A lipase mutant tolerant to organic solvents and its encoding gene and application

Abstract

The present invention discloses a lipase mutant tolerant to organic solvents, its encoding gene and application. The present invention screens and obtains a lipase mutant G266C with significantly improved organic solvent tolerance and higher specific activity by performing site-directed mutagenesis on wild-type Rhizopus oryzae lipase. Experiments show that after incubation for 30 min in methanol, ethanol, isopropanol and acetone at a final concentration of 50% (v / v), the residual enzyme activity of the lipase mutant G266C is 3.1, 2.9, 2.1 and 2.6 times that of the wild-type Rhizopus oryzae lipase, respectively. The organic solvent tolerance is significantly improved, and the enzyme activity is better than that of the wild-type, and it can be applied to the synthesis of biodiesel such as fatty acid methyl esters, and is more suitable for application in the industrial field.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering, and in particular relates to an organic solvent-tolerant lipase mutant and a coding gene and application thereof. Background Art

[0002] Lipases (EC 3.1.1.3) are an important class of biocatalysts, widely used in oil processing, food, medicine, feed additives and environmental management. Lipases can hydrolyze in water, and catalyze esterification, acidolysis, alcoholysis and transesterification in organic solvents. Enzyme biotransformation in organic solvent media has always been a hot topic in the field of synthetic chemistry, because it has many advantages over traditional biotransformation in water media, including increasing the solubility of hydrophobic substrates, reducing microbial contamination and inhibiting water-dependent metabolic side reactions. However, under the action of organic solvents, enzymes are easily inactivated, which is not conducive to the reaction. In actual biocatalytic processes, how to maintain the high activity of lipases in organic media and at the same time utilize the wide diversity of their catalytic substrates is a major technical challenge currently faced.

[0003] Rhizopus oryzae lipase (ROL) can be used to enrich polyunsaturated fatty acids in fish oil and prepare biodiesel, and these reaction systems all contain organic solvents. It was found during use that due to the poor tolerance of the enzyme to organic solvents, it is easy to be inactivated during the application process, resulting in an increase in production costs and is not conducive to industrial application. Therefore, improving the organic solvent tolerance of Rhizopus oryzae lipase can lay the foundation for its industrial application. When looking for lipases with better organic solvent tolerance, due to the large number of mutagenic sites and the complexity of the protein structure, it is not easy to take into account both organic solvent tolerance and good enzyme activity, and enzymes that can adapt to industrial use are not easy. Summary of the invention

[0004] Aiming at the shortcoming that Rhizopus oryzae lipase has poor organic solvent tolerance, the present invention provides a lipase mutant with improved organic solvent tolerance, a coding gene and application thereof.

[0005] The first object of the present invention is to provide a lipase mutant, wherein the lipase mutant is the lipase mutant G266C, and the amino acid sequence is shown in SEQ ID NO.3.

[0006] The second object of the present invention is to provide a gene encoding the lipase mutant, characterized in that the nucleic acid sequence of the gene encoding the lipase mutant is shown in SEQ ID NO.4.

[0007] The third object of the present invention is to provide a recombinant expression vector containing the coding gene of the lipase mutant.

[0008] The fourth object of the present invention is to provide an engineered bacterium containing the recombinant expression vector.

[0009] The fifth object of the present invention is to provide the use of the lipase mutant or the encoding gene of the lipase mutant in the synthesis of fatty acid methyl esters.

[0010] The present invention has the following beneficial effects:

[0011] The present invention screens and obtains a lipase mutant G266C with significantly improved organic solvent tolerance and higher specific activity by performing site-directed mutagenesis on a wild-type Rhizopus oryzae lipase. Compared with the wild-type, under the same conditions, after the lipase mutant G266C obtained by the present invention is treated in methanol, ethanol, isopropanol and acetone with a final concentration of 50% (v / v) for 30 min, its residual enzyme activity is 3.1, 2.9, 2.1 and 2.6 times that of the wild-type Rhizopus oryzae lipase, respectively, which has a significant improvement; and the specific activity of the lipase mutant G266C is increased by 36% relative to that of the wild-type. Therefore, the lipase mutant G266C obtained by the present invention can be applied to biodiesel synthesis and is more suitable for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a graph showing the results of screening residual enzyme activity for organic solvent-tolerant mutants.

[0013] Figure 2 The tolerance of wild-type ROL and lipase mutant G266C in different organic solvents.

