Acid-resistant mutant of Candida antarctica lipase A and application thereof

Abstract

The invention discloses an acid-resistant mutant of candida antarctica lipase A and application of the acid-resistant mutant. In order to solve the problem that the Candida antarctica lipase A is easy to inactivate in an acid environment, the acid-resistant mutant of the Candida antarctica lipase A is obtained by mutating the 45th or 161th amino acid of the Candida antarctica lipase A. Compared with candida antarctica lipase A, the acid-resistant mutant has the advantages that the optimum reaction temperature, pH and specific activity of the acid-resistant mutant are not obviously changed, but the residual enzyme activity of the acid-resistant mutant is obviously improved after the acid-resistant mutant is incubated for the same time in an acid environment and is improved by 10-26%, and the yield of biodiesel prepared by catalyzing acid oil within the same time is also obviously improved. The mutant disclosed by the invention can be used for preparing biodiesel under an acidic condition, and is beneficial to recycling of acid oil.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering and biodiesel preparation technology. More specifically, it relates to an acid-resistant mutant of Candida antarctica lipase A and its applications. Background Technology

[0002] Lipase (EC 3.1.1.3), also known as triacylglycerol acyl hydrolase, is an important class of biocatalysts. It has multiple functions such as catalyzing the hydrolysis, esterification and transesterification of carboxylic acid esters and plays an important role in the food, energy, detergent and pharmaceutical industries.

[0003] In the enzymatic preparation of biodiesel, lipases possess unique multifunctional catalytic activity, enabling the simultaneous transesterification of triglycerides and the esterification of fatty acids. This highly efficient synergistic catalytic characteristic provides core technological support for the enzymatic preparation of biodiesel, making lipases a highly anticipated enzyme preparation type in current industrial biodiesel production. In the biodiesel industry, raw material costs account for a large proportion of production costs; therefore, selecting economically suitable raw materials directly relates to the economic feasibility of the production process and is a core consideration for large-scale industrial development. Currently, biodiesel production has experimented with various raw materials, including edible vegetable oils, non-edible vegetable oils, waste oils, and animal fats. However, to reduce costs, low-cost unrefined oils (such as various waste oils and non-edible vegetable oils) are considered more promising alternative raw materials. Among these, soapstock, as a major byproduct generated during vegetable oil refining, has become one of the most valuable biodiesel raw materials due to its stable source, ease of collection, and low cost. Soap residue itself is a heavy alkaline aqueous emulsion composed of fatty acids, triglycerides, and other lipid components. To effectively recover the fatty acids and hydrolyze residual oils, sulfuric acid is typically used industrially to acidify the raw soap residue. The product obtained through this treatment is called acidified oil, characterized by its high acid value. Due to the high acid value of acidified oil, it easily deactivates lipases. When using acidified oil as a raw material to produce biodiesel, lipases with excellent acid resistance and high catalytic efficiency are required to ensure the smooth progress of the reaction.

[0004] Although researchers have made significant progress in recent years through enzyme engineering techniques such as rational design and directed evolution in improving the thermal stability, catalytic activity, and substrate specificity of lipases, attempts to improve their acid resistance have been relatively limited. This imbalance in research focus has resulted in a severe shortage of acid-resistant lipases with industrial application value, directly hindering the large-scale application of low-cost, high-acid-value feedstocks such as acidified oils in biodiesel production, and limiting the achievement of cost control and sustainable development goals in the biodiesel industry.

[0005] Antarctic Candida lipase A (CALA) has advantages such as high catalytic activity, strong thermal stability and tolerance to organic solvents, but its catalytic activity is significantly reduced under acidic reaction conditions, making it difficult to meet the needs of acidified oil-based biodiesel production. This problem has become a key obstacle restricting the application of low-cost feedstocks. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides an acid-resistant mutant of Candida antarctica lipase A, which can be used to catalyze the production of biodiesel from acidified oil, thereby improving the yield of biodiesel.

[0007] The first objective of this invention is to provide an acid-resistant mutant of Candida antarcticis lipase A.

[0008] A second objective of this invention is to provide a gene encoding the acid-resistant mutant.

[0009] A third objective of this invention is to provide a polynucleotide construct containing the said gene.

[0010] A fourth object of the present invention is to provide a genetically engineered host cell containing the said gene or the said polynucleotide construct.

