Organic-solvent-resistant high-catalytic-activity lipase mutant and application thereof

Abstract

The invention discloses an organic-solvent-resistant high-catalytic-activity lipase mutant and application thereof. The mutant is obtained by replacing at least one amino acid in the 58th site, the 116th site, the 130th site and the 210th site of an amino acid sequence as shown in SEQ ID No.1; the 58th-site serine Ser is mutated into lysine Lys, the 116th-site serine Ser is mutated into methionine Met, the 130th-site aspartic acid Asp is mutated into arginine Arg, and the 210th-site glutamic acid Glu is mutated into serine Ser. The mutant obtained by carrying out directed mutation on the wild type lipase derived from thermomyces lanuginosus has improved organic solvent tolerance compared with the wild type lipase, can be applied to synthesis of biodiesel such as fatty acid methyl ester, and is more suitable for application in the industrial field.

Description

Technical Field

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

[0002] Lipases, as an important class of biocatalysts, have become core tools in green chemistry and biomanufacturing due to their efficient catalysis of esterification, transesterification, hydrolysis, and chiral synthesis reactions in non-aqueous media. Especially in organic solvent systems, the application of lipases can significantly expand the substrate range, alter reaction equilibrium, simplify product separation, and achieve chemical transformations that are difficult to perform in aqueous phases. However, the stability and activity of enzymes in organic solvents often decrease significantly, which has become a key bottleneck restricting their industrial applications. Dimethyl sulfoxide (DMSO), ethanol, and isopropanol represent polar aprotic, polar small-molecule alcohols, and moderately polar branched-chain alcohol solvents, respectively, and serve as model systems for studying the solvent tolerance of lipases. Although DMSO can dissolve poorly soluble drug intermediates, its strong dehydration and osmotic effects can destroy the "structural water layer" and internal hydrogen bonds of the enzyme protein, leading to irreversible inactivation. Ethanol, as a green solvent and reaction substrate, exhibits dehydration effects and hydroxyl groups that interfere with the microenvironment of the catalytic triplet and can alter the enzyme's secondary structure, reducing the α-helix content. Isopropanol has a relatively mild denaturing effect, but its hydrophobic isopropyl group may perturb the hydrophobic core of the enzyme protein, inducing conformational relaxation. The inactivation mechanisms of these three solvents encompass disruption of the surface hydration layer, interference with the internal hydrogen bond network, and perturbation of hydrophobic interactions. Simultaneous study of these three solvents helps to elucidate the adaptation mechanisms of lipases in complex organic media, providing a theoretical basis for establishing universal enzyme tolerance modification strategies.

[0003] Traditional strategies to improve the organic solvent tolerance of lipases include physical immobilization, chemical modification, and media engineering, but these methods often suffer from drawbacks such as high cost, limited effectiveness, or poor versatility.

[0004] In recent years, breakthroughs have been made in enzyme molecular modification strategies based on rational protein design and directed evolution. By deeply elucidating the structural basis of lipase inactivation in organic solvents (such as increased molecular flexibility, breakage of key hydrogen bonds or salt bridges, and impaired opening and closing mechanisms of the active site cap), targeted mutation sites can be designed. Therefore, the development of lipase mutants with excellent organic solvent tolerance is of great significance for their application. Summary of the Invention

[0005] Objectives of the invention: The first objective of this invention is to provide a highly catalytically active lipase mutant that is resistant to organic solvents and has improved tolerance to organic solvents; the second objective of this invention is to provide applications of the aforementioned highly catalytically active lipase mutant that is resistant to organic solvents.

[0006] Technical solution: The organic solvent-resistant, highly catalytically active lipase mutant of the present invention is characterized by the substitution of at least one amino acid at positions 58, 116, 130, and 210 of the amino acid sequence shown in SEQ ID No. 1; the serine at position 58 (Ser) is mutated to lysine (Lys), the serine at position 116 (Ser) is mutated to methionine (Met), the aspartic acid at position 130 (Asp) is mutated to arginine (Arg), and the glutamate at position 210 (Glu) is mutated to serine (Ser).

