A molecular modification design method for improving lipase solvent tolerance and its mutants

Abstract

This invention provides a rational design method for improving the solvent tolerance of lipases. The *Candida antarcticis* lipase B mutant of this invention exhibits a maximum tolerance of 5.5 times that of the mean lipase (WT) in 10% (v / v) pyridine solvent, and its tolerance in 50% (v / v) methanol, 67.7% (v / v) DMSO, and 50% (v / v) isopropanol solutions is also higher than that of the WT. The dominant mutants (D145N-I285F, D145N-I285D, and T42K-D145N-I285D) as catalysts show improved synthesis efficiency in different enzyme-catalyzed reactions. Specifically, the synthesis efficiency of vitamin E succinate is increased by 14.6% compared to WT, the synthesis efficiency of phytosterol esters is increased by up to 18.0% compared to WT, the synthesis efficiency of sucrose laurate is increased by up to 17.0% compared to WT, and the synthesis efficiency of glucose laurate is increased by 19.5% compared to WT.

Description

Technical Field

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

[0002] Ester compounds, as important substances widely used in food, pharmaceuticals, daily chemicals, and biofuels, have long relied on traditional acid- or base-catalyzed chemical synthesis methods for industrial production. While this method offers advantages such as ease of operation and high yield, it also suffers from drawbacks including high energy consumption, numerous byproducts, difficulties in separation and purification, and chemical catalyst residues. 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. Industrial production typically uses highly polar organic solvents as reaction media, such as methanol, isopropanol, pyridine, and DMSO. However, these highly polar solvents can severely affect enzyme performance and even lead to enzyme inactivation. Generally, highly polar organic solvents reduce enzyme activity by altering enzyme conformation and stripping essential water molecules from the enzyme surface, thereby affecting synthesis efficiency. Although enzyme immobilization and chemical modification techniques have been attempted to improve enzyme performance, the properties of enzymes, especially their solvent tolerance, cannot be significantly improved by these methods. Summary of the Invention

[0003] This invention provides a molecular modification design method to improve the solvent tolerance of lipases. *Candida antarcticus* lipase B possesses excellent stereoselectivity and broad substrate adaptability, making it an important biocatalyst in the field of lipase-catalyzed synthesis. The channels and pockets of *Candida antarcticus* lipase B were initially identified using Fpocket and CaverDock software, and then overly conserved sites were eliminated using ConSurf software, ultimately determining the modification sites. A mutant library was then constructed using site-directed saturation mutagenesis with NNK degenerate codons, and superior mutants with improved pyridine tolerance were screened using a spectrophotometric microplate screening method. The *Candida antarcticus* lipase B mutant of this invention exhibits a maximum tolerance increase of 5.5 times compared to the WT in 10% (v / v) pyridine solvent, and higher tolerance than the WT in 50% (v / v) methanol, 67.7% (v / v) DMSO, and 50% (v / v) isopropanol solutions. Using the superior mutant as a catalyst, the 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 18.0% higher than that of WT, the catalytic synthesis efficiency of sucrose laurate was 17.0% higher than that of WT, and the catalytic synthesis efficiency of glucose laurate was 19.5% higher than that of WT.

[0004] To solve the technical problem of this invention, the proposed technical solution includes the following steps: a molecular modification design method for improving lipase solvent tolerance, characterized by including the following steps:

[0005] (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.

[0006] (2) The channels and pockets of Candida antarcticis lipase B were determined using Fpocket and CaverDock software, and then the sites with excessively high conservation were excluded using ConSurf software, and the modification sites were finally determined.

[0007] (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 pyridine tolerance of the modified lipase was determined using the p-nitrophenol method to screen for single mutants with improved pyridine tolerance.

[0008] (4) The single mutants screened in step (3) were subjected to double recombination, and the double mutants were subjected to pyridine tolerance test.

[0009] (5) The double mutants screened in step (4) are further recombined to obtain triple mutants. Based on this, pyridine tolerance is determined by the p-nitrophenol method.

