Candida antarctica lipase B mutant with high transesterification activity and application thereof

Abstract

The invention discloses a Candida antarctica lipase B mutant with high transesterification activity and application thereof. The nucleotide sequence of the Candida antarctica lipase B mutant is shown as SEQ ID NO.1, and the amino acid sequence of the expression protein of the Candida antarctica lipase B mutant is shown as SEQ ID NO.2. The invention further discloses a preparation method of the Candida antarctica lipase B mutant. Compared with an original enzyme, the mutant has higher fat hydrolysis activity and transesterification activity, the specific enzyme activity of the fat hydrolysis activity reaches 480.39 U / mg, the specific enzyme activity of the transesterification activity reaches 40.67 U / mg, the biodiesel conversion rate at the temperature of 30 DEG C can reach 92%, low-temperature high catalytic activity is shown, and the mutant can be used for enzymatic preparation of biodiesel and has potential industrial application value.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a Candida antarctica lipase B mutant with high transesterification activity, and a preparation method and application thereof. Background Art

[0002] Biodiesel has comparable performance to petroleum diesel and is considered a potential substitute for the latter. The preparation methods of biodiesel are mainly divided into chemical and biological methods, and the use of microbial lipid conversion is an important research direction. Traditional biodiesel synthesis methods are very costly on an industrial scale, and compared with harsh alkaline or acid conversion at high temperatures, enzymatic transesterification has become an environmentally friendly method for biodiesel production.

[0003] As a good biocatalyst, the production and utilization of lipase is a good substitute for chemical catalysts and can be used in the production of biodiesel. Candida antarctic lipase B (CalB) is one of them. This lipase is different from other lipases in molecular structure. It has no lid structure and does not show interfacial activation. The active center is exposed to the outside, which easily allows the substrate to enter the catalytic pocket. In addition, the oxygen ion hole is an important catalytic element near the active center of the lipase, which is used to stabilize the transition state structure of the negatively charged deprotonated oxygen.

[0004] Chinese patent CN114107250A discloses a lipase CALBgold and its preparation method and application. It aims at the modification of Antarctic Candida lipase B, and improves its transesterification ability and stability by modifying the amino acid positions 105, 110, 154, 173, 188 and 260 of the enzyme molecule; Chinese patent CN117070496A discloses a Candida Antarcticus lipase B mutant and its preparation method and application. While improving the activity, it pays attention to the change of its stereospecificity. However, both lipase mutants are expressed by Pichia pastoris, and cannot be produced and screened as quickly as Escherichia coli. In addition, although Chinese patent CN114107250A mentions that the transesterification rate of lipase CALBgold for olive oil can be as high as 94%, it needs to react at the optimal temperature of 40°C, which is relatively high.

[0005] At present, the expression level of CalB in Escherichia coli is low and the application cost is high. A large amount of CalB is required to make the transesterification reaction conversion rate reach more than 80%. Therefore, it is still necessary to improve the transesterification activity of the lipase; single-point or multi-point mutations are performed on some amino acid sites that affect the transesterification activity of the enzyme to obtain CalB mutants with better catalytic properties, thereby reducing the cost of industrial applications and building a green industrial system, which has good application prospects. Summary of the invention

[0006] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a Candida antarctica lipase B mutant with high transesterification activity. The Candida antarctica lipase B mutant has significantly improved transesterification activity and can be used in the efficient enzymatic preparation of biodiesel, thus having potential industrial application value.

[0007] The present invention also provides application of the Candida antarctica lipase B mutant with high transesterification activity.

[0008] Technical solution: In order to achieve the above-mentioned object, the present invention provides a Candida antarctica lipase B mutant with high transesterification activity, and the nucleotide sequence of the encoding gene of the Candida antarctica lipase B mutant with high transesterification activity is shown in SEQ ID NO.1.

[0009] Furthermore, the high transesterification activity Antarctic Candida lipase B mutant includes the amino acid sequence shown in SEQ ID NO.2, or a derivative protein with high transesterification activity of Antarctic Candida lipase B formed by replacing, deleting or adding one or more amino acid residues in the amino acid sequence SEQ ID NO.2.