[0011] A fifth object of the present invention is to provide a composition containing the acid-resistant mutant and / or the genetically engineered host cell.

[0012] A sixth object of the present invention is to provide the use of the acid-resistant mutant, the genetically engineered host cell, or the composition in the production of biodiesel from catalytically acidified oil.

[0013] A seventh object of the present invention is to provide the use of the acid-resistant mutant, the gene, the polynucleotide construct, the genetically engineered host cell, or the composition in the preparation of formulations for the catalytic production of biodiesel from acidified oil.

[0014] The eighth object of the present invention is to provide a method for improving the tolerance of Candida antarctica lipase A to acidic environments.

[0015] The above-mentioned objective of this invention is achieved through the following technical solution: This invention obtains an acid-resistant mutant of Candida antarctic lipase A by mutating amino acids at positions 45 or 161. Compared to Candida antarctic lipase A, the optimal reaction temperature, pH, and specific activity of the acid-resistant mutant are not significantly altered, but its residual enzyme activity after incubation for the same time under acidic conditions is significantly increased, and the yield of biodiesel produced from acidified oil is also significantly increased. Therefore, this invention seeks protection for the acid-resistant mutant and its applications.

[0016] This invention seeks to protect an acid-resistant mutant of Candida antarcticus lipase A. Specifically, the mutant is obtained by mutating the aspartic acid at position 45 of Candida antarcticus lipase A to methionine, or by mutating the aspartic acid at position 161 of Candida antarcticus lipase A to asparagine; the amino acid sequence of the Candida antarcticus lipase A is shown in SEQ ID NO. 1.

[0017] In a specific embodiment of the present invention, the gene sequence encoding the Antarctic Candida lipase A is shown in SEQ ID NO.2.

[0018] The present invention also seeks protection for the gene encoding the acid-resistant mutant.

[0019] Specifically, the acid-resistant mutant obtained by mutating the 45th amino acid of Candida antarctica lipase A is denoted as D45M, and its amino acid sequence is shown in SEQ ID NO.3. The gene sequence encoding this acid-resistant mutant is shown in SEQ ID NO.4.

[0020] Specifically, the acid-resistant mutant obtained by mutating the 161st amino acid of Candida antarctica lipase A is denoted as D161N, and its amino acid sequence is shown in SEQ ID NO.5. The gene sequence encoding this acid-resistant mutant is shown in SEQ ID NO.6.

[0021] The present invention also seeks protection for polynucleotide constructs containing the said gene.

[0022] Optionally, the polynucleotide construct is a cloning vector or expression vector containing the gene.

[0023] The present invention also seeks protection for genetically engineered host cells containing the said gene or the said polynucleotide construct.

[0024] Optionally, the host cell includes Escherichia coli and Pichia pastoris.

[0025] In a specific embodiment of the present invention, the host cell is Escherichia coli.

[0026] More specifically, the Escherichia coli is Escherichia coli BL21(DE3) strain.

[0027] The present invention also claims protection for a composition comprising the acid-resistant mutant and / or the genetically engineered host cell.

[0028] Compared to Candida antarcticis lipase A, the acid-resistant mutant of this invention exhibits significantly increased residual enzyme activity after incubation for the same period in an acidic environment, and its yield of biodiesel from catalytic acidified oil is also significantly increased. Therefore, this invention also claims protection for the use of the acid-resistant mutant, the genetically engineered host cell, or the composition in the catalytic acidified oil production of biodiesel.

[0029] The present invention also claims protection for the use of the acid-resistant mutant, the gene, the polynucleotide construct, the genetically engineered host cell, or the composition in the preparation of formulations for the catalytic production of biodiesel from acidified oil.

[0030] Specifically, the acidified oil is a plant-based soap residue acidified oil.

[0031] In a specific embodiment of the present invention, the acidified oil is acidified soybean oil.

[0032] Specifically, the preparation is an enzyme catalyst.

[0033] In addition, the present invention provides a method for improving the tolerance of Candida antarcticis lipase A to acidic environment, wherein the method is: mutating the aspartic acid at position 45 of Candida antarcticis lipase A to methionine, or mutating the aspartic acid at position 161 of Candida antarcticis lipase A to asparagine.

