A lipase mutant and its use in the synthesis of aspirin

Abstract

The present application relates to the technical field of genetic engineering and enzyme engineering, in particular to a kind of lipase mutant and its application in the synthesis of aspirin.The lipase mutant provided by the present application is obtained by joint mutation of the 125th and 198th positions on the amino acid sequence shown in wild-type lipase.The lipase mutant constructed by the present application exhibits ultra-high activity in the synthesis of aspirin, and can be applied to the preparation of aspirin.The present application also provides a green high-yield preparation process of aspirin, which uses the lipase mutant to synthesize aspirin in a non-aqueous phase or a solvent-free system, and the substrate conversion rate can reach 99.9%, without by-product, and the product has high purity, which can meet the growing demand of market for aspirin, and has good industrial popularization value.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, specifically to a lipase mutant and its application in the synthesis of aspirin. Background Technology

[0002] Aspirin, also known as acetylsalicylic acid, is an organic compound with the chemical formula C9H8O4. Aspirin is widely used due to its broad pharmacological effects, rapid onset of action, and stable efficacy. Initially, it was primarily used for antipyretics, analgesia, and antirheumatic purposes. With further research, scholars have discovered that aspirin can also be used for the prevention and treatment of various other diseases, such as cardiovascular and cerebrovascular diseases, colorectal cancer, lung cancer, breast cancer, esophageal cancer, bile duct cancer, and pancreatic cancer. In agriculture, aspirin also promotes robust plant growth, enhances stress resistance, increases the survival rate of tree stumps, and prolongs the lifespan of cut flowers.

[0003] Traditionally, aspirin is produced by the acylation reaction of acetic anhydride and salicylic acid under concentrated sulfuric acid catalysis. While the concentrated sulfuric acid catalytic method is a mature technology, the product yield is low, typically 65%–70%, and it produces numerous byproducts such as aspirin anhydride, salicylic acid polymers, and aspirin-salicylic acid dimers, leading to complex subsequent separation and purification processes. Furthermore, concentrated sulfuric acid severely corrodes equipment and generates large amounts of waste acid, which is also environmentally unfriendly.

[0004] With the increasing clinical application of aspirin, its synthetic process is constantly being innovated and optimized. Patent document CN108727188A discloses a method for the tyrosine-catalyzed synthesis of aspirin. This method uses tyrosine as a catalyst to catalyze the esterification reaction of acetic anhydride and salicylic acid, synthesizing aspirin under ultrasonic conditions. Experimental results show that under the conditions of an ultrasonic power of 500 W, a molar ratio of acetic anhydride to salicylic acid of 2:1, an ultrasonic reaction time of 20 min, a reaction temperature of 60 ℃, and a catalyst dosage of 8% of the mass of salicylic acid, the product yield is 93.43%, which is not high overall.

[0005] Patent document CN118561688A discloses a method for synthesizing aspirin using inexpensive mineral functional materials as catalysts. This method uses zeolite, sepiolite, attapulgite, or kaolin as catalysts to catalyze the esterification reaction of salicylic acid and acetic anhydride. After the reaction, the catalyst is filtered, the solvent is recovered by vacuum distillation, recrystallized with ethanol and water, filtered again, and dried in an oven to obtain the aspirin. This method has the advantages of low cost, no environmental pollution, high catalyst activity, recyclability after use, and mild reaction conditions. However, the mineral functional materials in this method require water washing, acid washing, and alkali washing activation steps before use, and the subsequent purification steps are complex, resulting in numerous procedures and complicated operations.