[0010] Furthermore, the high transesterification activity Antarctic Candida lipase B mutant is mutated at positions 11, 13, 15, 18, 24, 25, 58, 74, 88, 96, 98, 99, 126, 139, 141, 144, 163, 168, 189, 206, 208, 242, 243, 244, 249, 250, 263, 278, 281, 282, 285, 286, 301, 305, and 307 using the amino acid sequence shown in SEQ ID NO.3 as a template to obtain the mutant shown in SEQ ID NO.2.

[0011] The recombinant vector of the present invention comprises a gene encoding a Candida antarctica lipase B mutant with high transesterification activity.

[0012] The recombinant bacteria of the present invention comprises a coding gene of a Candida antarctica lipase B mutant with high transesterification activity or the recombinant vector.

[0013] Furthermore, the starting strain of the recombinant bacteria is Escherichia coli.

[0014] The method for expressing and purifying the Candida antarctica lipase B mutant with high transesterification activity of the present invention comprises the following steps:

[0015] (1) connecting the coding gene of the Candida antarctica lipase B mutant with high transesterification activity to an expression plasmid to form a mutant recombinant expression plasmid;

[0016] (2) Transforming the successfully constructed recombinant expression plasmid into Escherichia coli expression cells, selecting positive clones, culturing, and then adding inducers;

[0017] (3) The supernatant of the Escherichia coli lysate in which the mutant is induced to be expressed is collected and purified to obtain a highly pure Antarctic Candida lipase B mutant protein with high transesterification activity.

[0018] The invention discloses an application of the Candida antarctica lipase B mutant with high transesterification activity in improving the conversion rate of biodiesel.

[0019] Furthermore, the application process is: soybean oil or olive oil or rapeseed oil or peanut oil, tert-butyl alcohol and water are mixed, methanol is added, and then the Antarctic Candida lipase B mutant is added for transesterification.

[0020] Furthermore, the molar ratio of methanol to soybean oil is 1:1-6:1, and the transesterification reaction temperature is 20-40°C.

[0021] Preferably, the molar ratio of methanol to soybean oil is 4:1, and the transesterification reaction temperature is 30°C.

[0022] Among them, in the simulated molecular structure of the expressed protein, while ensuring that the catalytic active pocket is larger, the distance between its substrate triolein and the active pocket is closer, and the distance between the active pocket and the oxygen ion hole is farther.

[0023] The invention discloses an application of the Candida antarctica lipase B mutant with high transesterification activity in the preparation of biodiesel by enzymatic method.

[0024] The present invention uses a database comparison, takes the conserved sequence in the Antarctic Candida lipase B sequence as the skeleton, selects the non-conserved sites of its active pocket and other amino acid sites with higher B values ​​according to the simulated three-dimensional structure, and randomly combines them. Through molecular docking and molecular dynamics simulation, the Antarctic Candida lipase B mutant gene with the highest binding free energy with the ligand is selected, and the gene is artificially synthesized and expressed to obtain a mutant enzyme with better activity. The target mutant gene is obtained and expressed in a shorter time and with convenient manpower, thereby improving the hydrolysis activity and transesterification activity of the enzyme.

[0025] The present invention provides a completely new mutant, which is different from the mutation point and sequence of the existing patent and has the guiding significance of molecular space. It shortens the time of molecular design through semi-rational design and obtains a mutant with high transesterification activity.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0027] The present invention provides a new Antarctic Candida lipase B mutant gene, the modified gene expression protein has the highest enzyme activity under the condition of 30°C, the fat hydrolysis activity specific enzyme activity reaches 480.39U / mg, and the transesterification specific enzyme activity reaches 40.67U / mg; the enzyme has a high enzyme activity in the temperature range of 20-40°C. Compared with the original Antarctic Candida lipase B (referred to as the original CalB), the Antarctic Candida lipase B mutant gene expression protein of the present invention has an increase of about 45% in transesterification activity and about 16% in hydrolysis activity, and the biodiesel conversion rate can reach 92% at 30°C, showing high catalytic activity at low temperature. The above characteristics make the Antarctic Candida lipase B mutant expression protein obtained by the present invention have greater advantages than the original CalB, and is suitable for carrying out transesterification reactions and hydrolysis reactions with the same effect with a smaller amount of enzyme, and can be used in the efficient enzymatic preparation of biodiesel, with potential industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the SDS-PAGE protein electrophoresis of Candida antarctica lipase B mutant CM-1;