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

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

[0012] Preferably, (1) Lipase B from Candida antarctica was selected as the target for modification; firstly, the active pockets were predicted using Fpocket software; based on this, we determined the final target from the 7 active pockets given by the software by combining the position of the Candida antarctica lipase B catalytic triplet; the amino acids in the pockets are: 39Gly, 40Thr, 42Thr, 47Lys, 104Trp, 105Ser, 134Asp, 138Thr, 144Leu, 154Val, 157Gln, 188Glu, 189Ile, 190Val, 224His, 281Ala, 282Ala, 285Ile;

[0013] (2) Channel sites of Candida antarctica lipase B were predicted using CaverDock software. Using the catalytic triplet (Ser105-His224-Asp187) and the oxygen anion hole (Thr40, Gln106) as 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... Lower loci: 39Gly, 40Thr, 104Trp, 105Ser, 138Thr, 154Val, 157Gln, 189Ile, 190Val, 224His, 282Ala, 285Ile; The third channel includes the following loci: 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 Candida antarctica lipase B to avoid the destruction of the modified enzyme structure. Based on this, sites in the active pocket and channels were screened, and amino acid sites with conservation ≥6 were excluded, i.e., highly conserved sites were excluded. Eight sites were obtained: 42Thr, 47Lys, 144Leu, 145Asp, 154Val, 189Ile, 282Ala, and 285Ile.

[0015] Preferably, the channels and pockets of Candida antarctica lipase B were determined using Fpocket and CaverDock software, and then highly conserved sites were eliminated using ConSurf software to finally determine the modification sites. For the modification sites, primers were designed using NNK degenerate codons to perform site-directed saturation mutagenesis, and the pyridine tolerance of the modified lipase was determined using the p-nitrophenol method to screen for single mutants with improved pyridine tolerance. Based on the single-site screening, recombination was performed, and the resulting double and triple mutants were tested for pyridine tolerance, methanol tolerance, DMSO tolerance, and isopropanol tolerance. Finally, the selected dominant mutants were used as catalysts in the synthesis of vitamin E succinate, phytosterol esters, sucrose laurate, and glucose laurate.

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

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

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

[0019] 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);

[0020] 2) The channels and pockets of Candida antarctica lipase B were determined using Fpocket and CaverDock software, and then sites with excessive conservation (>6) were excluded using ConSurf software, finally determining the modification sites;

[0021] 3) For the potential target sites identified in step 2), site-directed saturation mutagenesis was performed using NNK degenerate codons, and the solvent tolerance of the modified lipase was determined using the p-nitrophenol method to screen for single mutants with improved pyridine tolerance.

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

[0023] 5) The double mutants screened in step 4) were further recombinated to obtain triple mutants. Based on this, pyridine tolerance was determined using the p-nitrophenol method.

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

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

[0026] Beneficial effects:

[0027] This invention utilizes computer-aided technology to modify *Candida antarcticus* lipase B, enhancing its solvent tolerance and enabling its application in ester synthesis. First, the active pockets and channel sites of *Candida antarcticus* lipase B were predicted using Fpocket and CaverDock software. Overly conserved sites were then eliminated using ConSurf software, ultimately determining the modification sites. Based on this, a saturated mutant library was established through primer design, plasmid extraction, PCR, gel extraction, transformation, and culture experiments, followed by pyridine tolerance screening. Subsequent recombination identified mutants with excellent pyridine tolerance. Finally, the pyridine-tolerant mutant was expressed separately and used as a catalyst in ester synthesis, improving its synthetic efficiency.