[0006] Lipase (EC 3.1.1.3) is an important industrial enzyme that catalyzes the hydrolysis of oils and fats, as well as the synthesis of esters or transesterification. It exhibits high selectivity, mild catalytic conditions, and environmental friendliness, and is widely used in food, pharmaceuticals, and biofuels. Therefore, researching and exploring the application of lipase in aspirin preparation is of great significance for improving aspirin production efficiency and product quality, and reducing impurity formation and environmental pollution. Summary of the Invention

[0007] To improve the quality of aspirin and achieve the goals of impurity-free, high-yield, low-consumption, and environmentally friendly production, this invention provides a lipase mutant with high catalytic efficiency and high conversion rate. Furthermore, this invention proposes a process for the enzymatic biosynthesis of aspirin using this lipase mutant. This lipase mutant can catalyze the acetylation of salicylic acid and acetic anhydride to aspirin in a non-aqueous or solvent-free system. This method has the advantages of high conversion rate, low substrate residue, low impurities, and high purity. It not only simplifies the separation and purification process of aspirin but is also environmentally friendly and pollution-free, laying the foundation for the application of lipases in the pharmaceutical field.

[0008] To achieve the above objectives, the present invention discloses the following technical solutions: The lipase provided by this invention is derived from Candida antarcticis ( Candida antarctica The lipase B (CALB) of wild-type Candida antarctica, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0009] In a first aspect, the present invention provides a lipase mutant, wherein the lipase mutant is an Antarctic Candida lipase B mutant, which is obtained by jointly mutating the 125th and 198th positions of the amino acid sequence shown in SEQ ID NO.2.

[0010] Furthermore, the lipase mutant is specifically obtained by mutating valine at position 125 of the amino acid sequence of wild-type Candida antarctica lipase B to asparagine and proline at position 198 to tryptophan.

[0011] Furthermore, the amino acid sequence of the lipase mutant is shown in SEQ ID NO.4.

[0012] In addition, the present invention also provides a nucleic acid molecule that encodes the above-mentioned lipase mutant.

[0013] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.3.

[0014] Furthermore, the present invention provides a recombinant vector comprising the above-mentioned nucleic acid molecules.

[0015] Furthermore, the present invention also provides a recombinant cell comprising the above-mentioned nucleic acid molecule or the above-mentioned recombinant vector.

[0016] Secondly, the present invention also claims protection for the use of the lipase mutant, the nucleic acid molecule, the recombinant vector, or the recombinant cell in the synthesis of aspirin.

[0017] Thirdly, the present invention also provides a method for preparing aspirin, wherein the method uses salicylic acid and acetic anhydride as raw materials and carries out an acetylation reaction under the catalysis of a lipase mutant to generate aspirin.

[0018] Specifically, the present invention provides a method for preparing aspirin in a non-aqueous phase system, comprising the following steps: Aspirin is produced by acetylation of salicylic acid and acetic anhydride as raw materials and n-hexane as solvent under the catalysis of a lipase mutant at a temperature of 40-60 °C.

[0019] Furthermore, in the method for preparing aspirin in a non-aqueous phase system, the ratio of salicylic acid to acetic anhydride is 1 g: 1.2 mL, the ratio of salicylic acid to n-hexane is 1 g: 2 mL, and the mass ratio of salicylic acid to lipase mutant is 10:1.

[0020] Furthermore, the present invention also provides a solvent-free method for preparing aspirin, comprising the following steps: Using salicylic acid and acetic anhydride as raw materials, and under the catalysis provided in this invention, an acetylation reaction is carried out at a temperature of 40~60 °C to produce aspirin.

[0021] Furthermore, in the solvent-free aspirin preparation method, the ratio of salicylic acid to acetic anhydride is 1 g: 1.5 mL, and the mass ratio of salicylic acid to lipase mutant is 10:1.

[0022] To address the problems of numerous reaction byproducts, complex separation and purification processes, and low production efficiency in existing aspirin production technologies, this invention provides a novel enzymatic biosynthesis process for aspirin suitable for industrial production. This invention utilizes error-prone PCR screening to identify a lipase mutant, specifically obtained by mutating valine at position 125 of the wild-type *Candida antarcticus* lipase B amino acid sequence to asparagine and proline at position 198 to tryptophan. The inventors have found that this *Candida antarcticus* lipase B mutant exhibits extremely high activity in aspirin synthesis. In a non-aqueous or solvent-free system, the substrate conversion rate reaches 99.9% within 30 minutes of reaction, with no byproducts and high product purity. This is an environmentally friendly, pollution-free, high-quality, and high-efficiency enzymatic biosynthesis process for aspirin.

