Molecular modification design method for improving lipase solvent tolerance and mutant thereof

Abstract

The invention provides a rational design method for improving lipase solvent tolerance. The tolerance of the Candida antarctica lipase B mutant in a 10% (v / v) pyridine solvent is maximally increased to 5.5 times that of WT, and the tolerance in a 50% (v / v) methanol solution, a 67.7% (v / v) DMSO solution and a 50% (v / v) isopropanol solution is higher than that of WT. The dominant mutation (D145N-I285F, D145N-I285D and T42K-D145N-I285D) is used as a catalyst, and the synthesis efficiency is improved in different enzyme catalytic reactions. Wherein the catalytic synthesis efficiency of vitamin E succinate is improved by 14.6% compared with WT, the catalytic synthesis efficiency of phytosterol ester is improved by 18.0% at most compared with WT, the catalytic synthesis efficiency of sucrose laurate is improved by 17.0% at most compared with WT, and the catalytic synthesis efficiency of glucose laurate is improved by 19.5% compared with WT.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme engineering, and specifically relates to a lipase modification and design method based on structural analysis, thereby improving the organic solvent tolerance of the lipase and applying it to the synthesis of ester compounds. Background Art

[0002] Ester compounds, important substances widely used in food, pharmaceuticals, daily chemicals, and biofuels, have long relied on traditional acid- or base-catalyzed chemical synthesis for their industrial production. While these methods offer advantages such as ease of operation and high yields, they suffer from drawbacks such as high energy consumption, numerous byproducts, difficulty in separation and purification, and residual chemical catalysts. To meet the demands of green and sustainable development, enzymatic synthesis methods with mild reaction conditions and high selectivity have become a new research hotspot. However, the efficiency of enzymatic synthesis is highly dependent on the reaction solvent. Highly polar organic solvents such as methanol, isopropanol, pyridine, and DMSO are commonly used as reaction media in industrial production. However, these highly polar solvents can severely affect enzyme performance and even lead to enzyme inactivation. Typically, highly polar organic solvents reduce enzyme activity by altering enzyme conformation and depriving the enzyme of essential water molecules on its surface, thereby affecting synthesis efficiency. Although enzyme immobilization and chemical modification techniques have been attempted to improve enzyme performance, these methods have not been able to significantly improve enzyme properties, particularly solvent tolerance. Summary of the Invention

[0003] The present invention provides a molecular engineering design method for improving the solvent tolerance of lipases. Antarctic Candida lipase B, with its excellent stereoselectivity and broad substrate adaptability, is an important biocatalyst in the field of lipase catalytic synthesis. The channel and pocket of Antarctic Candida lipase B were initially identified using Fpocket and CaverDock software. Overly conserved sites were then eliminated using ConSurf software, ultimately identifying the sites for modification. Site-directed saturation mutagenesis using NNK degenerate codons was then used to establish a mutant library. A spectrophotometric microplate screening method was then used to identify superior mutants with improved pyridine tolerance. The Antarctic Candida lipase B mutants of the present invention exhibited tolerance improvements of up to 5.5 times that of the WT in 10% (v / v) pyridine solvent. Their tolerances were also higher than the WT in 50% (v / v) methanol, 67.7% (v / v) DMSO, and 50% (v / v) isopropanol. Using these superior mutants as catalysts, synthesis efficiency was improved in various enzyme-catalyzed reactions. Among them, the catalytic synthesis efficiency of vitamin E succinate was 14.6% higher than that of WT, the catalytic synthesis efficiency of phytosterol ester was the highest, increasing by 18.0% compared with WT, the catalytic synthesis efficiency of sucrose laurate was the highest, increasing by 17.0% compared with WT, and the catalytic synthesis efficiency of glucose laurate was 19.5% higher than that of WT.

[0004] In order to solve the technical problems of the present application, the technical scheme is proposed: including the following steps: a molecular modification design method for improving the solvent tolerance of lipase, characterized by comprising the following steps:

[0005] (1) Selecting the lipase B from Candida antarctica as the object of modification; obtaining the crystal structure and amino acid sequence of the lipase to be modified from the protein database PDB;

[0006] (2) Using Fpocket and CaverDock software to determine the channels and pockets of Candida antarctica lipase B, and then excluding the sites with too high conservation by ConSurf software to finally determine the modification sites;

[0007] (3) For the potential target sites determined in step (2), NNK degenerate codon primers are designed for site-directed saturation mutation, and pyridine tolerance of the modified lipase is determined by p-nitrophenol method to screen single mutants with improved pyridine tolerance;

[0008] (4) Double recombination is carried out on the single mutants screened in step (3), and the pyridine tolerance of the double mutants is determined;

[0009] (5) Further recombination is carried out on the double mutants screened in step (4) to obtain triple mutants, and on this basis, the pyridine tolerance is determined by p-nitrophenol method.

[0010] Preferably, the solvent tolerance determination includes the determination of relative enzyme activity and residual enzyme activity.

[0011] Preferably, the dominant mutants screened are used as catalysts in the synthesis of vitamin E succinate, phytosterol ester, sucrose laurate, and glucose laurate.