[0028] This invention provides a rational design method for improving the solvent tolerance of lipases. The channels and pockets of *Candida antarcticus* lipase B were initially determined using Fpocket and CaverDock software, and then highly conserved sites were eliminated using ConSurf software, ultimately determining the modification sites. A mutant library was then constructed using site-directed saturation mutagenesis with NNK degenerate codons, and superior mutants with improved pyridine tolerance were screened using a spectrophotometric microplate screening method. The *Candida antarcticus* lipase B mutant of this invention showed a maximum tolerance increase of 5.5 times that of the WT in 10% (v / v) pyridine solvent, and higher tolerance than the WT in 50% (v / v) methanol, 67.7% (v / v) DMSO, and 50% (v / v) isopropanol solutions. The dominant mutants (D145N-I285F, D145N-I285D, and T42K-D145N-I285D) improved the synthesis efficiency in various enzyme-catalyzed reactions when used as catalysts. Specifically, the synthesis efficiency of vitamin E succinate was increased by 14.6% compared to the mean lipase (WT), phytosterol esters by up to 18.0%, sucrose laurate by up to 17.0%, and glucose laurate by up to 19.5%. This demonstrates that the strategy of improving enzyme catalytic efficiency by enhancing lipase tolerance is feasible, providing a new approach for other enzyme-catalyzed reactions. Attached Figure Description

[0029] Figure 1 Solvent tolerance selection strategy diagram;

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

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

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

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

[0034] Figure 6 : Synthesis yields of different enzymatic reactions (WT: wild-type Candida antarcticis lipase B, ND: Candida antarcticis lipase B mutant D145N-I285D, NF: D145N-I285F, NDK: T42K-D145N-I285D). Detailed implementation method:

[0035] The present invention will be further described below with reference to specific embodiments, but is not limited thereto. Unless otherwise specified, the reagent raw materials described in the following embodiments are all commercially available common raw materials, and the reagents are prepared using conventional methods. Methods not detailed in the embodiments are all conventional operations in the art.

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

[0037] (1) Lipase B from *Candida antarcticus* was selected as the target for modification. First, the active pockets were predicted using Fpocket software. Based on this, we determined the final target from the seven active pockets provided by the software, considering the position of the *Candida antarcticus* lipase B catalytic triplet. The amino acids in the pockets were: 39Gly, 40Thr, 42Thr, 47Lys, 104Trp, 105Ser, 134Asp, 138Thr, 144Leu, 154Val, 157Gln, 188Glu, 189Ile, 190Val, 224His, 281Ala, 282Ala, and 285Ile.

[0038] (2) Channel sites for Candida antarctica lipase B were predicted using CaverDock software. Three channels were obtained using the catalytic triplet (Ser105-His224-Asp187) and the oxygen anion hole (Thr40, Gln106) as starting sites. 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 pathway includes the following sites: 39Gly, 40Thr, 104Trp, 105Ser, 134Asp, 138Thr, 154Val, 157Gln, 189Ile, 190Val, 224His, 282Ala, and 285Ile.

[0039] (3) ConSurf software was used to identify the evolutionary conservation of amino acid residues in Candida antarctica lipase B to avoid disrupting the modified enzyme structure. Based on this, sites in the active pocket and channels were screened, excluding amino acid sites with a conservation ≥6, i.e., highly conserved sites. Finally, eight sites were selected: 42Thr, 47Lys, 144Leu, 145Asp, 154Val, 189Ile, 282Ala, and 285Ile. The results of the comprehensive site selection strategy are as follows: Figure 1 As shown.

[0040] The amino acid sequence of Candida antarcticis lipase B (SEQ ID NO.1):

[0041] LPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMVNAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAGPLDALAVSAPSVWQQT TGSALTTALRNAGGLTQIVPTTNLYSATDEIVQPQVSNSPLDSSYLFNGKNVQAQAVCGPLFVIDHAGSLTSQFSYVVGRSALRSTTGQARSADYGITDCNPLPANDLTPEQKVAAAALLAPAAAAIVAGPKQNCEPDLMPYARPFAVGKRTCSGIVTP

[0042] Nucleotide sequence of Candida antarcticis lipase B (SEQ ID NO.2):