[0023] Compared with the prior art, the lipase mutant provided by the present invention has the following advantages: (1) The lipase mutant provided by the present invention exhibits extremely high activity in the synthesis of aspirin and can be applied to the preparation of aspirin, laying the foundation for the application of lipase in the pharmaceutical field.

[0024] (2) The enzymatic biosynthesis process for aspirin suitable for industrial production provided by the present invention has the advantages of mild reaction conditions, high production efficiency, high product purity and environmental protection and no pollution. The lipase mutant synthesizes aspirin in a non-aqueous phase or solvent-free system with a substrate conversion rate of up to 99.9%, no by-products, and high product purity, which can meet the growing market demand for aspirin and has great industrial promotion value. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the construction of the recombinant plasmid pPIC9K-CALB.

[0026] Figure 2 This is the HPLC chromatogram of aspirin in the synthesis of aspirin catalyzed by a lipase mutant in a non-aqueous system.

[0027] Figure 3 This is an HPLC chromatogram of the synthesis of salicylic acid in aspirin catalyzed by a lipase mutant in a non-aqueous system.

[0028] Figure 4 This is an HPLC chromatogram of the reaction sample in the solvent-free system catalyzed by a lipase mutant for the synthesis of aspirin.

[0029] Figure 5 The diagram shows the reaction scheme for the synthesis of aspirin catalyzed by a lipase mutant. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are not intended to limit the invention, but only to illustrate it. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the raw materials and reagents used in the following embodiments are commercially available. The materials used in the embodiments of the present invention include: salicylic acid, acetic anhydride, aspirin, and n-hexane reagent, purchased from Maclean's.

[0031] Example 1: Construction of a lipase mutant library 1. Genes synthesized from the entire genome: The company commissioned Qingke Biotechnology to obtain Candida antarcticis (Candida) using whole genome synthesis techniques. Candida antarctica The complete gene of the CALB lipase (GenBank: CAA83122.1) was extracted and linked between the EcoRI and NotI restriction sites of the vector pPIC9K to form the recombinant plasmid pPIC9K-CALB, as shown in the diagram. Figure 1 As shown in the figure. The nucleic acid sequence of this gene was optimized by codons to obtain the nucleotide sequence shown in SEQ ID NO.1, and the amino acid sequence encoded by this gene is shown in SEQ ID NO.2.

[0032] (1) Nucleotide sequence of the wild-type lipase CALB encoding gene (SEQ ID NO.1): TTGCCAAGTGGATCTGATCCAGCTTTCTCTCAACCTAAATCCGTTCTGGATGCCGGTCTGACTTGTCAAGGTGCATCTCCATCTAGTGTTTCCAAGCCTATTCTTCTGGTTCCAGGTACTGGTACGACTGGACCTCAATCTTTTGATTCTAACTGGATTCCATTGTCTACTCAATTGGGTTACACTCCATGCTGGATCTCTCCACCACCATTCATGTTGAATGATACACAAGTCAATACTGAGTACATGGTCAACGCAATTACCACCTTGTATGCTGGCTCTGGTAACAACAAACTGCCAGTTCTGACTTGGTCACAAGGTGGACTGGTTGCTCAATGGGGTTTGACTTTCTTCCCTTCCATCAGATCCAAAGTTGACAGATTGATGGCTTTCGCACCTGACTATAAGGGTACAGTTCTGGCTGGTCCACTGGATGCTTTGGCTGTCTCTGCACCTTCTGTTTGGCAACAAACCACTGGTTCTGCTCTAACTACTGCTCTGAGAAATTCTGGTGGTTTGACTCAAATCGTTCCAACCACTAACCTGTACAGTGCTACTGATGAGATTGTTCAACCTCAAGTCTCTAATTCTCCACTGGACTCCAGTTATCTGTTTAACGGAAAGAACGTCCAAGCTCAGGCTGTTTGTGGTCCACTGTTCGTTATCGATCATGCTGGTTCTTTGACCTCTCAATTCTCTTATGTTGTTGGTAGAAGTGCTTTGAGATCCACTACCGGACAAGCCAGATCAGCTGATTATGGAATTACAGATTGTAATCCATTGCCTGCTAATGACCTGACTCCAGAACAGAAGGTTGCTGCTGCTGCACTGTTGGCTCCAGCTGCTGCTGCAATCGTCGCTGGTCCAAAGCAGAACTGTGAACCAGACTTGATGCCTTACGCTAGACCATTTGCAGTCGGTAAGAGAACTTGTTCTGGTATTGTCACTCCACATCACCATCATCATCATTGA。