[0012] Preferably, (1) the lipase B from Candida antarctica is selected as the object of modification; first, the active pocket is predicted by Fpocket software; on this basis, we determine the final target from the 7 active pockets given by the software in combination with the position of the catalytic triad of Candida antarctica lipase B; the amino acids in the pocket are: 39Gly, 40Thr, 42Thr, 47Lys, 104Trp, 105Ser, 134Asp, 138Thr, 144Leu, 154Val, 157Gln, 188Glu, 189Ile, 190Val, 224His, 281Ala, 282Ala, 285Ile;

[0013] (2) The channel sites of Antarctic Candida lipase B were predicted by CaverDock software. With the catalytic triad (Ser105-His224-Asp187) and the oxygen anion hole (Thr40, Gln106) as the starting sites, three channels were obtained. The first channel contains the following sites: 40Thr, 134Asp, 138Thr, 144Leu, 145Asp, 154Val, 157Gln, 189Ile, 224His, 285Ile; the second channel contains the following sites: The lower site: 39Gly, 40Thr, 104Trp, 105Ser, 138Thr, 154Val, 157Gln, 189Ile, 190Val, 224His, 282Ala, 285Ile; the third channel contains the following sites: 39Gly, 40Thr, 104Trp, 105Ser, 134Asp, 138Thr, 154Val, 157Gln, 189Ile, 190Val, 224His, 282Ala, 285Ile;

[0014] (3) ConSurf software was used to identify the evolutionary conservation of amino acid residues in Antarctic Candida lipase B to avoid damage to the enzyme structure after modification. Based on this, sites in the active pocket and channel were screened, and amino acid sites with a conservation of ≥6, that is, highly conserved sites, were excluded. Eight sites were screened: 42Thr, 47Lys, 144Leu, 145Asp, 154Val, 189Ile, 282Ala, and 285Ile.

[0015] Preferably, the channel and pocket of Antarctic Candida lipase B are determined using Fpocket and CaverDock software, and then the overly conserved sites are excluded using ConSurf software to finally determine the modification site; for the modification site, primers are designed using NNK degenerate codons to perform site-directed saturation mutagenesis, and the modified lipase is subjected to pyridine tolerance determination using the p-nitrophenol method to screen single mutants with improved pyridine tolerance; recombination is performed on the basis of single-point screening, and the obtained double and triple mutants are subjected to pyridine tolerance, methanol tolerance, DMSO tolerance and isopropanol tolerance determination; finally, the screened dominant mutants are used as catalysts in the synthesis of vitamin E succinate, phytosterol esters, sucrose laurate and glucose laurate.

[0016] Preferably, the Candida antarctica lipase B mutant is used in the synthesis of vitamin E succinate, phytosterol esters, glucose laurate and sucrose laurate.

[0017] Preferably, for the synthesis of vitamin E succinate, the catalytic activity of the mutant ND is increased by 12.7% compared with the wild type, the catalytic activity of D145N-I285F is increased by 14.6% compared with the wild type, and the catalytic activity of T42K-D145N-I285D is increased by 8.5% compared with the wild type; for the synthesis of phytosterol esters, the catalytic activity of the mutant ND is increased by 14.1% compared with the wild type, the catalytic activity of D145N-I285F is increased by 18.0% compared with the wild type, and the catalytic activity of T42K-D145N-I285D is increased by 10.5% compared with the wild type. %; for the glucose laurate synthesis reaction, the catalytic activity of the mutant ND was 11.5% higher than that of the wild type, the catalytic activity of D145N-I285F was 19.5% higher than that of the wild type, and the catalytic activity of T42K-D145N-I285D was 8.5% higher than that of the wild type; for the sucrose laurate synthesis reaction, the catalytic activity of the mutant ND was 17.0% higher than that of the wild type, the catalytic activity of D145N-I285F was 13.6% higher than that of the wild type, and the catalytic activity of T42K-D145N-I285D was 13.0% higher than that of the wild type.

[0018] Preferably, the amino acid sequence of the Candida antarctica lipase B mutant ND is shown in SEQ ID NO.3, and the nucleotide sequence thereof is shown in SEQ ID NO.4.

[0019] 1) Selecting lipase B from Candida antarctica as the target for modification; obtaining the crystal structure and amino acid sequence of the lipase to be modified from the Protein Data Bank (PDB);

[0020] 2) The channel and pocket of Candida antarctica lipase B were determined using Fpocket and CaverDock software. Highly conserved sites (>6) were then excluded using ConSurf software to determine the modification sites.

[0021] 3) performing site-directed saturation mutagenesis on the potential target sites identified in step 2) using the NNK degenerate codon, and performing solvent tolerance testing on the modified lipase using the p-nitrophenol method to screen for single mutants with improved pyridine tolerance;

[0022] 4) performing double recombination on the single mutants screened in step 3), and performing pyridine tolerance test on the double mutants;

[0023] 5) The double mutants screened in step 4) are further recombined to obtain triple mutants, and pyridine tolerance is determined using the p-nitrophenol method.

[0024] The molecular modification design method for improving the solvent tolerance of lipase, and the solvent tolerance determination include the determination of relative enzyme activity and residual enzyme activity.

[0025] The advantageous mutants screened by the molecular modification design method for improving the solvent tolerance of lipase are used as catalysts in the synthesis of vitamin E succinate, phytosterol esters, sucrose laurate and glucose laurate.

[0026] Beneficial effects:

[0027] The present invention transforms Antarctic Candida lipase B based on computer-aided technology to improve the solvent tolerance of Antarctic Candida lipase B, and then applies it to the synthesis of ester compounds. First, the active pocket and channel site of Antarctic Candida lipase B are predicted using Fpocket and CaverDock software. Sites with high conservatism are eliminated using ConSurf software, and the modification sites are finally determined. On this basis, a saturated mutant library is established through experiments such as primer design, plasmid extraction, PCR, gel recovery, transformation, and cultivation, and pyridine tolerance screening is performed on this library. Subsequent recombination is carried out to find mutants with excellent pyridine tolerance. Finally, the mutants with good pyridine tolerance are expressed separately and used as catalysts for the synthesis of ester compounds, thereby improving their synthesis efficiency.