[0029] Figure 2 is a comparison of the Candida antarctica lipase B mutant CM-1 and the original CalB;

[0030] Figure 3 This is a comparison of the hydrolysis activities of the Candida antarctica lipase B mutant CM-1 and the original CalB on an emulsion plate;

[0031] Figure 4 is a comparison of the translipidation activity of Candida antarctica lipase B mutant CM-1 and original CalB;

[0032] Figure 5 This is the gas chromatogram of the product after 24h transesterification reaction of the protein CM-1 expressed by the mutant gene of Candida antarctica lipase B;

[0033] Figure 6 This is an analysis of the molecular mechanism of high enzyme activity of the Antarctic Candida albicans lipase B mutant CM-1, where (A) is the distance between the original CalB and CM-1 active pocket and the ligand triolein; (B) is the distance between the original CalB and CM-1 active pocket and the oxygen ion hole; (C) is the change in the size of the CM-1 active pocket compared with the original CalB. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with the embodiments and drawings.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0036] Taq PCR Mix premix, PCR product recovery kit, T4 DNA ligase, agar powder, isopropyl-β-D-thiogalactoside, kanamycin, Ni 2+ Affinity chromatography column, potassium dihydrogen phosphate, disodium hydrogen phosphate: products of Shanghai Sangon Biotechnology Co., Ltd.;

[0037] Polyvinyl alcohol (PVA, average molecular weight 1750±50), tert-butyl alcohol, methanol: products of Sinopharm Chemical Reagent Co., Ltd.;

[0038] Tributyrin: a product of Tokyo Chemical Industry Co., Ltd.;

[0039] NdeⅠ, XhoⅠ restriction endonucleases: products of Bio-Rad Biotechnology (Beijing) Co., Ltd.;

[0040] Escherichia coli BL21 (DE3), Escherichia coli DH5α: products of Novagan, USA;

[0041] The soybean oil in the examples was purchased from JD.com COFCO Group Fortune Flagship Store, product number: 1523698;

[0042] The soybean oil in the present invention may also be common commercially available soybean oil.

[0043] The plasmids used in all examples are the universal plasmid pET-28a produced by Novagan, USA.

[0044] The determination protocol of the transesterification specific enzyme activity of Candida antarctica lipase B was carried out and measured according to the method in the literature: Carbon-based magnetic nano-particle utilizing nano-biochar as core and its immobilizing lipase for biodiesel preparation, DOI: 10.1016 / j.indcrop.2024.119693.

[0045] The NaOH titration method used in the determination of the hydrolytic activity of Candida antarctica lipase B was carried out according to the method in the literature: Enhancing the methanol tolerance of Candida antarctica lipase B by saturation mutagenesis for biodiesel preparation, DOI: 10.1007 / s13205-021-03095-x.

[0046] Example 1

[0047] Obtaining a mutant gene of lipase B from Candida antarctica

[0048] The amino acid sequence of Candida antarctic lipase B (SEQ ID NO.3) (GenBank accession number Z30645) was used as a template, and its base sequence was SEQ ID NO.4. Through molecular docking, Antarctic Candida lipase B was docked with triolein and tributyrin respectively to obtain amino acids with more contact times and located near the active center: positions 139, 141, 144, 189, 278, 281, 282, 285, and 286; combined with B value sorting and RMSF analysis of molecular dynamics simulation, the final modified amino acids were determined: positions 11, 13, 15, 18, 24, 25, 58, 74, 88, 96, 98, 99, 126, 163, 168, 206, 208, 242, 243, 244, 249, 250, 263, 301, 305, and 307; the amino acids near the active center The positions were replaced with amino acids that can expand the active pocket, namely V139I, A141G, L144V, I189L, L278G, A281G, A282L, I285P, and V286M; other amino acids were replaced with amino acids of similar charge and properties, namely Q11N, K13R, V15R, A18G, G24A, A25G, Q58N, N74Q, T88S, N96Q, K98R, L99I, D126E, L163I, R168K, N206Q, K208R, R242K, S243T, T244S, R249K, S250T, A263G, A301G, A305G, and G307A. Finally, the parameters of the modified enzyme were set, and the amino acid sequence was optimized according to the codon preference of Escherichia coli, that is, the bases were changed to the coding preferred by Escherichia coli, and the CM-1 mutant encoding gene was prepared by artificial synthesis. The mutant encoding gene sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0049] Example 2