[0034] Specifically, the amino acid sequence of the Candida antarcticis lipase A is shown in SEQ ID NO.1.

[0035] The present invention also provides a method for preparing biodiesel, the method comprising the step of synthesizing biodiesel using the acid-resistant mutant, the genetically engineered host cell, or the composition.

[0036] Specifically, the method includes the step of carrying out an ester exchange reaction using acidified oil and organic alcohol as raw materials in the presence of water and the acid-resistant mutant.

[0037] Specifically, the acidified oil is a plant-based soap residue acidified oil.

[0038] In a specific embodiment of the present invention, the acidified oil is acidified soybean oil, and the organic alcohol is methanol.

[0039] Beneficial effects: This invention addresses the problem of easy inactivation of *Candida antarcticus* lipase A in acidic environments by mutating amino acids at positions 45 or 161 of *Candida antarcticus* lipase A, resulting in an acid-resistant mutant. Compared to *Candida antarcticus* lipase A, the optimal reaction temperature, pH, and specific activity of the acid-resistant mutant remain largely unchanged. However, its residual enzyme activity after incubation for the same time in an acidic environment is significantly increased, by 10%–26%, and its yield of biodiesel from acidified oil within the same time period is also significantly increased. The mutant described in this invention can be used for the preparation of biodiesel under acidic conditions, which is beneficial for the recycling of acidified oil. Attached Figure Description

[0040] Figure 1 The results of PCR amplification and electrophoresis detection of the expression vector of Candida antarcticis lipase A mutant are shown in the figure; lanes 1 to 6 in the figure are mutants D45M, R60N, G144P, D161N, G387P and G403P, respectively.

[0041] Figure 2 The results of SDS-PAGE analysis of purified samples of Candida antarcticis lipase A (CALA) and its mutants are shown in the figure; lanes 1 to 7 are CALA and its mutants D45M, R60N, G144P, D161N, G387P and G403P, respectively.

[0042] Figure 3 The figure shows the residual enzyme activity of CALA and its mutants D45M, R60N, G144P, D161N, G387P, and G403P after incubation at pH 4 for 6 h; p <0.05.

[0043] Figure 4 The residual enzyme activity of CALA and its mutants D45M and D161N after incubation at different pH conditions for 24 h is shown; WT in the figure represents CALA.

[0044] Figure 5 Changes in product yields for the preparation of biodiesel from soybean oil and methanol catalyzed by CALA and its mutants D45M and D161N. Detailed Implementation

[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0046] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0047] The amino acid sequence of Candida antarcticis lipase A (CALA) described in this embodiment of the invention is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding CALA is shown in SEQ ID NO.2.

[0048] Example 1: Preparation of CALA and its mutants To improve CALA's tolerance to acidic environments and enable it to catalyze the production of biodiesel from acidified oil, this invention combines the structural analysis results of CALA with the inventors' research and development experience to perform site-directed mutagenesis on six amino acid sites in CALA, namely positions 45, 60, 144, 161, 387, and 403, thereby constructing CALA mutants D45M, R60N, G144P, D161N, G387P, and G403P.

[0049] The specific process for constructing the CALA mutant is as follows: 1. Construction of CALA expression vector The construction of the CALA expression vector pET-22b(+)-CALA was commissioned to Sangon Biotech (Shanghai) Co., Ltd. The specific process was as follows: first, the gene encoding CALA was synthesized in its entirety; then, the vectors introduced at both ends were used... Nde I and Not The CALA gene was cloned into the pET-22b(+) vector by I restriction site, thus constructing the CALA expression vector pET-22b(+)-CALA.

[0050] 2. Construction of mutant expression vector This invention uses the CALA expression vector pET-22b(+)-CALA as a template and employs a PCR site-directed mutagenesis strategy to construct a CALA mutant expression vector through PCR amplification. The nucleotide sequences of the primers used to construct the CALA mutant expression vector are shown in Table 1, and the primers are designed according to the target mutation site.

[0051] Table 1 Primers used to construct CALA mutant expression vectors

[0052] The PCR amplification reaction system was as follows: 50 ng DNA template (pET-22b(+)-CALA), 10 μL PrimeSTAR Max Premix (2×), 1 μL each of upstream and downstream primers, and ddH2O to a final volume of 20 μL.