[0028] The present invention provides a rational design method for improving the solvent tolerance of lipase. The channel and pocket of Antarctic Candida lipase B were preliminarily determined by Fpocket and CaverDock software, and then the overly conserved sites were eliminated by ConSurf software, and the modification sites were finally determined. Then, NNK degenerate codons were used for site-directed saturation mutagenesis to establish a mutant library, and excellent mutants with improved pyridine tolerance were screened out based on the microplate screening method of spectrophotometry. The Antarctic Candida lipase B mutant of the present invention has a maximum tolerance of 5.5 times that of WT in 10% (v / v) pyridine solvent, and its tolerance is higher than WT in 50% (v / v) methanol solution, 67.7% (v / v) DMSO solution and 50% (v / v) isopropanol solution. The dominant mutations (D145N-I285F, D145N-I285D, and T42K-D145N-I285D) acted as catalysts, improving the synthesis efficiency of various enzyme-catalyzed reactions. The synthesis efficiency of vitamin E succinate increased by 14.6% compared to WT, while the highest efficiency for phytosterol esters was 18.0% higher than WT. The highest efficiency for sucrose laurate was 17.0% higher than WT, and the efficiency for glucose laurate increased by 19.5% compared to WT. This demonstrates the feasibility of improving enzyme catalytic efficiency by increasing lipase tolerance, providing new insights for other enzyme-catalyzed reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Solvent tolerance point selection strategy diagram;

[0030] Figure 2 : Residual enzyme activity of single point mutants and pyridine resistance relative to the wild-type enzyme;

[0031] Figure 3 : Residual enzyme activity of the double mutant and pyridine resistance relative to the wild-type enzyme;

[0032] Figure 4 : Residual enzyme activity of the triple mutant and pyridine resistance relative to the wild-type enzyme;

[0033] Figure 5 : Resistance of multiple mutants relative to the wild-type enzyme in different solvents (WT: Candida antarctica lipase B wild type, ND: Candida antarctica lipase B mutant D145N-I285D, NF: D145N-I285F, NDK: T42K-D145N-I285D);

[0034] Figure 6 : Synthesis yield of different enzymatic reactions (WT: wild type of Candida antarctica lipase B, ND: mutant D145N-I285D of Candida antarctica lipase B, NF: D145N-I285F, NDK: T42K-D145N-I285D). DETAILED DESCRIPTION

[0035] The present invention will be further described below with reference to specific examples, but is not limited thereto. Unless otherwise indicated, the reagent raw materials described in the following examples are all commercially available common raw materials, and the preparation of the reagents adopts conventional methods. The methods not described in detail in the examples are all conventional operations in the art.

[0036] Example 1: Method for improving the solvent tolerance of Candida antarctica lipase B.

[0037] (1) Lipase B from Antarctic Candida was selected as the target for modification. First, the active pocket was predicted using the Fpocket software. On this basis, we determined the final target from the seven active pockets given by the software in combination with the position of the catalytic triad of Antarctic Candida lipase B. The amino acids in the pocket are: 39Gly, 40Thr, 42Th, 47Lys, 104Trp, 105Ser, 134Asp, 138Thr, 144Leu, 154Val, 157Gln, 188Glu, 189Ile, 190Val, 224His, 281Ala, 282Ala, 285Ile.

[0038] (2) The channel sites of Antarctic Candida lipase B were predicted using CaverDock software. With the catalytic triad (Ser105-His224-Asp187) and the oxygen anion hole (Thr40, Gln106) as the starting sites, three channels were obtained. The first channel contains the following sites: 40Thr, 134Asp, 138Thr, 144Leu, 145Asp, 154Val, 157Gln, 189Ile, 224His, 285Ile. The second channel contains the following sites: 39Gly, 40Thr, 104Trp, 105Ser, 138Thr, 154Val, 157Gln, 189Ile, 190Val, 224His, 282Ala, 285Ile. The third channel contains the following sites: 39Gly, 40Thr, 104Trp, 105Ser, 134Asp, 138Thr, 154Val, 157Gln, 189Ile, 190Val, 224His, 282Ala, 285Ile.

[0039] (3) ConSurf software was used to identify the evolutionary conservation of amino acid residues in Antarctic Candida lipase B to avoid damage to the enzyme structure after modification. On this basis, the sites in the active pocket and channel were screened, and amino acid sites with conservation ≥6, that is, highly conserved sites, were excluded. Finally, 8 sites were screened: 42Thr, 47Lys, 144Leu, 145Asp, 154Val, 189Ile, 282Ala, and 285Ile. The results of the comprehensive site selection strategy are as follows: Figure 1 shown.