[0043] CTGCCTAGTGGTAGCGATCCGGCCTTTAGCCAGCCGAAAAGCGTGCTGGATGCAGGTCTGACCTGCCAGGGCGCCAGTCCGAGCAGCGTGAGCAAACCGATTCTGCTGGTGCCGGGTACAGGCACCACCGGTCCGCAGAGCTTTGATAGCAATTGGATTCCGCTGAGCACCCAGCTGGGTTATACCCCGTGTTGGATTAGCCCGCCGCCGTTTATGCTGAATGATACCCAGGTTAATACCGAATATATGGTGAATGCAATTACCGCACTGTATGCCGGCAGTGGCAATAATAAGCTGCCGGTTCTGACCTGGAGCCAGGGCGGTCTGGTTGCCCAGTGGGGTCTGACCTTTTTCCCGAGCATTCGCAGTAAAGTTGATCGCCTGATGGCCTTTGCACCGGATTATAAAGGTACAGTTCTGGCCGGCCCGCTGGATGCCCTGGCAGTTAGCGCACCGAGCGTGTGGCAGCAGACCACCGGTAGTGCCCTGACCACCGCCCTGCGTAATGCAGGTGGTCTGACCCAGATTGTTCCGACCACCAATCTGTATAGTGCAACCGATGAAATTGTGCAGCCGCAGGTGAGTAATAGTCCGCTGGATAGCAGCTATCTGTTTAATGGTAAAAATGTTCAGGCCCAGGCCGTTTGCGGCCCGCTGTTTGTGATTGATCATGCAGGTAGTCTGACCAGTCAGTTTAGTTATGTTGTTGGCCGCAGCGCCCTGCGCAGCACCACAGGTCAGGCACGCAGTGCAGATTATGGTATTACCGATTGCAATCCGCTGCCGGCAAATGATCTGACCCCGGAACAGAAAGTGGCAGCAGCAGCCCTGCTGGCCCCGGCAGCAGCAGCGATTGTTGCAGGCCCGAAACAGAATTGCGAACCGGATCTGATGCCGTATGCCCGTCCGTTTGCAGTTGGTAAACGTACCTGCAGTGGCATTGTTACCCCG

[0044] Example 2: Expression of Candida antarctica lipase B.

[0045] (1) Using the lipase B gene from Candida antarctica as a template, the enzyme was named CALB and inserted into the pET25b(+) vector with EcoRI-NcoI as the cloning site. The wild recombinant expression plasmid pET25b-CALB was synthesized.

[0046] (2) The above plasmid was transformed into BL21(DE3) competent cells by heat shock. 500 μL of LB medium without any antibiotics was added and incubated at 37°C for 60 min. After a brief centrifugation at 3000 rpm, the remaining 100 μL of supernatant was mixed with the cells. An appropriate amount was spread on an LB plate containing 100 mg / L ampicillin and cultured overnight at 37°C until a clear single colony grew.

[0047] (3) Pick a single colony from the overnight plate into 5 mL of LB liquid medium containing 100 mg / L ampicillin resistance, and culture it in a shaker at 37°C for 12 h to preserve the bacteria and sequence them.

[0048] (4) Select the successfully verified single clones and culture them in LB liquid medium containing 100 mg / L ampicillin resistance for 12-16 h at 37 °C and 220 rpm. Transfer 1-2% of the clones to 50 mL of TB medium containing 100 mg / L ampicillin resistance and culture them at 37 °C and 220 rpm for 2-4 h until the OD reaches 0.6-0.8. Add IPTG to a final concentration of 0.1 mmol and induce for 24 h at 18 °C and 220 rpm.

[0049] (5) Centrifuge the fermentation broth at 4℃ and 6000rpm for 20min to collect the cells. Add 10mL of PBS buffer to each 1g of cells to resuspend the cells. Perform sonication at 3s-pause-5s and 30% power for 20min of each 10mL of PBS buffer. Centrifuge the disrupted broth at 4℃ and 6000rpm for 20min to remove cell debris and collect the supernatant.