[0014] Figure 3 The product yields of wild-type ROL and lipase mutant G266C in synthetic biodiesel (fatty acid methyl esters). DETAILED DESCRIPTION

[0015] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0016] Materials and reagents used in the following examples: plasmid extraction kit purchased from Omega Trading Co., Ltd.; Primer STAR kit purchased from TAKARA; Sypro Orange dye purchased from Sigma; TOP10 Escherichia coli competent cells purchased from Tiangen Biotechnology Co., Ltd.; mutation primers synthesized by Shanghai Shenggong Biotechnology Co., Ltd.; Pme Ⅰ restriction endonuclease purchased from New England Biolabs; PCR product purification and recovery kit purchased from Dalian Bao Biotechnology Co., Ltd.; electroporator purchased from Bio-Rad; LLB, LLB+Zeocin, YPD, BMGY, and BMMY culture media were prepared according to the Invitrogen Pichia pastoris expression kit operation manual, and the remaining reagents were of analytical grade purchased at home and abroad.

[0017] The lipase mutants in the following examples refer to site-directed mutations in wild-type ROL, and "original amino acid-position-replaced amino acid" is used to represent the mutated amino acid in the lipase mutant, such as the lipase mutant G266C, that is, the mutation site of the lipase mutant G266C is Gly266C.

[0018] Example 1: Construction of mutant lipase expression vector

[0019] The expression vector pPICZαA-ROL of ROL was constructed in the early stage of this experiment, that is, the gene of ROL (its nucleic acid sequence is shown in SEQ ID NO.2, and the encoded amino acid sequence is shown in SEQ ID NO.1) was inserted into the vector pPICZαA, and the restriction enzyme sites were EcoR Ⅰ and Sal Ⅰ. The crystal structure of ROL (PDB ID: 1LGY) was analyzed. Based on the accumulated experience, the crystal structure of the enzyme was observed using PyMOL software. After repeated analysis, 5 mutants were finally designed, namely lipase mutant I90N, lipase mutant I90T, lipase mutant I254T, lipase mutant L258T or lipase mutant G266C (its amino acid sequence is shown in SEQ ID NO.3, which is a mutation of glycine Gly to cysteine ​​Cys at position 266 of ROL; the nucleic acid sequence of its encoding gene is shown in SEQ ID NO.4).

[0020] Primers for lipase mutants were designed on the QuikChange primer design website (http: / / www.genomics.agilent.com / primerDesignProgram.jsp). Then, PCR reaction was performed using pPICZαA-ROL as a template to construct expression vectors for lipase mutant I90N, lipase mutant I90T, lipase mutant I254T, lipase mutant L258T or lipase mutant G266C.

[0021] PCR amplification conditions were: 98°C for 3 min; 98°C for 10 s, 55°C for 30 s, 72°C for 5 min, 30 cycles; 72°C extension for 5 min. Reaction system: 1 μL of each primer, 12.5 μL of Primer Star (2X), 1 μL of template, and 9.5 μL of ddH2O. The amplified product was digested with Dpn Ⅰ enzyme, and the mutant plasmid was obtained after the size of the mutant band was detected by agarose gel electrophoresis. The mutant plasmid was then transferred into E. coli TOP10 competent cells were plated on LLB (Zeocin concentration was 25 μg / mL) solid plates and cultured overnight at 37°C. Positive transformants were selected for plasmid sequencing.

[0022] Example 2: Construction, expression and purification of mutant lipase expression strains

[0023] After the positive transformants with correct sequencing were amplified and cultured overnight in LLB liquid medium (Zeocin concentration was 25 μg / mL), the plasmid was extracted, linearized with Pme Ⅰ endonuclease and purified, and a total of 5 μg of the plasmid linearization product was mixed with X-33 Pichia pastoris competent cells for electroporation transformation. The preparation of Pichia pastoris competent cells was carried out according to the Invitrogen operating manual, and the program was set according to the parameters recommended by Bio-Rad.

[0024] After electroporation, add 1 mL of 1 mol / L sorbitol solution immediately. Incubate the bacterial solution at 30°C for 1 h to recover, then evenly spread it on YPDS+Zeocin (Zeocin concentration is 100 μg / mL) resistance plates for screening. After culturing for 72 h, select positive transformants.

[0025] A single colony of the positive transformant was inoculated into 50 mL of BMGY medium and cultured at 30°C 250 rpm to an OD of 0.8-1.0. A 10% inoculum was inoculated into 400 mL of BMMY medium and fermented at 30°C 250 rpm for 96 h. The culture was induced every 24 h with a final concentration of 1% methanol.

[0026] The fermentation broth was centrifuged at 4℃ and 7000 r / min for 40 min, and the supernatant was collected. The supernatant was filtered with a 0.45 μm filter membrane and purified by a nickel affinity chromatography column. The column was first equilibrated with a phosphate buffer (pH 7.4) containing 20 mM imidazole at a flow rate of 4 mL / min. After the equilibration, the supernatant was combined with the column, and then the impurities were eluted with a phosphate buffer (pH 7.4) containing 20 mM imidazole, and then the target protein was eluted with a phosphate buffer (pH 7.4) containing 300 mM imidazole. The eluted target protein was further desalted with a G-25 desalting column to obtain the purified enzyme solution of wild-type ROL and lipase mutants.