[0007] The wild-type lipase is the enzyme corresponding to NCBI ID: XP_069271870.1, derived from *Thermomyces lanuginosus*, with the amino acid sequence SEQ ID NO.1 and the gene sequence SEQ ID NO.2.

[0008] The mutants are S58L, MS116M, E210S, S116M / E210S, or S116M / D190R / E210S.

[0009] The mutant S58L is a mutation where the 58th serine (Ser) is mutated to lysine (Lys).

[0010] The mutant S116M is a mutation of the 116th serine (Ser) to methionine (Met).

[0011] The mutant E210S is a mutation where the glutamic acid Glu at position 210 is mutated to serine Ser.

[0012] The mutant S116M / E210S has a serine (serine) at position 116 mutated to methionine (Met), and a glutamic acid (glutamate) at position 210 mutated to serine (serine).

[0013] The mutant S116M / D130R / E210S has the following mutations: Serine at position 116 is mutated to Methionine at position 116, Aspartic acid at position 130 is mutated to Arginine at position 130, and Glutamic acid at position 210 is mutated to Serine at position 210.

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

[0015] The encoding gene of the lipase mutant of the present invention was obtained by means of the following method: using the recombinant plasmid pET28b(+)-TlLipase derived from the wild-type lipase gene of Thermomyces lanuginosus as a template, PCR amplification was performed using site-directed mutagenesis primers, and the target mutant gene was screened.

[0016] The recombinant vector of the present invention contains the aforementioned nucleic acid.

[0017] Preferably, the original vector of the recombinant vector is a PET series expression vector.

[0018] The recombinant cells of the present invention comprise the recombinant vector described above.

[0019] Preferably, the cells are Escherichia coli.

[0020] The method for constructing the recombinant cells includes the following steps:

[0021] (1) Construction of recombinant plasmid pET28b(+)-TlLipase: The lipase gene Lipase was ligated with the enzyme-digested plasmid pET28b(+) to obtain the recombinant expression vector pET28b(+)-TlLipase;

[0022] (2) Construction of recombinant E. coli BL21(DE3) / pET28b(+)-TlLipase: The constructed recombinant expression vector pET28b(+)-TlLipase was heat-transformed into Escherichia coli BL21(DE3) competent cells, and the recombinant E. coli BL21(DE3) / pET28b(+)-TlLipase was obtained by culturing and screening.

[0023] The product of the present invention comprises the lipase mutant, the nucleic acid, the recombinant vector, or the recombinant cell.

[0024] Preferably, the product comprises immobilized enzymes or immobilized cells made using immobilization technology.

[0025] The application of the lipase mutant, the nucleic acid, the recombinant vector, the recombinant cell, or the product described in this invention as a catalyst in biodiesel.

[0026] Preferably, the biodiesel is a fatty acid methyl ester.

[0027] Invention Mechanism:

[0028] In this invention, using the sequence and structural information of publicly reported lipases, a number of potential enzyme genes were screened through non-redundant searches in databases such as NCBI, based on principles such as protein structural similarity, conserved site analysis, and host origin diversity. These genes were functionally expressed in an E. coli expression system and then purified to obtain pure enzymes. Preferred lipases were derived from *Thermomyces lanuginosus*, which possess certain catalytic activity.