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

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

[0052] Mutant construction: Specifically, using pET25b-CALB as the amplification template, 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 using whole-plasmid PCR. Specific amplification parameters are shown in Table 2, and the PCR reaction conditions are as follows:

[0053]

[0054] The amplified products were digested and then gel-recovered. The recovered products were transformed into BL21(DE3) competent cells and replicated and expressed therein. The cells were plated, cultured, and single clones were selected for sequencing. After successful sequencing verification, saturated mutants of the target site were obtained.

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

[0056]

[0057]

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

[0059]

[0060] Mutant Expression: Mutants were cultured and expressed in 96-well plates. Specifically, single clones were picked and placed in 96-well plates, each well containing 500 μL of LB liquid medium containing 100 mg / L ampicillin resistance. Five wild-type single clones were included in each well as a negative control. The plates were incubated overnight at 37°C and 300 rpm. 20 μL of the mutant was then transferred to 500 μL of TB liquid medium containing 100 mg / L ampicillin resistance and incubated at 37°C and 220 rpm for 2-4 hours until OD (Organic Growth Rate) was reached. 600 Add 20 μL of IPTG to a final IPTG concentration of 0.1 mmol, and induce for 24 h at 18 °C and 300 rpm.

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

[0062] Add 20 μL of lysozyme (10 mg / mL) solution to each well using a multichannel pipette to achieve a final concentration of 0.4 mg / mL. Vortex to fully suspend the bacterial cells in the 96-well plate, incubate on ice for 30 min, then heat shock in a 37°C water bath for 5 min. Afterward, incubate on ice for 30 min to release the crude enzyme solution from the cells, and centrifuge at 4000 rpm for 30 min. Collect the supernatant enzyme solution for later use.

[0063] Solvent tolerance of the mutant was assessed using a 10% (v / v) pyridine solution. First, 40 μL of enzyme was mixed with 20 μL of prepared pyridine aqueous solution and incubated for 18 min at 40°C and 500 rpm in a 96-well plate shaker. Immediately after incubation, 180 μL of a mixture of pNPP A and B was added, and the reaction was carried out for 10 min at 40°C and 500 rpm. OD 410 Absorbance was measured. This set of data represents the residual activity of lipase. After replacing the pyridine aqueous solution with the same volume of TB medium for subsequent incubation and other operations, the measured data represent the initial activity of lipase (Table 3).

[0064] Lipase solvent tolerance can be expressed by the pNPP hydrolase activity measured above, as shown in the following formula:

[0065]

[0066] The solvent tolerance of lipases can be characterized by their residual enzyme activity (RRv) relative to the wild-type enzyme, as shown below:

[0067]

[0068] Among them, R V It is the residual activity of the mutant, R WT It is the residual activity of wild-type enzymes.

[0069] Finally, mutants with significantly improved solvent tolerance were selected for further screening to confirm their performance. After further confirmation of solvent tolerance, the strains were cultured in LB medium and sequenced. The strains were then mixed with 50% glycerol and stored at -80°C for long-term use in subsequent studies.

[0070] Table 3: Methods for determining pyridine tolerance

[0071]