[0040] Amino acid sequence of Candida antarctica lipase B (SEQ ID NO. 1):

[0041] LPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMVNAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAGPLDALAVSAPSVWQQT TGSALTTALRNAGGLTQIVPTTNLYSATDEIVQPQVSNSPLDSSYLFNGKNVQAQAVCGPLFVIDHAGSLTSQFSYVVGRSALRSTTGQARSADYGITDCNPLPANDLTPEQKVAAAALLAPAAAAIVAGPKQNCEPDLMPYARPFAVGKRTCSGIVTP

[0042] The nucleotide sequence of Candida antarctica lipase B (SEQ ID NO.2):

[0043] CTGCCTAGTGGTAGCGATCCGGCCTTTAGCCAGCCGAAAAGCGTGCTGGATGCAGGTCTGACCTGCCAGGGCGCCAGTCCGAGCAGCGTGAGCAAACCGATTCTGCTGGTGCCGGGTACAGGCACCACCGGTCCGCAGAGCTTTGATAGCAATTGGATTCCGCTGAGCACCCAGCTGGGTTATACCCCGTGTTGGATTAGCCCGCCGCCGTTTATGCTGAATGATACCCAGGTTAATACCGAATATATGGTGAATGCAATTACCGCACTGTATGCCGGCAGTGGCAATAATAAGCTGCCGGTTCTGACCTGGAGCCAGGGCGGTCTGGTTGCCCAGTGGGGTCTGACCTTTTTCCCGAGCATTCGCAGTAAAGTTGATCGCCTGATGGCCTTTGCACCGGATTATAAAGGTACAGTTCTGGCCGGCCCGCTGGATGCCCTGGCAGTTAGCGCACCGAGCGTGTGGCAGCAGACCACCGGTAGTGCCCTGACCACCGCCCTGCGTAATGCAGGTGGTCTGACCCAGATTGTTCCGACCACCAATCTGTATAGTGCAACCGATGAAATTGTGCAGCCGCAGGTGAGTAATAGTCCGCTGGATAGCAGCTATCTGTTTAATGGTAAAAATGTTCAGGCCCAGGCCGTTTGCGGCCCGCTGTTTGTGATTGATCATGCAGGTAGTCTGACCAGTCAGTTTAGTTATGTTGTTGGCCGCAGCGCCCTGCGCAGCACCACAGGTCAGGCACGCAGTGCAGATTATGGTATTACCGATTGCAATCCGCTGCCGGCAAATGATCTGACCCCGGAACAGAAAGTGGCAGCAGCAGCCCTGCTGGCCCCGGCAGCAGCAGCGATTGTTGCAGGCCCGAAACAGAATTGCGAACCGGATCTGATGCCGTATGCCCGTCCGTTTGCAGTTGGTAAACGTACCTGCAGTGGCATTGTTACCCCG

[0044] 实施例2:南极假丝酵母脂肪酶B的表达。

[0045] (1) The lipase B gene from Candida antarctica was used as a template, and the enzyme was named CALB. It was inserted into the pET25b(+) vector using the EcoRI-NcoI cloning site, and the wild-type recombinant expression plasmid pET25b-CALB was obtained by gene synthesis.

[0046] (2) Transform the above plasmid into BL21(DE3) competent cells by heat shock method, add 500 μL of LB medium without any resistance, and incubate at 37°C for 60 min. Centrifuge at 3000 rpm, and mix the remaining 100 μL of supernatant with the cells. Spread an appropriate amount on LB plates containing 100 mg / L ampicillin resistance and culture at 37°C overnight until single colonies are clearly grown.

[0047] (3) Pick a single colony from the overnight plate and transfer it to 5 mL of LB liquid medium containing 100 mg / L ampicillin resistance, culture it in a shaking incubator at 37°C for 12 h, and then perform bacterial maintenance and sequencing.

[0048] (4) Successfully verified monoclones were selected and cultured in LB liquid medium containing 100 mg / L ampicillin at 37°C and agitation at 220 rpm for 12-16 h. A 1-2% transfer of the clones was performed to 50 mL of TB medium containing 100 mg / L ampicillin at 37°C and agitation at 220 rpm for 2-4 h to an OD of 0.6-0.8. IPTG was then added to a final concentration of 0.1 mmol and induced at 18°C ​​and agitation at 220 rpm for 24 h.

[0049] (5) The fermentation broth was centrifuged at 4°C and 6000 rpm for 20 min to collect the cells. 10 mL of PBS buffer was added per 1 g of cells to resuspend the cells. The cells were ultrasonically disrupted under the conditions of ultrasonication for 3 s, pause for 5 s, and 30% power. Ultrasonication was performed for 20 min per 10 mL. The disrupted bacterial solution was centrifuged at 4°C and 6000 rpm for 20 min to remove cell debris, and the supernatant was collected.

[0050] (6) Determination of lipase activity: Solution A: 15 mg of p-nitrophenyl palmitate (pNPP) was dissolved in 10 mL of DMSO and stored in a brown bottle at 4°C. Solution B: 1.51 g of tris (hydroxymethyl)aminomethane (Tris) was dissolved in 200 mL of water, 1 mL of Triton-X100, 0.25 g of gum arabic, and 0.72 mL of concentrated hydrochloric acid were added, stirred evenly, the pH was adjusted to 7.5, the volume was adjusted to 250 mL, and stored at 4°C. A and B mixed solution: A and B solutions were mixed in a volume ratio of 1:9 and incubated at 40°C for use. 4.5 mL of substrate solution was added to 0.1 mL of the obtained crude wild-type lipase solution. 4.5 mL of substrate solution was added to 0.1 mL of blank culture medium as a control. The reaction was kept in a water bath at 40°C for 5 min, and 1 mL of acetone was added to terminate the reaction. Lipase hydrolyzes pNPB to release p-nitrophenol pNP, and the solution of p-nitrophenol is yellow-green under alkaline conditions. Enzyme activity is defined as: 1 mL of lipase hydrolyzes pNPB to release 1 μmol of p-nitrophenol in 1 min, which is 1 enzyme activity unit (U), μmol / mL -1 min -1 .

