obtain the target mutant gene (i.e., the nucleic acid encoding the lipase mutant T25M).

[0040] The PCR primers for the nucleic acid encoding the lipase mutant T25M are:

[0041] T25M upstream primer: GCCGATGCAATGGCTATT ATG CTGTATAGCTATC;

[0042] T25M downstream primer: CTATGATATGTC GTA TTATCGGTAACGTAGCCG.

[0043] The PCR reaction system for the whole plasmid PCR is shown in Table 1.

[0044] Table 1 PCR Reaction System

[0045]

[0046] The PCR reaction procedure for the whole plasmid PCR is shown in Table 2.

[0047] Table 2 PCR reaction procedure

[0048]

[0049] After PCR amplification of the target fragment, the amplification products were detected by 1% agarose gel electrophoresis. The results showed that the amplification products were single bands, each approximately 6000 bp in size. The amplification products were then purified and recovered using a DNA gel purification kit.

[0050] Finally, the pET28b plasmid of the lipase mutant T25M was obtained, which is the expression vector of the lipase mutant T25M.

[0051] Step 2: Construct host cells for transfection with the expression vector.

[0052] Using E. coli BL21(DE3) strain as the expression host (i.e., host cell), the successfully sequenced recombinant plasmid was transformed into E. coli BL21(DE3) to construct the recombinant mutant expression strain E. coli BL21(DE3) / pET28b-Kan-PfLipase.

[0053] Specifically, the purified gene fragment was digested with EasyCut endonuclease-DpnI to remove the template, and then recombined with recombinase. The recombinant product was transformed into E. coli DH5α competent cells, plated on LB solid medium containing 30 mg / mL kanamycin sulfate, and incubated at 37°C for 12 h. Single colonies were then transferred to LB liquid medium and cultured at 37°C with shaking at 220 rpm for 12 h. After incubation, a portion of the bacterial culture was added with sterile glycerol to a final concentration of 25%, numbered, and stored at -80°C for later use to obtain the recombinant mutant plasmid clone strain E. coli DH5α / pET28b-Kan-PfLipase. Another portion of the bacterial culture was centrifuged at 4,000 rpm for 10 min and the cells were collected. The plasmid was extracted from E. coli DH5α / pET28b-Kan-PfLipase using a high-purity plasmid miniprep kit, and the correctness of the mutation site was verified by sequencing.

[0054] Finally, the recombinant mutant plasmid clone of lipase mutant T25M and the corresponding recombinant mutant plasmid were obtained, namely the host cells transfected with the expression vector of lipase mutant T25M.

[0055] Step 3: Construct a recombinant mutant protein expression strain to obtain a culture containing the lipase mutant T25M.

[0056] The successfully sequenced pET28b plasmid was transformed into E. coli BL21(DE3) as the expression host (i.e., host cell) to construct the recombinant mutant protein expression strain E. coli BL21(DE3) / pET28b-Kan-PfLipase.

[0057] Specifically, the successfully constructed recombinant mutant plasmid was transformed into E. coli BL21(DE3) competent cells, plated onto plates containing kanamycin sulfate at a final concentration of 30 mg / mL, and then a single colony was picked and inoculated into a culture tube containing 5 mL of LB liquid medium containing 30 mg / mL kanamycin sulfate. The culture was then incubated at 37°C and 220 rpm for 12 h with shaking to obtain the recombinant mutant protein expression strain E. coli BL21(DE3) / pET28b-Kan-PfLipase. This was then inoculated into 500 mL of resistant LB medium containing 30 mg / mL kanamycin sulfate at an OD rate of 3 mL. 600When the concentration reaches approximately 0.6, add 0.5 mL of 0.5 M IPTG (isopropyl-β-D-thiopyranoside) to make the final IPTG concentration 0.5 mM. Induce at 20℃ for about 16 h to obtain a culture containing the lipase mutant T25M.

[0058] After induction, the cells were obtained by centrifugation, resuspended in buffer, and sonicated under ice bath conditions (3 s working time, 6 s interval, working time 20 min). The supernatant was collected by centrifugation at 12,000 rpm / min for 20 min at 4℃, which yielded the crude enzyme solution of the lipase mutant T25M.

[0059] Example 3: Preparation of lipase mutants T25M, D283P, T25M / D283P, T25M / S242A / D283P, and T25M / S242G / D283P.

[0060] The preparation method of Example 1 was used. PCR primers are shown in Table 3.

[0061] Table 3 Primers for constructing mutants

[0062]

[0063] Example 4: Enzyme activity of wild-type lipase and lipase mutant in catalyzing the production of p-nitrophenol from p-nitrophenol palmitate

[0064] A catalyst for the production of p-nitrophenol from p-nitrophenol palmitate was obtained from the cultures containing lipase mutants obtained in Examples 2 and 3.