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

[0073] According to the solvent tolerance test method in Example 4, 42Thr, 47Lys, 144Leu, 145Asp, 154Val, 189Ile, 282Ala, and 285Ile were screened, and the results are as follows: Figure 2 As shown, 20 point mutations exhibited improved pyridine solvent tolerance among the 8 selected sites. After incubation with 10% (v / v) pyridine aqueous solution for 18 min, all showed higher residual enzyme activity than the WT (wt) level. Although all screened mutants showed improved tolerance, there were still some differences in solvent tolerance between different mutants, and between different mutants at the same site. Notably, the solvent tolerance at sites 42 (T42K, T42Q) and 285 (I285F, I285S, I285N, I285D, I285G) was significantly higher than the residual enzyme activity at other single sites, reaching up to 2.0 times that of the WT residual enzyme activity. Furthermore, the pyridine resistance at sites 144 (L144P, L144Q) and 145 (D145T, D145N, D145I, D145A) was second only to these, with residual enzyme activity reaching a maximum of 29.0%, 1.6 times that of the WT. However, among the nine selected sites, sites 47, 189, and 281, while yielding pyridine-resistant mutants through 96-well plate screening, showed little difference in efficacy compared to the WT data. Based on the results of these single-site screenings, this invention discarded sites 47, 189, and 282, selecting sites 42, 144, 145, 154, and 285 as starting sites for recombination. The aim is to identify superior recombinants, thereby further improving the pyridine tolerance of Candida antarctica lipase B.

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

[0075] Considering the high efficiency of recombination, sites 42, 144, 145, 154, and 285 were selected, along with the frequency of mutation sites. Ultimately, 42K, 42Q, 144P, 145T, 145N, 154A, 285F, and 285N were chosen as the initiation sites and amino acids for recombination. Figure 3 It can be seen that, compared with the results of single-point mutations, most duplex mutants showed a significant improvement in pyridine tolerance, especially the recombination at sites 145 and 285. Among them, mutants D145N-I285F (NF) and D145N-I285D (ND) exhibited the best pyridine tolerance. Compared with the residual enzyme activity of WT, they were increased by 78.6% and 80.7%, respectively, meaning that in a 10% (v / v) pyridine solution, their solvent tolerance was 5.4 and 5.5 times that of WT. In addition, the duplex mutant D145T-I285F (TF) also showed good pyridine tolerance, improved to 3.7 times that of WT. Although other recombinant mutants at sites 145 and 285 did not show the same excellent solvent tolerance as the above mutants, they were still improved compared with the single-point mutants at sites 145 and 285. For example, the residual enzyme activity of D145T-I285D(TD) was 51.0%, which was 22.7% and 19.8% higher than that of D145T and I285D, respectively. These results indicate that sites 145 and 285 have an epistatic effect on pyridine solvent tolerance. Furthermore, the recombination of sites 145 and 285 with other mutant sites was analyzed: other double mutants of 145 did not resist pyridine damage to the enzyme, especially the mutant superimposed with 42Q, whose residual enzyme activity was even lower than that of the single site. Relatively speaking, the double mutant superimposed with I285F had a greater advantage, with T42K-I285F(KF) and L144P-I285F(PF) showing the highest improvement in tolerance by up to 3.0 times. Similarly, the superposition states of different mutants at the same site were completely different, which was perfectly demonstrated at sites 42 and 285, with a maximum difference in residual enzyme activity of 27.4%. To further explore the effect of the selected modification sites on the pyridine tolerance of Candida antarcticis lipase B, the modification was further superimposed on the basis of the double mutant.

[0076] Example 7: Tolerance screening of triple mutant of Candida antarctic lipase B mutant.

[0077] The solvent tolerance results of the duplex mutants show that the superposition of sites 145 and 285 has a better effect, while the superposition of sites 285 with other sites has a lesser effect. To further explore whether the superposition of the selected modified sites improves tolerance to pyridine, this invention selected the top four duplex mutants (NF, ND, TF, PF) with the highest tolerance results from the above screening as starting points, and continued to superimpose the remaining three sites. Figure 4It is known that the pyridine tolerance of most mutants decreased significantly after triple superposition. The residual enzyme activity of the triple mutant starting at NF was as low as 22.3%. This indicates that the superposition of the third site negatively impacted the pyridine tolerance of NF. Although mutants starting from ND also showed a significant decrease, the T42K-D145N-I285D (KND) mutant maintained its original advantage, with residual enzyme activity comparable to that before superposition, 5.4 times that of WT. A similar situation occurred in the TF series of triple mutants, where most residual enzyme activity decreased, while some retained the solvent resistance advantage of the duplex mutant. Finally, the data shows that the triple mutant starting at PF also failed to achieve an epistatic effect, thus failing to synergistically improve the enzyme's pyridine tolerance. It is noteworthy that both ND and TF showed losses when superimposed on sites 144 and 154, while only superposition with site 42 maintained good pyridine tolerance. Based on this data, it is inferred that when the mutation at position 42 is changed to K, the tolerance to the double mutant has a neutral effect. 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 in superior mutants.