[0033] (2) Wild-type lipase CALB amino acid sequence (SEQ ID NO.2): LPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMVNAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAGPLDALAVSAPSVWQQTTGSA LTTALRNAGGLTQIVPTTNLYSATDEIVQPQVSNSPLDSSYLFNGKNVQAQAVCGPLFVIDHAGSLTSQFSYVVGRSALRSTTGQARSADYGITDCNPLPANDLTPEQKVAAAALLAPAAAAIVAGPKQNCEPDLMPYARPFAVGKRTCSGIVTPHHHHHH.

[0034] 2. Error-prone PCR library construction: The target gene was modified using the error-prone PCR method. Primers were: Upstream primer: ACGTAGAATTCTTACCTTCTGGTT (SEQ ID NO.5).

[0035] Downstream primer: TGCCGGCCGCCTAGTGGTGGTG (SEQ ID NO.6).

[0036] The error-prone PCR reaction system is shown in Table 1, and the PCR amplification conditions are shown in Table 2.

[0037] Table 1 PCR reaction system reaction system volume 25 mmol / L dATP, dGTP 2 µL 25 mmol / L dCTP, dTTP 5 µL upstream primer 0.5 µL Downstream primer 0.5 µL Template DNA 1 µL Taq DNA polymerase 1 µL <![CDATA[25 mmo1 / L Mg 2+ ]]> 15 µL <![CDATA[5 mmo1 / L Mn 2+ ]]> 2 µL <![CDATA[ddH2O]]> 23 µL Total volume 50 µL Table 2 PCR amplification conditions

[0038] After purification using a DNA purification kit, the error-prone PCR amplification products and plasmid pPIC9K were digested, ligated, and transformed into *E. coli* DH5α competent cells using restriction endonucleases. The transformed cells were plated on LB agar plates containing kanamycin (100 mg / L) and incubated overnight at 37 °C. All transformants were then transferred to LB liquid medium to obtain the mutant plasmid. The mutant plasmid was linearized with SacI and electroporated into *Pichia pastoris* GS115 competent cells. The cells were then plated on MD agar plates and incubated at 30 °C for 2 days to construct the mutant library.

[0039] Example 2: Preliminary screening of lipase mutants Transformants from the MD plates in Example 1 were inoculated into 48-well plates containing BMGY medium and cultured at 30 °C and 220 rpm for 24 h. After centrifugation at 5000 rpm, the plates were resuspended in BMGY medium and induced to express at 28 °C and 220 rpm. After induction for 96 h, the supernatant was collected by centrifugation. 5 µL of the supernatant was added to 195 µL of p-nitrophenol ester (final concentration 1 mmol / L) to form a ELISA plate, and the mixture was shaken to mix. Strains that rapidly turned a distinct yellow color within 3 min were then screened again.

[0040] The color development principle is as follows: lipase catalyzes the reaction of p-nitrophenol ester to produce p-nitrophenol, which turns yellow under alkaline conditions.

[0041] Example 3: Screening of lipase mutants for efficient aspirin synthesis The strain obtained in Example 2 was inoculated into 5 mL of BMGY medium and cultured at 30 °C and 220 rpm for 24 h. After centrifugation at 5000 rpm, it was resuspended in 20 mL of BMGY medium and induced to express at 28 °C and 220 rpm. After induction for 96 h, the supernatant was collected by centrifugation and concentrated to 2 mL. 0.3 g of ABA resin was added to the concentrate, and the mixture was immobilized at 25 °C and 150 rpm for 4 h. After washing, it was dried at 30 °C for 1 h. The immobilized lipase was added to a centrifuge tube containing 0.2 g of salicylic acid and 1 mL of acetic anhydride and reacted at 40–60 °C and 220 rpm for 20 min. 50 μL of the reaction sample was then added dropwise to 200 μL of 1% ferric chloride solution, and the color change was observed.