[0053] The PCR amplification reaction program was as follows: 98℃ for 3 min; 98℃ for 10 s, 55℃ for 30 s, 72℃ for 5 min, for 30 cycles; extension at 72℃ for 5 min.

[0054] After PCR amplification, the amplification products were detected by 1% agarose gel electrophoresis, and the results are as follows: Figure 1 As shown. The target strip size is approximately 6700 bp, composed of... Figure 1 As can be seen, the amplified bands obtained by this invention are single and their size is consistent with expectations, indicating that the six CALA mutant expression vectors have been successfully constructed.

[0055] 3. Recombinant expression of CALA and its mutants The amplification products were purified and recovered using... Dpn I enzyme was used to treat at 37°C for 1 h to remove methylated template DNA. The reaction system was: 1 μL Dpn 1. 1 μL reaction buffer and 8 μL amplification product. The digested amplification product (digestion product) was introduced into *E. coli* Top10 competent cells via heat shock. The specific procedure was as follows: 50 μL of Top10 competent cells were mixed with the digestion product, incubated on ice for 30 min, then heat-shocked at 42℃ for 60 s, followed by cooling on ice for 2 min; 500 μL of LB liquid medium was added, and the cells were cultured at 37℃ and 200 rpm for 60 min with shaking; 250 μL of the bacterial culture was evenly spread on the surface of LB solid medium containing 100 μg / mL ampicillin, and incubated upside down at 37℃ overnight; single colonies were picked and sent to Sangon Biotech (Shanghai) Co., Ltd. for gene sequencing verification.

[0056] Expression vectors were extracted from transformants whose sequences were verified to be correct by gene sequencing and transformed into *E. coli* BL21(DE3) strain using the heat shock method. The transformed vectors were evenly spread on LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37°C. Single colonies were picked and transferred to 5 mL of LB liquid medium containing 100 μg / mL ampicillin and incubated overnight at 37°C and 200 rpm. 1 mL of the bacterial culture was inoculated into 100 mL of LB liquid medium and incubated for 3 h at 37°C and 200 rpm. 50 mL of the bacterial culture was inoculated into 350 mL of LB liquid medium and incubated at 37°C and 200 rpm. When the OD600 of the bacterial culture reached 0.6–0.8, IPTG was added for induction, with a final IPTG concentration of 0.5 mM. After induction for 18 h at 16°C and 200 rpm, the fermentation broth was centrifuged at 5000 × g at 4°C for 30 seconds. After centrifugation, collect the bacterial culture. Add buffer (20 mM phosphate, pH 8.0, containing 30 mM imidazole and 300 mM NaCl) at a ratio of bacterial weight to Buffer A (g / mL) of 1:10. Disrupt the sample using ultrasound, and centrifuge the disrupted solution at 10000×g at 4°C. After centrifugation for 30 min, collect the supernatant for further purification.

[0057] The target protein was purified using Ni-NTA. After equilibration with Buffer A, the crude enzyme solution was loaded at a rate of 5 mL / min, followed by rinsing with Buffer A to remove impurities. The sample was then eluted with Buffer B (20 mM phosphate, pH 8.0, containing 300 mM imidazole and 300 mM NaCl), and the eluent was collected. After purity verification by 10% SDS-PAGE, the imidazole was removed using a 10 kDa ultrafiltration tube with 20 mM phosphate (pH 8.0) to obtain recombinant CALA and its mutant samples.

[0058] SDS-PAGE results of purified CALA and its mutant samples are as follows: Figure 2 As shown, lanes 1 through 7 are, in order, CALA and its mutants D45M, R60N, G144P, D161N, G387P, and G403P. Figure 2 It can be seen that the purity of the recombinant CALA and its mutant samples obtained by this invention is greater than 90%, which meets the requirements of subsequent experiments.

[0059] Example 2: Determination of the acid resistance of CALA and its mutants The recombinant CALA and its mutant (purified enzyme) samples obtained in Example 1 were diluted with 20 mM citrate buffer (pH 4.0) to a final concentration of 0.2 mg / mL. After incubation at 4°C for 6 h, the residual enzyme activity was determined by pNP colorimetry to rapidly screen for CALA mutants that are relatively more acid-resistant.