[0051] Example 3: Establishment of a solvent-tolerant mutant library of Candida antarctica lipase B.

[0052] Mutant construction: Specifically, pET25b-CALB was used as the amplification template, and primers were designed based on the sites obtained in Example 1 (42Thr, 47Lys, 144Leu, 145Asp, 154Val, 189Ile, 282Ala, 285Ile), as shown in Table 1. A saturated mutant library was constructed by whole-plasmid PCR. The specific amplification parameters are shown in Table 2. The PCR reaction conditions are as follows:

[0053]

[0054] The amplified product was digested and then recovered by gel recovery. The recovered product was transformed into BL21 (DE3) competent cells and replicated and expressed therein. The plate was coated and cultured. After the monoclonal product was selected for sequencing, the saturation mutant of the target site was obtained after successful sequencing verification.

[0055] Table 1: Primer design for Candida antarctica lipase saturation mutants

[0056]

[0057]

[0058] Table 2: Mutant amplification system (50 μL)

[0059]

[0060] Mutant expression: The cultivation and expression of mutants were carried out in 96-deep well plates. Specifically, single colonies were picked and placed in 96-deep well plates, each containing 500 μL of LB liquid medium containing 100 mg / L ampicillin resistance, and 5 wild-type single colonies were included as negative controls in the 96-well plate; the plate was incubated at 37°C, 300 rpm overnight; 20 μL was transferred to 500 μL of TB liquid medium containing 100 mg / L ampicillin resistance, and incubated at 37°C, 220 rpm for 2-4 h to OD 600 0.6-0.8, 20 μL of IPTG was added to a final concentration of 0.1 mmol, and induced at 18°C, 300 rpm for 24 h.

[0061] Example 4: Determination of solvent tolerance screening method for Candida antarctica lipase B mutants.

[0062] Using a multichannel pipette, 20 μL of lysozyme (10 mg / mL) solution was added to each well to a final concentration of 0.4 mg / mL. Vortex, suspend the bacteria in the 96-deep well plate, ice bath for 30 min, heat shock in a 37°C water bath for 5 min. After ice bath for 30 min, release the crude enzyme solution from the cells, centrifuge at 4000 rpm for 30 min. Take out the upper enzyme solution for use.

[0063] Using 10% (v / v) pyridine solution, the solvent tolerance of the mutants was evaluated. First, 40 μL of enzyme was mixed with 20 μL of prepared pyridine aqueous solution, and incubated in a 96-well plate shaker at 40°C, 500 rpm for 18 min. Immediately after incubation, 180 μL of pNPP A, B mixed solution was added, and reacted at 40°C, 500 rpm for 10 min, OD 410 Determination of absorbance. This group of data was determined for the residual activity of lipase. Replace the pyridine aqueous solution with the same volume of TB medium for subsequent incubation and other operations, and the data determined is the initial activity of the lipase (Table 3).

[0064] The solvent tolerance of lipase can be represented by the pNPP hydrolytic enzyme activity determined above, as follows:

[0065]

[0066] The solvent tolerance of lipase can be characterized as the residual enzyme activity (RRv) relative to the wild-type enzyme, as follows:

[0067]

[0068] wherein R V is the residual activity of the mutant, and R WT is the residual activity of the wild-type enzyme.

[0069] Finally, mutants with significantly improved solvent tolerance were selected and rescreened to further confirm their performance. After further confirmation of solvent tolerance, the strains were cultured in LB medium and sequenced. The strains were mixed with 50% glycerol and stored in a -80°C freezer for long-term storage for future research.

[0070] Table 3: Pyridine tolerance determination method

[0071]

[0072] Example 5: Screening of solvent tolerance of single-point mutants of Candida antarctica lipase B.

[0073] 42Thr, 47Lys, 144Leu, 145Asp, 154Val, 189Ile, 282Ala, and 285Ile were screened according to the solvent tolerance determination method in Example 4. The results are as follows: Figure 2 As shown, among the eight sites screened, 20 point mutations emerged that improved pyridine solvent tolerance. After incubation with 10% (v / v) pyridine aqueous solution for 18 minutes, all exhibited higher residual enzyme activity than the WT. Although all the selected mutants demonstrated improved tolerance, there were still some differences in solvent tolerance between different mutants, and between different mutants at the same site. Notably, sites 42 (T42K, T42Q) and 285 (I285F, I285S, I285N, I285D, I285G) exhibited far higher solvent tolerance than the other single sites, with the highest reaching 2.0-fold the WT residual enzyme activity. Furthermore, sites 144 (L144P, L144Q) and 145 (D145T, D145N, D145I, D145A) were second only in pyridine resistance, with residual enzyme activity reaching up to 29.0%, 1.6-fold that of the WT. However, among the nine sites screened, although pyridine-resistant mutants were identified at sites 47, 189, and 281 using a 96-well plate, the results were minimally different from the WT data. Based on these single-site screening results, the present invention discarded sites 47, 189, and 282 and selected sites 42, 144, 145, 154, and 285 as initiation sites for recombination. This aims to identify superior recombinants and further improve the pyridine tolerance of Candida antarctica lipase B.

[0074] Example 6: Tolerance screening of double mutants of Candida antarctica lipase B mutants.