[0065] Lipase hydrolyzes p-nitrophenol palmitate to release p-nitrophenol, which is yellow-green in alkaline conditions. According to Table 4, 3 mg of p-nitrophenol was dissolved in 1 mL of isopropanol, and then 9 mL of ultrapure water was added to prepare a stock solution. This stock solution was then serially diluted using 100 mM, pH 7.5 potassium dihydrogen phosphate / dipotassium hydrogen phosphate buffer to prepare p-nitrophenol solutions of different concentrations. The absorbance at 405 nm was measured. A standard curve was plotted with absorbance (y) on the ordinate and standard solution concentration (x) on the abscissa, using a blank buffer solution as a control. Enzyme activity was calculated based on the standard curve. The standard curve is shown below. Figure 1 As shown. Enzyme activity is defined as follows: 1 μmol of p-nitrophenol is released from 1 ml of lipase hydrolyzing p-nitrophenol palmitate within 1 min, which is defined as 1 enzyme activity unit (μmol·ml). -1 ·min -1 .

[0066] Table 4. Preparation of Standard Curve using the p-nitrophenol Method

[0067]

[0068] Lipase activity was determined using the p-nitrophenol ester method. 30 mg of p-nitrophenol palmitate was dissolved in 10 mL of isopropanol, and then 90 mL of ultrapure water was added to prepare the substrate solution. The supernatant from whole-cell bacterial lysis was used as the catalyst. The reaction system consisted of 50 μL OD0.05. 600 After lysing whole-cell bacterial culture of 40, the supernatant and 250 μL of p-nitrophenol palmitate solution were added and reacted in a 40℃ water bath for 15 min. The reaction was terminated by adding 500 mL of anhydrous ethanol. The reaction solution was centrifuged at 1,2000 rpm for 3 min, and 300 μL of the supernatant was transferred to an ELISA plate. The absorbance of the product p-nitrophenol at 405 nm was detected by an ELISA reader to compare enzyme activity.

[0069] The results are as follows Figure 2 As shown, compared to wild-type lipase (corresponding to Figure 2 The lipase mutants (in terms of WT) exhibit increased enzyme activity, significantly enhancing the efficiency of catalyzing the conversion of p-nitrophenol palmitate to p-nitrophenol. In particular, the lipase mutant T25M / S242G / D283P demonstrates a catalytic efficiency 1.6 times that of the wild-type lipase. In conclusion, the lipase mutants exhibit greater activity in catalyzing the conversion of p-nitrophenol palmitate to p-nitrophenol than the wild-type lipase.

[0070] Example 3: Determination of residual enzyme activity of lipase mutant after high-temperature tolerance treatment

[0071] This embodiment uses a lipase mutant to study the residual enzyme activity of the lipase mutant in the catalytic reaction of p-nitrophenol palmitate to p-nitrophenol after a high-temperature tolerance treatment at 95°C for 30 min. Residual enzyme activity is defined as the ratio of the enzyme activity measured after the 95°C 30-min high-temperature tolerance treatment to the initial enzyme activity of the untreated enzyme at room temperature.

[0072] The reaction system consisted of 50 μL of supernatant from the lysis of whole-cell bacterial culture after high-temperature tolerance treatment (OD 600 = 40), and 250 μL of p-nitrophenol palmitate solution. The mixture was placed in a 40°C water bath for 15 min, and 500 mL of anhydrous ethanol was added to terminate the reaction. The reaction solution was centrifuged at 1,2000 rpm for 3 min, and 300 μL of the supernatant was transferred to an ELISA plate. The absorbance of the p-nitrophenol product at 405 nm was detected using an ELISA reader to compare the residual enzyme activity after high-temperature tolerance treatment.

[0073] The results are as follows Figure 3As shown in the figure. Experiments have shown that after high-temperature tolerance treatment at 95℃ for 30 min, the residual enzyme activity of wild-type lipase in the catalytic reaction of p-nitrophenol palmitate to p-nitrophenol remained at 19%, while the residual enzyme activity of the optimal lipase mutant T25M / S242G / D283P was 30%, demonstrating good application potential.

Claims

1. A high-temperature resistant, highly active lipase mutant, characterized in that, The mutant is formed by replacing at least one amino acid at positions 25, 242, and 283 of the amino acid sequence shown in SEQ ID No. 1; threonine at position 25 is mutated to methionine, serine at position 242 is mutated to glycine or alanine, and aspartic acid at position 283 is mutated to proline.

2. The lipase mutant according to claim 1, characterized in that, The mutants are T25M, D283P, T25M / D283P, T25M / S242A / D283P, or T25M / S242G / D283P.

3. A nucleic acid, characterized in that, Encodes the lipase mutant of claim 1 or 2.

4. A recombinant vector, characterized in that, It includes the nucleic acid described in claim 3.

5. The recombinant vector according to claim 4, characterized in that, The original vector for the recombinant vector is a PET series expression vector.

6. A recombinant cell, characterized in that, It includes the recombinant vector as described in claim 4 or 5.

7. The recombinant cells according to claim 6, characterized in that, The cells in question are Escherichia coli.

8. A product characterized in that, The product comprises the lipase mutant of claim 1, the nucleic acid of claim 3, the recombinant vector of claim 4, or the recombinant cell of claim 6.

9. The product according to claim 8, characterized in that, The products include immobilized enzymes or immobilized cells made using immobilization technology.

10. The use of a lipase mutant of claim 1, a nucleic acid of claim 3, a recombinant vector of claim 4, a recombinant cell of claim 6, or a product of claim 8 in the food industry, feed processing, wastewater treatment, or oil processing.

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