[0029] This invention amplifies the above-mentioned wild-type lipase and systematically modifies the tolerance of the target lipase to three key organic solvents: dimethyl sulfoxide, ethanol, and isopropanol. Ultimately, a lipase mutant with significantly improved tolerance to organic solvents is obtained, thereby enabling its application in chemical processing.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The present invention obtains a mutant with improved organic solvent tolerance by directional mutation of wild-type lipase derived from *Thermophilus sparsely cottony*; (2) After incubation for 30 min in dimethyl sulfoxide, ethanol and isopropanol at a final concentration of 30% (v / v) of the lipase mutant S116M / D130R / E210S, the catalytic efficiency is 2.3, 2.2 and 2.7 times that of wild-type lipase, respectively, and the residual enzyme activity is 1.6, 1.2 and 1.3 times that of wild-type lipase, respectively; (3) The lipase mutant of the present invention has improved organic solvent tolerance and can be applied to the synthesis of biodiesel such as fatty acid methyl esters, and is more suitable for industrial applications. Attached Figure Description

[0031] Figure 1 The enzyme activities of the lipase mutants in Examples 1-5;

[0032] Figure 2 Residual enzyme activity of wild-type and lipase mutant (S116M / D130R / E210S) under different organic solvent conditions;

[0033] Figure 3 Enzyme activities of wild type and lipase mutant (S116M / D130R / E210S) in other organic solvent environments. Detailed Implementation

[0034] The technical solution of the present invention will be further described below with reference to the embodiments.

[0035] Example 1

[0036] The organic solvent-resistant, highly catalytically active lipase mutant of the present invention is prepared by the following method:

[0037] 1. Construction of lipase mutant plasmids

[0038] (1) Obtaining pET28b(+)-TlLipase

[0039] The wild-type lipase gene from *Thermomyces lanuginosus* was synthesized by Genewiz (Suzhou) Co., Ltd. and constructed into the pET28b(+) vector (provided by Genewiz). The vector was then transformed into *E. coli* DH5α strain (purchased from Sangon Biotech (Shanghai) Co., Ltd.). The recombinant strain *E. coli* DH5α / pET28b(+)-TlLipase was inoculated into 5 mL culture tubes and cultured at 37°C with shaking at 220 rpm for 12 h. After culture, the cells were centrifuged at 12,000 rpm for 1 min and collected. Using a high-purity plasmid miniprep kit, plasmids were extracted from *E. coli* DH5α / pET28b(+)-TlLipase* as templates for iterative mutagenesis to construct the pET28b(+)-TlLipase mutant.

[0040] (2) Construction of recombinant Escherichia coli BL21(DE3) / pET28b(+)-TlLipase mutant

[0041] Gene mutation was performed using whole plasmid PCR to obtain the target mutant gene. Taking S116M as an example, the required primers were designed. Other mutants were designed using the same principle and single-point iterative mutations were performed.

[0042] S58L upstream primer: TAGTTTCGAAGAT CTG GGCGTGGGCGATG;

[0043] S58L downstream primer: CATCGCCCACGCC CAG ATCTTCGAAACTA.

[0044] The PCR system is shown in Table 1.

[0045] Table 1 PCR reaction system

[0046]

[0047] The PCR reaction conditions are shown in Table 2.

[0048] Table 2 PCR reaction conditions

[0049]

[0050] After PCR amplification, the amplification products were detected by 0.9% 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 purification kit.

[0051] The purified gene fragment was digested with 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 μg / mL kanamycin, and incubated at 37°C for 12 h. Single colonies were picked and transferred to LB liquid culture. Positive transformants were identified by PCR, and the correctness of the mutation site was verified by sequencing. After verification, a portion of the cells were treated with sterile glycerol to a final concentration of 25%, numbered, and stored at -80°C for later use. Plasmids were extracted from the remaining cells using a plasmid extraction kit, and the recombinant plasmids were stored at -20°C.

[0052] The successfully sequenced recombinant expression plasmid pET28b(+)-TlLipase was transformed into E. coli BL21(DE3) (Escherichia coli, purchased from Sangon Biotech (Shanghai) Co., Ltd.) as the expression host to construct the recombinant mutant expression strain E. coliBL21(DE3) / pET28b(+)-TlLipase.