[0075] Considering the high efficiency of recombination, we selected sites 42, 144, 145, 154, and 285, and also considered the frequency of mutation sites. Finally, we selected 42K, 42Q, 144P, 145T, 145N, 154A, 285F, and 285N as the starting sites and amino acids for recombination. Figure 3 Compared to the results of single-point mutations, most double mutants showed significantly improved pyridine tolerance, particularly at sites 145 and 285. Among them, mutants D145N-I285F (NF) and D145N-I285D (ND) exhibited the best pyridine tolerance. Compared to the residual enzyme activity of the WT, their residual enzyme activity increased by 78.6% and 80.7%, respectively. In other words, in a 10% (v / v) pyridine solution, their solvent tolerance was 5.4 and 5.5 times that of the WT. Furthermore, the double mutant D145T-I285F (TF) also exhibited excellent pyridine tolerance, increasing by 3.7 times that of the WT. Although other recombinant mutants at sites 145 and 285 did not exhibit the same excellent solvent tolerance as the aforementioned mutants, they still showed improvements compared to the single-point mutants at 145 and 285. For example, the residual enzyme activity of D145T-I285D (TD) was 51.0%, a 22.7% and 19.8% increase over D145T and I285D, respectively. These results suggest that sites 145 and 285 have an epistatic effect on pyridine solvent tolerance. Furthermore, recombination analysis of 145 and 285 with other mutations revealed that other double mutants of 145 failed to resist pyridine damage, particularly those stacked with 42Q, whose residual enzyme activity was even lower than that of a single site. In contrast, double mutants stacked with I285F exhibited even greater advantages, with T42K-I285F (KF) and L144P-I285F (PF) showing up to a 3.0-fold increase in tolerance. Similarly, stacking different mutants at the same site exhibited completely different behaviors, a particularly striking example being sites 42 and 285, where residual enzyme activity differed by up to 27.4%. In order to continue to explore the effect of the selected modified sites on the pyridine tolerance of Candida antarctica lipase B, the double mutants were further superimposed.

[0076] Example 7: Tolerance screening of triple mutants of Candida antarctica lipase B mutants.

[0077] From the solvent tolerance results of the double mutants, it can be seen that the superposition effect of sites 145 and 285 is better, and the superposition effect of 285 and other sites is second. In order to further explore whether the tolerance to pyridine is improved after further superposition of the selected modified sites, the present invention selected the top four double mutants (NF, ND, TF, PF) with the best tolerance results as the starting points, and continued to superpose the remaining three sites. Figure 4It is clear that the pyridine tolerance of most mutants decreased significantly after triple stacking. The triple mutant starting with NF had the lowest residual enzyme activity, only 22.3%. This indicates that the addition of the third site adversely affected the pyridine tolerance of NF. Although mutants stacked starting with ND also experienced a significant decrease, T42K-D145N-I285D (KND) maintained its original advantage, with residual enzyme activity comparable to that before stacking and 5.4 times that of WT. A similar situation was observed in the TF series of triple mutants, with most experiencing a decrease in residual enzyme activity, while some maintained the solvent resistance advantage of the double mutant. Finally, the data show that the triple mutant starting with PF also failed to achieve an epistatic effect, thereby synergistically improving the enzyme's pyridine tolerance. Notably, both ND and TF suffered losses when stacked with sites 144 and 154, while stacking with site 42 alone retained good pyridine tolerance. Based on this data, it is speculated that when position 42 is mutated to K, the tolerance to the double mutant is neutral. Subsequently, this study cultured WT, NF, ND, and KND and used them as catalysts for the enzymatic synthesis of ester compounds.

[0078] Example 8: Expansion of solvent tolerance of dominant mutants.

[0079] In order to further verify whether the obtained multiple mutants also have improved tolerance to other solvents, we expanded the solvents for tolerance determination. The solvents finally selected were the three most common solvents for enzyme catalysis reactions - methanol, DMSO, and isopropanol, and the mutants selected were the three best performing ND, NF, and NDK. First, the WT residual enzyme activity in the three solvents was limited through preliminary experiments, and the concentrations of the three solvents were determined, which were 50% (v / v) methanol solution, 67.7% (v / v) DMSO solution, and 50% (v / v) isopropanol solution. After determining the solvent ratio, the tolerance was uniformly measured by pNPP hydrolase activity. By Figure 5 It can be seen that ND can still maintain good tolerance in 50% (v / v) methanol solution and 67.7% (v / v) DMSO solution, especially in DMSO, where it can be increased to 7.5 times that of WT. Although the tolerance of ND in isopropanol solvent is not much improved, it still exceeds that of the other two mutants. NF's tolerance among the three solvents is second only to that of ND. In methanol, NF's resistance is comparable to that of ND, but in DMSO and isopropanol, NF's resistance is half that of ND. Finally, the triple mutant NDK has poor tolerance. In the three solvents other than pyridine, its maximum resistance is only 1.8 times that of WT. In summary, the solvent tolerance of the double mutant obtained in this experiment is relatively wide, and it is expected that it can be further applied to different enzyme-catalyzed reactions.

[0080] Example 9: Application of the advantageous mutants in the synthesis of ester compounds.

[0081] The advantageous mutants (D145N-I285F, D145N-I285D, T42K-D145N-I285D) were applied as catalysts in different enzymatic reactions (vitamin E succinate, phytosterol esters, sucrose laurate, glucose laurate). The specific synthesis methods are as follows:

[0082] (1) Vitamin E 0.39 mmol, succinic anhydride 1.19 mmol were weighed into a 15 mL reaction bottle, and 400 μL of enzyme solution, 2 mL of DMSO were added respectively, and reacted at 40°C, 400 rpm on a magnetic stirrer for 24 h, and quantified by HPLC-UV.