[0050] Subcloning of the mutant gene CM-1 of lipase B from Candida antarctica

[0051] The following primer pairs were used to perform PCR amplification of the gene encoding the Candida antarctica lipase B mutant in Example 1 (shown in SEQ ID NO. 1):

[0052] CM-1-F: 5'-AACGATCATATGCTGCCTTCCGGTTCGGAC-3';

[0053] CM-1-R: 5'-AACGATCTCGAGGGGGGTGACGATGCCGGAG-3'.

[0054] When the above primers were synthesized, CM-1-F introduced an NdeⅠ restriction site, and CM-1-R introduced an XhoⅠ restriction site.

[0055] PCR reaction system: 1 μL of the Candida antarctic lipase B mutant encoding gene synthesized in Example 1 (shown in SEQ ID NO.1), 2 μL of CM-1-F (10 μmol / L), 2 μL of CM-1-R (10 μmol / L), 20 μL of ddH2O, and 25 μL of Taq PCR Mix premix.

[0056] PCR reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 59°C for 30 sec, extension at 72°C for 3 min, 30 cycles; extension at 72°C for 10 min; insulation at 12°C.

[0057] The yield and specificity of the PCR products were tested by 1% agarose gel electrophoresis and purified using a PCR product recovery kit.

[0058] Example 3

[0059] Recombinant cloning, construction and verification of expression vector pET-28a-CM-1

[0060] The purified PCR product (prepared in Example 2) and pET-28a vector were double-digested with NdeⅠ and XhoⅠ, respectively, and the digested PCR product and the large vector fragment were recovered by agarose electrophoresis. The target fragment and vector recovered after gel cutting were added with 1μL 10×Ligase Buffer and 1μL Ligase, and connected at 16°C overnight to construct the pET-28a-CM-1 recombinant expression vector. The successfully constructed plasmid was transferred into Escherichia coli DH5α competent cells, and then applied to a solid broth medium (plate) containing 100μg / mL kanamycin and cultured at 37°C for 12h.

[0061] Multiple single colonies were picked from the transformation plate for colony PCR verification, and plasmids were extracted using a plasmid mini-extraction kit. The obtained plasmid was double-digested and verified and the obtained recombinant plasmid was sequenced. The sequencing results showed that the cloned target fragment (nucleotide length of 951bp) was inserted into the pET-28a vector, and the DNA sequence of CM-1 was shown in SEQ ID NO.1, which represented the amino acid sequence of the Antarctic Candida lipase B mutant with high hydrolysis activity and high transesterification activity as shown in SEQ ID NO.2, and the protein was named CM-1.

[0062] Example 4

[0063] Using the heat shock transformation method, the recombinant expression vector pET-28a-CM-1 and the host Escherichia coli mixture were placed in an ice bath for 20 minutes, and then quickly heat-shocked in a 42°C water bath for 90 seconds, and then placed in an ice bath for 5 minutes, and the recombinant expression vector pET-28a-CM-1 (prepared in Example 3) was transformed into the host bacteria E. coli BL21 (DE3) to obtain recombinant bacteria containing recombinant plasmids. The recombinant bacteria of a single colony were inoculated into 10 mL of Luria-Bertani broth (LB) medium containing 100 μg / mL kanamycin, and cultured at 37°C and 220 rpm for 4 hours to obtain a recombinant bacterial solution. The above 8 mL of bacterial solution was transferred to 800 mL of LB medium, and cultured at 37°C and 200 rpm. When the absorbance OD 600 When the concentration reached 0.4–0.6, 800 μL of 50 mg / mL isopropyl-β-D-thiogalactoside (IPTG) was added, and the expression was induced at 20°C and 250 rpm for 15 h. The culture solution was centrifuged at 6000 rpm for 7 min at 4°C using a high-speed refrigerated centrifuge to collect the bacteria. The collected bacteria were washed with 50 mL of ultrapure water and centrifuged at 6000 rpm for 7 min at 4°C, and the bacteria were recovered and then resuspended with 20 mL of sterile ultrapure water. In an ice water bath, the bacterial cells were broken with an ultrasonic crusher, and the bacteria were centrifuged at 10000 rpm for 60 min at 4°C to precipitate the bacteria. The supernatant was collected to obtain a crude extract containing the Antarctic Candida lipase B mutant CM-1.