[0079] To further verify whether the obtained multiple mutants also exhibited increased tolerance to other solvents, we expanded the solvents used for tolerance testing. The solvents were ultimately selected from the three most commonly used solvents for enzyme-catalyzed reactions—methanol, DMSO, and isopropanol—while the mutants were chosen from the best-performing ND, NF, and NDK. First, preliminary experiments limited the residual WT enzyme activity in the three solvents, determining the concentrations as 50% (v / v) methanol, 67.7% (v / v) DMSO, and 50% (v / v) isopropanol solutions. After determining the solvent ratios, tolerance was uniformly measured using pNPP hydrolase activity. Figure 5 It was found that ND maintained good tolerance in 50% (v / v) methanol and 67.7% (v / v) DMSO solutions, especially in DMSO where its tolerance increased by up to 7.5 times that of WT. Although the increase in tolerance of ND in isopropanol was not significant, it still exceeded that of the other two mutants. NF showed the second-best tolerance in the three solvents. In methanol, NF's resistance was comparable to that of ND, but in DMSO and isopropanol, NF's resistance was half that of ND. Finally, the triple mutant NDK showed poor tolerance, with its resistance in the three solvents other than pyridine only reaching a maximum of 1.8 times that of WT. In summary, the dual mutants obtained in this experiment exhibited a wide range of solvent tolerance and are expected to be further applied to different enzyme-catalyzed reactions.

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

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

[0082] (1) Weigh 0.39 mmol of vitamin E and 1.19 mmol of succinic anhydride and add them to a 15 mL reaction flask. Add 400 μL of enzyme solution and 2 mL of DMSO respectively. React in a magnetic stirrer at 40 °C and 400 rpm for 24 h and quantify by HPLC-UV.

[0083] (2) Weigh 1 mmol of phytosterol and 6 mmol of oleic acid into a 15 mL reaction flask, and add 400 μL of enzyme solution and 2 mL of n-hexane respectively. React in a magnetic stirrer at 45 °C and 400 rpm for 24 h, and quantify by GC.

[0084] (3) Weigh 0.087 mmol of sucrose and 0.348 mmol of vinyl lauryl ester and add them to a 15 mL reaction flask. Add 400 μL of enzyme solution and 2 mL of pyridine respectively. React in a magnetic stirrer at 40 °C and 400 rpm for 24 h and quantify by HPLC-ELSD.

[0085] (4) Weigh 0.056 mmol of glucose and 0.166 mmol of vinyl lauryl ester and add them to a 15 mL reaction flask. Add 400 μL of enzyme solution and 2 mL of DMSO:2M2B (4:1) respectively. React in a magnetic stirrer at 40 °C and 400 rpm for 24 h and quantify by HPLC-ELSD.

[0086] like Figure 6As shown, the pyridine-tolerant Candida antarcticis lipase B mutant has also made significant progress in the synthesis of ester compounds. Compared with WT, both the duplex mutants NF and ND, as well as the triple mutant KND, improved the synthesis efficiency of ester compounds. In the synthesis of glucose laurate, we found that ND, with the highest tolerance in DMSO, achieved the best catalytic effect, improving by 19.5% compared to WT. Furthermore, this was also observed in the synthesis of vitamin E succinate, phytosterol esters, and sucrose laurate, where the catalytic efficiency of mutants with improved solvent tolerance also increased. For example, ND, with the highest tolerance, exhibited the highest catalytic synthesis efficiency for vitamin E succinate, improving by 14.6% compared to WT. In conclusion, we can observe a positive trend between the level of organic solvent tolerance and the yield of lipase-catalyzed reactions, indicating that the strategy of improving enzyme catalytic efficiency by increasing the organic solvent tolerance of lipases is feasible.