[0083] (2) Phytosterol 1 mmol, oleic acid 6 mmol were weighed into a 15 mL reaction bottle, and 400 μL of enzyme solution, 2 mL of n-hexane were added respectively, and reacted at 45°C, 400 rpm on a magnetic stirrer for 24 h, and quantified by GC.

[0084] (3) Sucrose 0.087 mmol, lauryl vinyl ester 0.348 mmol were weighed into a 15 mL reaction bottle, and 400 μL of enzyme solution, 2 mL of pyridine were added respectively, and reacted at 40°C, 400 rpm on a magnetic stirrer for 24 h, and quantified by HPLC-ELSD.

[0085] (4) Glucose 0.056 mmol, lauryl vinyl ester 0.166 mmol were weighed into a 15 mL reaction bottle, and 400 μL of enzyme solution, 2 mL of DMSO:2M2B (4:1) were added respectively, and reacted at 40°C, 400 rpm on a magnetic stirrer for 24 h, and quantified by HPLC-ELSD.

[0086] As Figure 6As shown, Candida antarctica lipase B mutants with high pyridine tolerance also achieved significant improvements in the synthesis of ester compounds. Compared to the WT, both the double mutants NF and ND, as well as the triple mutant KND, improved the efficiency of ester synthesis. In the synthesis of glucose laurate, we found that ND, which had the highest DMSO tolerance, achieved the best catalytic efficiency, with a 19.5% improvement over the WT. This was also demonstrated in the synthesis of vitamin E succinate, phytosterol esters, and sucrose laurate, where the catalytic efficiency of the dominant mutants with improved solvent tolerance also increased. For example, ND, with the highest tolerance, had the highest catalytic efficiency for vitamin E succinate synthesis, a 14.6% improvement over the WT. In summary, we observed a positive correlation between organic solvent tolerance and the yield of lipase-catalyzed reactions, suggesting that improving the organic solvent tolerance of lipases to enhance their catalytic efficiency is a feasible strategy.

[0087] The amino acid sequence of the mutant ND is shown in SEQ ID NO.3, and the nucleotide sequence thereof is shown in SEQ ID NO.4.

[0088] SEQ ID NO.3:

[0089] LPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLS

[0090] TQLGYTPCWISPPPFMLNDTQVNTEYMVNAITALYAGSGNNKLPVLTWSQGGL

[0091] VAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAGPLNALAVSAPSVWQQTTGSA

[0092] LTTALRNAGGLTQIVPTTNLYSATDEIVQPQVSNSPLDSSYLFNGKNVQAQAVC

[0093] GPLFVIDHAGSLTSQFSYVVGRSALRSTTGQARSADYGITDCNPLPANDLTPEQ

[0094] KVAAAALLAPAAAADVAGPKQNCEPDLMPYARPFAVGKRTCSGIVTPLESEQ ID NO.4:

[0095] CTGCCTAGTGGTAGCGATCCGGCCTTTAGCCAGCCGAAAAGCGTGCTGGAT

[0096] GCAGGTCTGACCTGCCAGGGCGCCAGTCCGAGCAGCGTGAGCAAACCGAT

[0097] TCTGCTGGTGCCGGGTACAGGCACCACCGGTCCGCAGAGCTTTGATAGCAA

[0098] TTGGATTCCGCTGAGCACCCAGCTGGGTTATACCCCGTGTTGGATTAGCCC

[0099] GCCGCCGTTTATGCTGAATGATACCCAGGTTAATACCGAATATATGGTGAA

[0100] TGCAATTACCGCACTGTATGCCGGCAGTGGCAATAATAAGCTGCCGGTTCT

[0101] GACCTGGAGCCAGGGCGGTCTGGTTGCCCAGTGGGGTCTGACCTTTTTCCC

[0102] GAGCATTCGCAGTAAAGTTGATCGCCTGATGGCCTTTGCACCGGATTATAA

[0103] AGGTACAGTTCTGGCCGGCCCGCTGTTAGCCCTGGCAGTTAGCGCACCGAG

[0104] CGTGTGGCAGCAGACCACCGGTAGTGCCCTGACCACCGCCCTGCGTAATGC

[0105] AGGTGGTCTGACCCAGATTGTTCCGACCACCAATCTGTATAGTGCAACCGA

[0106] TGAAATTGTGCAGCCGCAGGTGAGTAATAGTCCGCTGGATAGCAGCTATCT

[0107] GTTTAATGGTAAAAATGTTCAGGCCCAGGCCGTTTGCGGCCCGCTGTTTGT

[0108] GATTGATCATGCAGGTAGTCTGACCAGTCAGTTTAGTTATGTTGTTGGCCG

[0109] CAGCGCCCTGCGCAGCACCACAGGTCAGGCACGCAGTGCAGATTATGGTA

[0110] TTACCGATTGCAATCCGCTGCCGGCAAATGATCTGACCCCGGAACAGAAA

[0111] GTGGCAGCAGCAGCCCTGCTGGCCCCGGCAGCAGCAGCGGATGTTGCAGG

[0112] CCCGAAACAGAATTGCGAACCGGATTCTGATGCCGTATGCCCGTCCGTTTGC

[0113] AGTTGGTAAACGTACCTGCAGTGGCATTGTTACCCCG

[0114] The present invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent replacement are within the protection scope required by the present invention.