[0060] The specific procedure for the pNP colorimetric method is as follows: Dissolve 4-nitrophenyl myristate in anhydrous ethanol to prepare a 10 mM 4-nitrophenyl hexanoate solution; mix 10 µL of the 4-nitrophenyl hexanoate solution with 80 µL of 20 mM Tris-HCl buffer (pH 8.0), add 10 µL of enzyme sample to start the reaction, incubate at 40℃ for 5 min, and immediately add 100 μL of isopropanol to terminate the reaction. Measure the absorbance of the system at 405 nm using a UV-Vis spectrophotometer.

[0061] The residual enzyme activity of CALA and its mutants D45M, R60N, G144P, D161N, G387P and G403P after incubation at pH 4 for 6 h is as follows: Figure 3 As shown. By Figure 3 It was found that the residual enzyme activity of CALA after incubation in citrate buffer at pH 4.0 for 6 h was 67%, while the residual enzyme activities of mutants D45M (the amino acid sequence of which is shown in SEQ ID NO.3, and the nucleotide sequence of the gene encoding the mutation is shown in SEQ ID NO.4) and D161N (the amino acid sequence of which is shown in SEQ ID NO.5, and the nucleotide sequence of the gene encoding the mutation is shown in SEQ ID NO.6) were 84% and 76%, respectively, significantly higher than that of CALA. p The value <0.05 indicates that its tolerance to acidic environments is significantly better than that of CALA.

[0062] Subsequent examples will further investigate the mutants D45M and D161N.

[0063] Example 3: Stability determination of CALA and its mutants D45M and D161N under different pH conditions To investigate the stability of mutants D45M and D161N under different pH conditions, this invention further determined the residual enzyme activity of CALA and its mutants D45M and D161N under different pH conditions. Following the method used in Example 2, the purified enzyme samples were diluted with buffers of different pH values, incubated at 4°C for 24 h, and the residual enzyme activity was determined using the pNP colorimetric method. The 20 mM citrate buffer (pH 4.0) was replaced with a buffer of the corresponding pH. In this example, the buffers for different pH values ​​were as follows: 20 mM sodium citrate buffer for pH 4.0–5.0; 20 mM sodium phosphate buffer for pH 6.0–7.0; 20 mM Tris-HCl buffer for pH 8.0–9.0; and 20 mM sodium carbonate-sodium bicarbonate buffer for pH 10.0.

[0064] The residual enzyme activity of CALA and its mutants D45M and D161N after incubation at different pH conditions for 24 h was as follows: Figure 4 As shown. By Figure 4 It was found that, compared with CALA, mutants D45M and D161N exhibited similar stability in neutral and alkaline environments (pH 7–10). At pH 4, the residual enzyme activities of CALA, mutant D45M, and mutant D161N were 17%, 34%, and 27%, respectively; at pH 5, the residual enzyme activities were 31%, 58%, and 44%, respectively; and at pH 6, the residual enzyme activities of lipases CALA, mutant D45M, and mutant D161N were 67%, 79%, and 83%, respectively. This indicates that under acidic conditions, the residual enzyme activities of mutants D45M and D161N were significantly higher than those of CALA, with mutants D45M and D161N showing a significant increase of 10%–26%.

[0065] Example 4: Determination of remaining enzymatic properties of CALA and its mutants D45M and D161N 1. Specific activity assay of enzymes The specific activity of the enzyme was determined by olive oil titration. 4% polyvinyl alcohol and olive oil were mixed at a mass ratio of 4:3 and homogenized for 8 min to prepare a substrate emulsion. 4 g of this substrate emulsion was placed in a 100 mL Erlenmeyer flask with 5 mL of 20 mM phosphate buffer (pH 8.0) and preheated at 40°C for 5 min. 1 mL of enzyme sample diluted with 20 mM phosphate buffer (pH 8.0) was added; the blank was replaced with this buffer. The reaction was carried out at 40°C for 10 min, and then terminated with 15 mL of 95% ethanol. The free fatty acid was titrated with 0.05 M NaOH using phenolphthalein as an indicator. The enzyme activity unit (U) was defined as the amount of enzyme required to release 1 μmol of fatty acid per minute under the above conditions.

[0066] 2. Determination of optimal reaction pH Replace the 20 mM phosphate buffer (pH 8.0) in the olive oil titration system with a buffer system of the corresponding pH value, and determine the specific activity of the enzyme to obtain the optimal reaction pH. The specific buffer preparations are as follows: 20 mM sodium citrate buffer for pH 2.0–5.0; 20 mM sodium phosphate buffer for pH 6.0–7.0; and 20 mM Tris-HCl buffer for pH 8.0–9.0.