[0042] The principle behind the color development is as follows: Salicylic acid contains phenolic hydroxyl groups, which react with ferric chloride solution to form a purple complex. If the solution turns purple, it indicates that the salicylic acid has not completely reacted; if the solution remains yellow, it proves that the salicylic acid has been completely consumed.

[0043] Example 4: Confirmation of the efficient synthesis of aspirin lipase mutant Using the genome of the mutant strain screened in Example 3 as a template, PCR amplification was performed using 5´AOX1 and 3´AOX1 primers. The PCR amplification products were sent for sequencing, which was completed by Qingke Biotechnology Co., Ltd. The nucleotide sequence is shown in SEQ ID NO.3. It was obtained by mutating valine at position 125 to asparagine and proline at position 198 to tryptophan, and named ASP01.

[0044] (1) Nucleotide sequence of the gene encoding the lipase mutant ASP01 (SEQ ID NO.3): TTGCCAAGTGGATCTGATCCAGCTTTCTCTCAACCTAAATCCGTTCTGGATGCCGGTCTGACTTGTCAAGGTGCATCTCCATCTAGTGTTTCCAAGCCTATTCTTCTGGTTCCAGGTACTGGTACGACTGGACCTCAATCTTTTGATTCTAACTGGATTCCATTGTCTACTCAATTGGGTTACACTCCATGCTGGATCTCTCCACCACCATTCATGTTGAATGATACACAAGTCAATACTGAGTACATGGTCAACGCAATTACCACCTTGTATGCTGGCTCTGGTAACAACAAACTGCCAGTTCTGACTTGGTCACAAGGTGGACTGGTTGCTCAATGGGGTTTGACTTTCTTCCCTTCCATCAGATCCAAAAACGACAGATTGATGGCTTTCGCACCTGACTATAAGGGTACAGTTCTGGCTGGTCCACTGGATGCTTTGGCTGTCTCTGCACCTTCTGTTTGGCAACAAACCACTGGTTCTGCTCTAACTACTGCTCTGAGAAATTCTGGTGGTTTGACTCAAATCGTTCCAACCACTAACCTGTACAGTGCTACTGATGAGATTGTTCAACCTCAAGTCTCTAATTCTTGGCTGGACTCCAGTTATCTGTTTAACGGAAAGAACGTCCAAGCTCAGGCTGTTTGTGGTCCACTGTTCGTTATCGATCATGCTGGTTCTTTGACCTCTCAATTCTCTTATGTTGTTGGTAGAAGTGCTTTGAGATCCACTACCGGACAAGCCAGATCAGCTGATTATGGAATTACAGATTGTAATCCATTGCCTGCTAATGACCTGACTCCAGAACAGAAGGTTGCTGCTGCTGCACTGTTGGCTCCAGCTGCTGCTGCAATCGTCGCTGGTCCAAAGCAGAACTGTGAACCAGACTTGATGCCTTACGCTAGACCATTTGCAGTCGGTAAGAGAACTTGTTCTGGTATTGTCACTCCACATCACCATCATCATCATTGA。

[0045] (2) The amino acid sequence of the lipase mutant ASP01 (SEQ ID NO.4): LPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMVNAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKNDRLMAFAPDYKGTVLAGPLDALAVSAPSVWQQTTGSA LTTALRNAGGLTQIVPTTNLYSATDEIVQPQVSNSWLDSSYLFNGKNVQAQAVCGPLFVIDHAGSLTSQFSYVVGRSALRSTTGQARSADYGITDCNPLPANDLTPEQKVAAAALLAPAAAAIVAGPKQNCEPDLMPYARPFAVGKRTCSGIVTPHHHHHH.