Claims

1. A molecular engineering design method for improving the solvent tolerance of lipase, characterized in that: The following steps are involved: (1) Lipase B from Candida antarctica was selected as the target for modification; the crystal structure and amino acid sequence of the lipase to be modified were obtained from the protein database (PDB); (2) The channel and pocket of Candida antarctica lipase B were determined using Fpocket and CaverDock software, and the highly conserved sites were eliminated using ConSurf software to finally determine the modification sites; (3) For the potential target sites identified in step (2), primers were designed using NNK degenerate codons to perform site-directed saturation mutagenesis, and the modified lipase was tested for pyridine tolerance using the p-nitrophenol method to screen single mutants with improved pyridine tolerance; (4) performing double recombination on the single mutants screened in step (3), and performing pyridine tolerance test on the double mutants; (5) The double mutants screened in step (4) were further recombined to obtain triple mutants. On this basis, pyridine tolerance was determined using the p-nitrophenol method.

2. The molecular modification design method for improving the solvent tolerance of lipase according to claim 1, characterized in that: The solvent tolerance determination includes the determination of relative enzyme activity and residual enzyme activity.

3. The molecular modification design method for improving the solvent tolerance of lipase according to claim 1, characterized in that: The screened advantageous mutants were used as catalysts in the synthesis of vitamin E succinate, phytosterol esters, sucrose laurate and glucose laurate.

4. The molecular modification design method for improving the solvent tolerance of lipase according to claim 1, characterized in that: (1) Lipase B from Antarctic Candida was selected as the target for modification. First, the active pocket was predicted using the Fpocket software. Based on this, the final target was determined from the seven active pockets given by the software based on the position of the catalytic triad of Antarctic Candida lipase B. The amino acids in the pocket are: 39Gly, 40Thr, 42Thr, 47Lys, 104Trp, 105Ser, 134Asp, 138Thr, 144Leu, 154Val, 157Gln, 188Glu, 189Ile, 190Val, 224His, 281Ala, 282Ala, 285Ile. (2) The channel sites of Antarctic Candida lipase B were predicted by CaverDock software. With the catalytic triad (Ser105-His224-Asp187) and the oxygen anion hole (Thr40, Gln106) as the starting sites, three channels were obtained. The first channel contains the following sites: 40Thr, 134Asp, 138Thr, 144Leu, 145Asp, 154Val, 157Gln, 189Ile, 224His, 285Ile; the second channel contains the following sites: The lower site: 39Gly, 40Thr, 104Trp, 105Ser, 138Thr, 154Val, 157Gln, 189Ile, 190Val, 224His, 282Ala, 285Ile; the third channel contains the following sites: 39Gly, 40Thr, 104Trp, 105Ser, 134Asp, 138Thr, 154Val, 157Gln, 189Ile, 190Val, 224His, 282Ala, 285Ile; (3) ConSurf software was used to identify the evolutionary conservation of amino acid residues in Antarctic Candida lipase B to avoid damage to the enzyme structure after modification. Based on this, sites in the active pocket and channel were screened, and amino acid sites with a conservation of ≥6, that is, highly conserved sites, were excluded. Eight sites were screened: 42Thr, 47Lys, 144Leu, 145Asp, 154Val, 189Ile, 282Ala, and 285Ile.

5. The molecular engineering design method for improving the solvent tolerance of lipase according to claim 1, characterized in that: Fpocket and CaverDock software were used to determine the channel and pocket of Candida antarctica lipase B, and then ConSurf software was used to eliminate the highly conserved sites, and finally the modification sites were determined; For the modified site, primers were designed using NNK degenerate codons to perform site-directed saturation mutagenesis, and the modified lipase was tested for pyridine tolerance using the p-nitrophenol method to screen single mutants with improved pyridine tolerance. Based on the single-point screening, recombination was performed, and the obtained double and triple mutants were tested for pyridine tolerance, methanol tolerance, DMSO tolerance, and isopropanol tolerance. Finally, the screened dominant mutants were used as catalysts in the synthesis of vitamin E succinate, phytosterol esters, sucrose laurate, and glucose laurate.

6. The use of the Candida antarctica lipase B mutant according to claim 5, characterized in that: Antarctic Candida antarctica lipase B mutants are used in the synthesis of vitamin E succinate, phytosterol esters, glucose laurate, and sucrose laurate.

7. The use of the Candida antarctica lipase B mutant according to claim 5, characterized in that: For the synthesis of vitamin E succinate, the catalytic activity of mutant ND increased by 12.7%, that of D145N-I285F increased by 14.6%, and that of T42K-D145N-I285D increased by 8.5% compared with the wild type. For the synthesis of phytosterol esters, the catalytic activity of mutant ND increased by 14.1%, that of D145N-I285F increased by 18.0%, and that of T42K-D145N-I285D increased by 10.5% compared with the wild type. For the glucose laurate synthesis reaction, the catalytic activity of the mutant ND was 11.5% higher than that of the wild type, the catalytic activity of D145N-I285F was 19.5% higher than that of the wild type, and the catalytic activity of T42K-D145N-I285D was 8.5% higher than that of the wild type; for the sucrose laurate synthesis reaction, the catalytic activity of the mutant ND was 17.0% higher than that of the wild type, the catalytic activity of D145N-I285F was 13.6% higher than that of the wild type, and the catalytic activity of T42K-D145N-I285D was 13.0% higher than that of the wild type.

8. The use of the Candida antarctica lipase B mutant according to claim 5, characterized in that: The amino acid sequence of the Candida antarctica lipase B mutant ND is shown in SEQ ID NO.3, and the nucleotide sequence thereof is shown in SEQ ID NO.4.

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