[0064] The original CalB protein supernatant was obtained by the same method as in Examples 1-4 of the present invention. The encoding DNA sequence of the original CalB is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.3.

[0065] The crude extract uses Ni 2+The affinity chromatography column was eluted and purified using 250 mM imidazole. The concentration of the enzyme protein was determined by the Bradford method, with bovine serum albumin as the standard. The purity of the purified enzyme was identified and the molecular weight was determined by SDS-PAGE. The concentration of the purified CM-1 protein was 32.79 μg / mL and the molecular weight was approximately 38.7 kDa. It was used as a pure enzyme in subsequent experiments. Figure 1 As shown, lane 1 represents the crude protein containing the Candida antarctica lipase B mutant CM-1, and lane 2 represents the purified CM-1 protein after elution, and its apparent molecular weight is close to the theoretical value.

[0066] The original CalB protein used as a control was eluted and purified in the same manner as mutant CM-1, and the concentration of the original CalB protein was 34.79 μg / mL.

[0067] Example 5

[0068] Detection of the hydrolytic activity of the recombinant Candida antarctica lipase B mutant gene expressed protein CM-1 and the original Candida antarctica lipase B (original CalB)

[0069] The purified mutant CM-1 and original CalB obtained in Example 4 were tested for lipase hydrolysis activity. The lipase hydrolysis activity was measured by NaOH titration: the amount of enzyme required for lipase to hydrolyze tributyrin and release 1 μmol of butyric acid per minute was one lipase hydrolysis activity unit (U). In a 50mM Na2HPO4-KH2PO4 buffer solution (pH 7.0), tributyrin and 1.0% (w / v) polyvinyl alcohol solution were added in a volume ratio of 1:7 and ultrasonically treated until the solution was mixed to form an emulsion. 9mL of the emulsified solution was taken and 1mL of the lipase (mutant CM-1 and original CalB) prepared in Example 4 was added, mixed and reacted at 30°C for 10min, and 15mL of ethanol was immediately added to terminate the reaction. At the same time, heat-inactivated lipase was used instead of normal active lipase as a control. The lipase hydrolysis activity was determined by titration with 50mM NaOH solution.

[0070] Experimental data such as Figure 2 As shown: the fat hydrolysis activity of the Antarctic Candida lipase B mutant CM-1 reached 480.39U / mg, and the fat hydrolysis activity of the original CalB reached 301.80U / mg; compared with the original CalB, its hydrolysis activity increased by about 16%. Figure 3 Shown: The hydrolysis circle of the mutant enzyme CM-1 is larger than that of the original lipase CalB.

[0071] Example 6

[0072] Transesterification activity of the recombinant Candida antarctica lipase B mutant gene expressed protein CM-1 and the original Candida antarctica lipase B (original CalB) and biodiesel conversion rate detection