[0053] 2. Cultivation of lipase mutants and preparation of crude enzyme solution

[0054] The successfully constructed recombinant mutant expression strain E. coli BL21(DE3) / pET28b(+)-TlLipase was plated onto agar plates containing kanamycin at a final concentration of 30 μg / mL. A single colony was picked and inoculated into 5 mL of LB medium containing antibiotic resistance and cultured overnight at 37 ℃ and 200 rpm / min. A 1% inoculum was then transferred to 500 mL of LB medium containing antibiotic resistance. 600 When the concentration reaches approximately 0.6, add IPTG to a final concentration of 0.5 mM and induce at 18 °C for approximately 14 h.

[0055] After centrifuging to obtain bacterial cells, the cells were resuspended in a buffer and sonicated in an ice bath (2 seconds per cycle, 5 seconds per cycle, for a total of 30 min). The cells were then centrifuged at 12,000 rpm / min for 20 min at 4°C. The supernatant was collected and filtered through a 0.22 μm aqueous filter to obtain the crude enzyme solution.

[0056] Example 2

[0057] Based on Example 1, in step (2) of the S116M mutant, the primers were changed while the other conditions remained the same. The primers are as follows:

[0058] S116M upstream primer: GCTTTACCAGT ATG TGGCGCAGTGTGG;

[0059] S116M downstream primer: CCACACTGCGCCA CATACTGGTAAAGC.

[0060] Example 3

[0061] Based on Example 1, in step (2) of the E210S mutant, the primers were changed while the other conditions remained the same. The primers are as follows:

[0062] E210S upstream primer: TGCCGCCGCGT AGT TTCGGTTACAGT;

[0063] E210S downstream primer: ACTGTAACCGAA GCT ACGCGGCGGCA.

[0064] Example 4

[0065] Based on Example 1, the S116M / E210S mutant uses the plasmid obtained in Example 2 as the DNA template in step (2), with the primers changed while keeping the other conditions unchanged. The primers are as follows:

[0066] S116M / E210S upstream primer: TGCCGCCGCGT AGT TTCGGTTACAGT;

[0067] S116M / E210S downstream primer: ACTGTAACCGAA GCT ACGCGGCGGCA.

[0068] Example 5

[0069] Based on Example 1, the S116M / D130R / E210S mutant was modified using the plasmid obtained in Example 4 as the DNA template in step (2), with the primers changed while all other conditions remained the same. The primers are as follows:

[0070] S116M / D130R / E210S upstream primer:

[0071] GACAGAAAGTTGAA AGA GCAGTGCGTGAACATC;

[0072] S116M / D130R / E210S downstream primers:

[0073] GATGTTCACGCACTGC TCT TTCAACTTTCTGTC.

[0074] Performance testing

[0075] 1. Plotting Standard Curves

[0076] Lipase hydrolyzes pNPP to release p-nitrophenol (pNP), which is yellow-green in alkaline conditions. PNP was dissolved in a 50 mM, pH 7.5 potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution to prepare solutions of different concentrations. 200 μL of each standard solution was added to a 96-well microplate, and 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. A solution with zero p-nitrophenol concentration was used as a blank control. Enzyme activity was calculated based on the standard curve: y = 0.0066x + 0.0733. Enzyme activity was defined as: 1 μmol of p-nitrophenol released by 1 mg of lipase hydrolyzing p-nitrophenol palmitate within 1 min, which is 1 enzyme activity unit (U), μmol / mg·min.

[0077] 2. Lipase activity detection

[0078] Add 0.5 mL of 50 mM p-nitrophenyl palmitate solution and 4 mL of 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution (pH 7.5), then add 0.5 mL of crude enzyme solution to complete the reaction system. Add 0.5 mL of heat-inactivated crude enzyme solution as a control. After incubating the reaction in a 40°C water bath for 5 min, add 1 mL of anhydrous ethanol to terminate the reaction. Mix well, cool, centrifuge at 12000 r / min for 3 min, and aspirate 200 μL of the supernatant into a 96-well microplate. Measure the absorbance at 405 nm. Calculate the enzyme activity using the above method. Enzyme activity is defined as: 1 μmol of p-nitrophenol released by the hydrolysis of p-nitrophenol palmitate by 1 mg of lipase in 1 min is 1 enzyme activity unit (U), μmol / mg·min. The original enzyme activity is defined as 100%. Further calculate the relative enzyme activity of the mutant enzyme.