[0067] 3. Determination of optimal reaction temperature The specific activity of the enzyme was measured by changing the temperature in the olive oil titration method to 55, 65, 70, 75 and 80 °C respectively, in order to obtain the optimal reaction temperature.

[0068] The specific activity, optimal reaction pH, and optimal reaction temperature of CALA and its mutants D45M and D161N are shown in Table 2.

[0069] Table 2. Specific activity, optimal reaction pH, and temperature of CALA and its mutants D45M and D161N

[0070] As shown in Table 2, the specific activities of CALA and its mutants D45M and D161N were 513.3, 532.9 and 501.1 U / mg, respectively. The specific activities of mutants D45M and D161N were similar to those of CALA, and the optimal reaction pH (8.0) and optimal reaction temperature (70℃) of the three were the same, indicating that mutants D45M and D161N did not change the original optimal reaction pH and optimal reaction temperature of the enzyme.

[0071] Example 5: Preparation of biodiesel using CALA mutants This embodiment uses a reaction system of waste acidified soybean oil and methanol as substrates to investigate the effects of mutants D45M and D161N on the synthesis of biodiesel (fatty acid methyl esters). The specific reaction system is as follows: acidified soybean oil (3 g, acid value 118 mg KOH / g) in a molar ratio of 7:1 to methanol, 1 mL of 0.5 mg / mL lipase solution, and water added (water accounts for 14% of the total system); the reaction was carried out at 38℃ for 36 h, and the changes in the content of products and reactants were detected by gas chromatography every 6 h, and the conversion rate was calculated. ; mFAME, mAG, mTAG, mDAG, and mFA represent the mass of lipase methyl ester, monoglyceride, diglyceride, triglyceride, and free fatty acid, respectively.

[0072] The yield changes of CALA and its mutants D45M and D161N in the catalytic acidification of soybean oil and methanol to produce biodiesel are as follows: Figure 5 As shown. By Figure 5 It was observed that the product yield of CALA gradually increased from 0 to 18 h, reaching 52.9% at 18 h, and then remained essentially constant. The product yield of mutant D161N gradually increased throughout the process, reaching 86.1% after 36 h; mutant D45M reached equilibrium after 30 h with a yield of 90.8%. These results indicate that mutants D45M and D161N are superior to wild-type CALA in the preparation of biodiesel.

[0073] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An acid-tolerant mutant of Candida antarctica lipase A, characterized in that, The mutant is obtained by mutating the aspartic acid at position 45 of Candida antarctica lipase A to methionine, or mutating the aspartic acid at position 161 of Candida antarctica lipase A to asparagine; the amino acid sequence of the Candida antarctica lipase A is shown in SEQ ID NO.

1.

2. A gene encoding the acid-tolerant mutant of claim 1.

3. A polynucleotide construct comprising the gene of claim 2.

4. A genetically engineered host cell comprising the gene of claim 2 or the polynucleotide construct of claim 3.

5. A composition characterized in that, 5. A composition comprising the acid-tolerant mutant of claim 1 and / or the genetically engineered host cell of claim 4.

6. Use of the acid-tolerant mutant of claim 1, the genetically engineered host cell of claim 4, or the composition of claim 5 in catalyzing the production of biodiesel from acidified oil.

7. Use of the acid-tolerant mutant of claim 1, the gene of claim 2, the polynucleotide construct of claim 3, the genetically engineered host cell of claim 4, or the composition of claim 5 in the preparation of a preparation for catalyzing the production of biodiesel from acidified oil.

8. Use according to claim 6 or 7, characterized in that, The acidified oil is plant soapstock acidified oil.

9. Use according to claim 7, characterized in that, The preparation is an enzyme catalyst.

10. A method of improving the tolerance of Candida antarctica lipase A to acidic environments, characterized in that, The method is: mutating the aspartic acid at position 45 of Candida antarctica lipase A to methionine, or mutating the aspartic acid at position 161 of Candida antarctica lipase A to asparagine; the amino acid sequence of the Candida antarctica lipase A is shown in SEQ ID NO. 1.

Images