[0027] Example 3: Determination of organic solvent tolerance of lipase mutants

[0028] First, the purified wild-type ROL and lipase mutant enzyme solutions obtained in Example 2 were treated and measured respectively using anhydrous methanol, that is, the purified enzyme solution was diluted to 0.2 mg / mL with 20 mM pH 7.0 phosphate buffer, and then added to methanol to make the final concentration of methanol 50% (v / v), and incubated at 30 °C for 30 min to obtain an enzyme solution treated with methanol; and the residual enzyme activity was measured by pNP colorimetry. pNP colorimetric method for residual enzyme activity: 24.23 µL of 4-nitrophenylcaprylate (p-Nitrophenylcaprylate, C8) was taken and dissolved in 10 mL of anhydrous ethanol, and vortexed to fully dissolve as a substrate. The reaction system is 10 µL 10 mM substrate, 10 µL enzyme solution treated with methanol, and 80 µL 20 mM pH 7.0 phosphate buffer. After incubation at room temperature for 5 min, 100 µL isopropanol was added to terminate the reaction, and the absorbance was measured at a wavelength of 405 nm. As Figure 1 As shown, the lipase mutant G266C had better tolerance to methanol, with a residual enzyme activity of 71%, which was 3.1 times that of the wild-type ROL (23%).

[0029] Then, the wild-type ROL or lipase mutant G266C was treated with ethanol, isopropanol or acetone in the same manner as above, except that the methanol was replaced, and the residual enzyme activity was determined. Figure 2 As shown in the figure, the lipase mutant G266C also showed better tolerance to other organic solvents. The residual enzyme activity of the lipase mutant G266C in ethanol, isopropanol and acetone was 2.9, 2.1 and 2.6 times that of the wild type ROL, respectively.

[0030] Example 4: Determination of specific activity of lipase

[0031] The enzyme activity of wild-type ROL and lipase mutant G266C was determined using an olive oil-rich emulsion as a substrate: 150 g of a 4% polyvinyl alcohol (PVA) aqueous solution and 50 g of olive oil were weighed and treated with a high-speed homogenizer for 6 min to prepare an emulsion. In a 100 mL conical flask, 4 g of the emulsion and 5 mL of 20 mM phosphate buffer (pH 7.5) were added and incubated at 30°C for 5 min. The purified wild-type ROL and lipase mutant G266C enzyme solutions obtained in Example 2 were diluted, 1 mL of the diluted enzyme solution was added to the conical flask for reaction for 10 min, and the reaction was terminated with 15 mL of 95% ethanol aqueous solution by volume. Phenolphthalein was added and titrated with 0.05 M sodium hydroxide solution to determine the amount of free fatty acids released during the reaction. Control group: The diluted enzyme solution added above was replaced with the same volume of 20 mM phosphate buffer (pH 7.5). The enzyme specific activity U is defined as the amount of enzyme consumed to produce 1 μmol of fatty acid per minute. The results of specific activity determination are shown in Table 1. Compared with the wild-type ROL, the specific activity of the lipase mutant G266C was increased by 36%.

[0032] Table 1 Specific activity assay results

[0033] Enzymes Specific activity (U / mg) Wild-type ROL 1180.2 Lipase mutant G266C 1605.1

[0034] Example 5: Application in the synthesis of fatty acid methyl esters

[0035] A reaction system with soybean oil and methanol solution as substrates was used to investigate the effect of lipase mutant G266C in synthesizing biodiesel (fatty acid methyl esters). The specific reaction system is as follows: waste soybean oil (3 g), methanol (the molar ratio of methanol: soybean oil is 4.5:1) and 1 mL of 0.5 mg / mL lipase (wild-type ROL or lipase mutant G266C) solution, reacted at 35°C for 24 hours. The changes in the content of products and reactants were detected, and the conversion rate was calculated. Figure 3 As shown, the yield of fatty acid methyl esters of the lipase mutant G266C reached about 92%, while that of the wild-type ROL was about 61%.

Claims

1. A lipase mutant, characterized in that: The lipase mutant is the lipase mutant G266C, and the amino acid sequence is shown in SEQ ID NO.

3.

2. A gene encoding the lipase mutant according to claim 1, characterized in that: The nucleic acid sequence of the encoding gene is shown in SEQ ID NO.

4.

3. A recombinant expression vector containing the gene encoding the lipase mutant according to claim 2.

4. An engineered bacterium containing the recombinant expression vector according to claim 3.

5. Use of the lipase mutant according to claim 1 or the gene encoding the lipase mutant according to claim 2 in the synthesis of fatty acid methyl esters.

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