[0079] 3. Effects of organic solvents on enzyme stability and catalytic efficiency

[0080] The lipase mutants and wild-type enzyme solutions obtained in Examples 1-5 were treated with dimethyl sulfoxide, ethanol, and isopropanol, respectively, to a final organic solvent concentration of 30% (v / v). The solutions were incubated at 40°C for 30 min, and then the substrate p-nitrophenol palmitate was added. Enzyme activity was determined by the pNP colorimetric method. Each reaction was repeated three times. The results are shown in Table 3 and [Table data missing]. Figure 1 As shown.

[0081] Table 3. Organic solvent resistance of lipase mutants

[0082]

[0083] As shown in Table 3, the catalytic conversion rates of all mutants under the conditions of 30% dimethyl sulfoxide, ethanol and isopropanol were higher than those of wild-type lipase. The catalytic efficiencies of mutants S116R / D130R / E210S under the above conditions were 2.3, 2.2 and 2.7 times that of wild-type lipase, respectively.

[0084] 4. Residual activity of lipase mutants (S116M / D130R / E210S)

[0085] The enzyme solutions of the lipase mutant S116M / D130R / E210S and the wild type were treated with dimethyl sulfoxide, ethanol, and isopropanol, respectively, to a final organic solvent concentration of 30% (v / v). The solutions were incubated at 40°C for 30 min, and then p-nitrophenol palmitate was added. Enzyme activity was determined by pNP colorimetry. Each reaction was repeated three times. The results are shown below. Figure 2 As shown.

[0086] like Figure 2 As shown, the residual enzyme activities of the lipase mutant S116M / D130R / E210S are 1.6% (dimethyl sulfoxide), 1.2% (ethanol), and 1.3% (isopropanol) of the wild-type lipase, respectively, indicating that the synergistic effect of these three sites confers higher structural stability to the mutant. Especially in reaction systems containing organic solvents, the mutant S116M / D130R / E210S can more effectively resist solvent-induced protein unfolding, maintaining the geometric structure of the active site, thus retaining residual activity far exceeding that of the wild type after organic solvent treatment.

[0087] 5. Lipase mutant (S116M / D130R / E210S) tolerance to other organic solvents

[0088] By replacing the organic solvents as described above, wild-type and lipase mutant S116M / D130R / E210S were treated with n-hexane, methanol, acetone, or acetonitrile, and their catalytic activity was determined.

[0089] like Figure 3 As shown, the lipase mutant S116M / D130R / E210S also exhibits superior tolerance to other organic solvents. The catalytic activities of the lipase mutant S116M / D130R / E210S in hexane, methanol, acetone, and acetonitrile are 1.6, 2.1, 1.8, and 2.2 times that of the wild type, respectively.

Claims

1. A lipase mutant with high catalytic activity resistant to organic solvents, characterized in that, The mutant is formed by replacing at least one amino acid at positions 58, 116, 130, and 210 of the amino acid sequence shown in SEQ ID No. 1; the serine at position 58 (Ser) is mutated to lysine (Lys), the serine at position 116 (Ser) is mutated to methionine (Met), the aspartic acid at position 130 (Asp) is mutated to arginine (Arg), and the glutamic acid at position 210 (Glu) is mutated to serine (Ser).

2. The organic solvent-resistant, highly catalytically active lipase mutant according to claim 1, characterized in that, The mutants are S58L, MS116M, E210S, S116M / E210S, or S116M / D190R / E210S.

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 were Escherichia coli.

8. A product characterized in that, The product comprises the lipase mutant of claim 1 or 2, 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 as a catalyst in biodiesel synthesis.

Images