[0073] The purified mutant CM-1 and original CalB obtained in Example 4 were tested for lipase transesterification activity: 5 g soybean oil, 3 mL tert-butyl alcohol, 150 μL water, 0.925 mL methanol were added to a 250 mL conical flask, and 10 mg freeze-dried mutant CM-1 and original CalB were added respectively (the purified enzyme solution CM-1 and original CalB obtained in Example 4 were directly freeze-dried and collected using a freeze dryer). After 24 h of conversion at 30° C. and 200 rpm in a shaking table, a GC-2030 gas chromatograph (Shimadzu Corporation, Japan) equipped with a capillary column (specifications 30 m×0.25 mm×0.25 μm, INNOWAX, USA) and a hydrogen flame ionization detector (FID) were used to quantitatively analyze fatty acid methyl esters. The lipase-catalyzed acyl transfer of triglycerides and methanol to produce biodiesel and glycerol, the amount of enzyme required to release 1 μmol of fatty acid methyl ester per minute is one lipase transesterification activity unit (U), and the transesterification specific enzyme activity is obtained. The experimental data are as follows Figure 4 As shown: the transesterification specific enzyme activity of the Antarctic Candida lipase B mutant CM-1 reached 40.67U / mg, and the transesterification specific enzyme activity of the original CalB was 28.11U / mg; compared with the original CalB, its transesterification activity was increased by about 45%.

[0074] After the transesterification experiment between mutant CM-1 and original CalB, the reaction products were extracted to calculate the biodiesel conversion rate. The formula for calculating the biodiesel conversion rate is shown in formula (1):

[0075]

[0076] Among them, W FAME is the mass of fatty acid methyl ester actually produced, W 油 is the weight of soybean oil before reaction, M FAME Average molecular weight of fatty acid methyl ester (289.7767 g / mol), M 油 is the average molecular weight of soybean oil (865.3 g / mol). Figure 4As shown, the biodiesel conversion rate of the mutant CM-1 was increased from 78% of the original CalB to 92%. In the actual reaction process, the reaction can reach equilibrium and achieve a conversion rate of more than 90% after 6-7 hours of reaction. Compared with the existing Chinese patent CN114107250A, which can only obtain a conversion rate of 94% at a high temperature of 40°C, the mutant of the present invention not only has significant differences in mutation sites, but also can be quickly and efficiently expressed in Escherichia coli, and a biodiesel conversion rate of 92% can be obtained at a reaction temperature of only 30°C. It has the advantages of low energy consumption, stable temperature and easy control, showing its high catalytic activity at low temperature. In addition, the mutant can not only perform transesterification reaction on soybean oil as a substrate, but also can achieve a biodiesel conversion rate of more than 90% after 6-7 hours when using other substrates such as olive oil, rapeseed oil, and peanut oil.

[0077] The reaction products were detected by gas chromatography, such as Figure 5 As shown in the figure: the four methyl ester peaks representing the biodiesel components generated by transesterification are peak 1 palmitic acid methyl ester, peak 2 linoleic acid methyl ester, peak 3 oleic acid methyl ester, and peak 4 stearic acid methyl ester, indicating that mutant CM-1 transesterified soybean oil and generated corresponding products.

[0078] Example 7

[0079] Comparison of transesterification activity of CM-1 protein expressed by recombinant Candida antarctica lipase B mutant gene at different temperatures

[0080] The purified mutant CM-1 obtained in Example 4 was tested for lipase transesterification activity: 5 g soybean oil, 3 mL tert-butyl alcohol, 150 μL water, 0.925 mL methanol were added to a 250 mL conical flask, and 10 mg freeze-dried mutant CM-1 was further added (the purified enzyme solution CM-1 obtained in Example 4 and the original CalB were directly freeze-dried and collected using a freeze dryer). After conversion at 20°C, 25°C, 30°C, 35°C, and 40°C, respectively, in a 200 rpm shaker for 30 min, a GC-2030 gas chromatograph (Shimadzu, Japan) equipped with a capillary column (specifications 30 m×0.25 mm×0.25 μm, INNOWAX, USA) and a hydrogen flame ionization detector (FID) was used to quantitatively analyze fatty acid methyl esters. Lipase was used to catalyze the acyl transfer of triglycerides and methanol to produce biodiesel and glycerol. The amount of enzyme required to release 1 μmol of fatty acid methyl ester per minute was one lipase transesterification activity unit (U), and the transesterification specific enzyme activity was obtained. The highest transesterification activity of the Antarctic Candida lipase B mutant CM-1 at 30°C was set as 100%. The transesterification activities at other temperatures between 20-40°C all decreased, but the high enzyme activity could still be maintained above 90%.