[0087] The amino acid sequence of mutant ND is shown in SEQ ID NO.3, and its nucleotide sequence 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. All technical solutions formed by equivalent substitutions are within the scope of protection claimed by the present invention.

Claims

1. A method for preparing an Antarctic Candida lipase B mutant with improved lipase solvent tolerance, characterized in that, Includes the following steps: (1) Antarctic Candida lipase B 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; the amino acid sequence of the Candida antarcticis lipase B is shown in SEQ ID NO.1; (2) The channels and pockets of Candida antarcticis lipase B were determined using Fpocket and CaverDock software, and then the sites with excessively high conservation were excluded using ConSurf software, and the modification sites were finally determined. (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 pyridine tolerance of the modified lipase was determined using the p-nitrophenol method to screen for single mutants with improved pyridine tolerance. (4) The single mutants selected in step (3) are subjected to double recombination, and the double mutants are subjected to pyridine tolerance test and screening. (5) Further recombination was performed on the double mutants screened in step (4) to obtain triple mutants, and on this basis, pyridine tolerance was determined by the p-nitrophenol method and screened. (6) The selected mutants have any one of the following amino acid mutations in the amino acid sequence shown in SEQ ID NO.1: D145N-I285F, D145N-I285D, T42K-D145N-I285D; the selected mutants are Candida antarcticis lipase B mutants.

2. The method for preparing the Antarctic Candida lipase B mutant with improved lipase solvent tolerance according to claim 1, characterized in that, The solvent tolerance test includes the determination of relative enzyme activity and residual enzyme activity.

3. The method for preparing the Antarctic Candida lipase B mutant with improved lipase solvent tolerance according to claim 1, characterized in that, The screened mutants were used as catalysts in the synthesis of vitamin E succinate, phytosterol esters, sucrose laurate, and glucose laurate.

4. The method for preparing the Antarctic Candida lipase B mutant with improved lipase solvent tolerance according to claim 1, characterized in that, (1) Antarctic Candida lipase B was selected as the target for modification. First, the active pockets were predicted using Fpocket software. Based on this, the final target was determined from the 7 active pockets given by the software, combined with the position of the Antarctic Candida lipase B catalytic triplet. The amino acids in the pockets were: 39Gly, 40Thr, 42Thr, 47Lys, 104Trp, 105Ser, 134Asp, 138Thr, 144Leu, 154Val, 157Gln, 188Glu, 189Ile, 190Val, 224His, 281Ala, 282Ala, 285Ile. (2) The channel sites of Candida antarctica lipase B were predicted using the CaverDock software. Using the catalytic triple Ser105-His224-Asp187 and the oxygen anion hole as 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, 104 The third channel contains the following sites: Trp, 105Ser, 138Thr, 154Val, 157Gln, 189Ile, 190Val, 224His, 282Ala, 285Ile; the oxygen negative ion holes are Thr40 and Gln106. (3) The evolutionary conservation of amino acid residues in Candida antarctica lipase B was identified using ConSurf software to avoid the destruction of the modified enzyme structure. Based on this, the sites in the active pocket and channels were screened to exclude amino acid sites with conservation ≥6, i.e., highly conserved sites were excluded. Eight sites were screened: 42Thr, 47Lys, 144Leu, 145Asp, 154Val, 189Ile, 282Ala, and 285Ile.

5. The application of the Candida antarcticis lipase B mutant according to claim 1 in the synthesis of vitamin E succinate, phytosterol ester, glucose laurate, and sucrose laurate.

6. The application of the Candida antarcticis lipase B mutant according to claim 5, characterized in that: The amino acid sequence of the Candida antarcticis lipase B mutant is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.4.

Images