[0081] Example 8

[0082] Molecular Mechanism Analysis of High Activity of Recombinant Candida antarctica Lipase B Mutant CM-1

[0083] The purified CM-1 obtained in Example 4 was subjected to homology modeling by Swiss-Model, and docked with the ligand triolein using Autodockvina (for usage, refer to the doctoral dissertation: Heterologous expression, heat resistance modification and substrate specificity analysis of Penicillium antarctica lipase), and then placed in PyMol to analyze its oxygen ion hole, the distance between the active center and the ligand triolein, and the size of the active pocket (for PyMol usage, refer to the paper: Enhancing the methanol tolerance of Candida antarctica lipase B by saturation mutagenesis for biodiesel preparation, DOI: 10.1007 / s13205-021-03095-x).

[0084] The results are as follows Figure 6 As shown: The size of the catalytic activity pocket of mutant CM-1 is significantly increased ( Figure 6 (C) shows that the distance between the substrate triolein and the catalytic active pocket is Shortened to ( Figure 6 (A) shows that the distance between the catalytic active pocket and the oxygen ion hole is Lengthened to ( Figure 6 (B)), which is more susceptible to hydrolysis and transesterification reactions. It can be concluded that these changes in spatial structure can improve the hydrolysis activity and transesterification activity of mutant CM-1.

Claims

1. A Candida antarctica lipase B mutant with high transesterification activity, characterized in that: The nucleotide sequence of the gene encoding the Candida antarctica lipase B mutant with high transesterification activity is shown in SEQ ID NO.

1.

2. The Candida antarctica lipase B mutant with high transesterification activity according to claim 1, characterized in that: The high transesterification activity Antarctic Candida lipase B mutant includes the amino acid sequence shown in SEQ ID NO.2, or a derivative protein with high transesterification activity of Antarctic Candida lipase B formed by replacing, deleting or adding one or more amino acid residues in the amino acid sequence of SEQ ID NO.

2.

3. The Candida antarctica lipase B mutant with high transesterification activity according to claim 1, characterized in that: The high transesterification activity Antarctic Candida lipase B mutant uses the amino acid sequence shown in SEQ ID NO.3 as a template, and mutates at positions 11, 13, 15, 18, 24, 25, 58, 74, 88, 96, 98, 99, 126, 139, 141, 144, 163, 168, 189, 206, 208, 242, 243, 244, 249, 250, 263, 278, 281, 282, 285, 286, 301, 305, and 307 to obtain the mutant shown in SEQ ID NO.

2.

4. A recombinant vector, characterized in that: The recombinant vector comprises the coding gene according to claim 1.

5. A recombinant bacterium, characterized in that: The recombinant bacteria comprises the coding gene according to claim 1 or the recombinant vector according to claim 4.

6. The recombinant bacterium according to claim 5, characterized in that The starting strain of the recombinant bacteria is Escherichia coli.

7. A method for expressing and purifying the Candida antarctica lipase B mutant with high transesterification activity according to claim 1, characterized in that: The steps include: (1) connecting the coding gene of the Candida antarctica lipase B mutant with high transesterification activity according to claim 1 to an expression plasmid to form a mutant recombinant expression plasmid; (2) Transforming the successfully constructed recombinant expression plasmid into Escherichia coli expression cells, selecting positive clones, culturing, and then adding inducers; (3) The supernatant of the Escherichia coli lysate in which the mutant is induced to be expressed is collected and purified to obtain a highly pure Antarctic Candida albicans lipase B mutant protein with high transesterification activity.

8. Use of the Candida antarctica lipase B mutant with high transesterification activity according to claim 1 in improving the conversion rate of biodiesel.

9. The use according to claim 8, characterized in that: The application process is: soybean oil or olive oil or rapeseed oil or peanut oil, tert-butyl alcohol and water are mixed, methanol is added, and then the Antarctic Candida lipase B mutant is added to carry out transesterification reaction.

10. The use according to claim 9, characterized in that: The molar ratio of the methanol to the soybean oil is 1:1-6:1, and the transesterification reaction temperature is 20-40°C.

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