[0046] Example 5: Preparation of immobilized lipase ASP01 1.5 L fermenter for producing lipase ASP01: (1) Seed culture: ① Primary seed culture: Pick a single colony of lipase ASP01 and inoculate it into a 5 mL YPD liquid culture medium test tube. Incubate at 30℃ and 180 rpm / min for 18~24 h.

[0047] ② Secondary seed culture: Inoculate the bacterial culture at a ratio of 2% into 200 mL of YPD liquid medium and incubate at 30 ℃ and 180 rpm / min for 20 h.

[0048] (2) Fermentation culture: Freshly cultured secondary seed culture was inoculated at a volume concentration of 5% into a fermentation medium containing 0.05% antifoaming agent (the fermentation medium consisted of: 2.5% 85% phosphate, 0.93 g / L calcium sulfate, 18.2 g / L potassium sulfate, 14.9 g / L magnesium sulfate heptahydrate, 4.13 g / L potassium hydroxide, 40 g / L glycerol, and 1.2% PTM trace salts; the PTM trace salts were: 6 g / L copper sulfate-5H2O, 0.08 g / L sodium iodide, 3 g / L manganese sulfate-H2O, 0.2 g / L sodium molybdate-2H2O, 0.02 g / L boric acid, 0.5 g / L cobalt chloride, 20 g / L zinc chloride, 65 g / L ferrous sulfate-7H2O, 0.2 g / L biotin, and 0.5% sulfuric acid). The temperature was controlled at 30 °C, and the fermentation pH was controlled with 25% ammonia. 5.0; During the process, dissolved oxygen (DO) was controlled to be greater than 20%, and dissolved oxygen was monitored. When dissolved oxygen rose, glycerol was added, and the glycerol feeding phase lasted for 4 hours, after which the glycerol feeding was stopped. One hour later, the fermenter temperature was adjusted to 25 °C, and methanol was added for induction. The fermentation was continued for 96 hours to obtain the fermentation broth; the broth was centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected.

[0049] 2. Immobilization of lipase ASP01: After decolorizing the collected supernatant with activated carbon, the enzyme solution was concentrated 5-10 times and used for immobilization experiments. The optimal immobilization conditions were: ABA resin: enzyme solution = 1:3 (m / V), pH 7.0, 30 ℃, shaking at 150 rpm for 4 h, followed by washing and drying at 30 ℃ for 1 h.

[0050] Example 6: Synthesis of aspirin catalyzed by lipase ASP01 in a non-aqueous system 1. Experimental Method: The immobilized lipase ASP01 obtained in Example 5 was used for a catalytic reaction. 10 g of salicylic acid, 12 mL of acetic anhydride, 20 mL of n-hexane, and 1 g of immobilized lipase ASP01 were mixed and reacted at 40–60 °C and 220 rpm. The reaction formula for the synthesis of aspirin catalyzed by the lipase mutant is as follows. Figure 5 As shown. Samples were taken after 30 min of reaction and analyzed by HPLC.

[0051] HPLC detection conditions: Column: Octadecylsilane-bonded silica gel (C18, 4.6 mm × 250 mm, 5 μm); Mobile phase A: Acetonitrile-tetrahydrofuran-glacial acetic acid-water = 20:5:5:70; Mobile phase B: Acetonitrile; Flow rate: 1 mL / min; Elution method: Gradient elution, as shown in Table 3; Column temperature: 30 ℃; Injection volume: 10 uL; Detector: UV detector, aspirin detection wavelength 276 nm, salicylic acid detection wavelength 303 nm.

[0052] Table 3 Gradient elution settings for the mobile phase Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 60 20 80 2. Experimental Results: Experimental results are as follows Figure 2 and Figure 3 As shown.

[0053] Figure 2 This is the HPLC chromatogram of aspirin in the synthesis of aspirin catalyzed by a lipase mutant in a non-aqueous system. Figure 3 This is an HPLC chromatogram of the synthesis of salicylic acid in aspirin catalyzed by a lipase mutant in a non-aqueous system.

[0054] Depend on Figure 2 and Figure 3 It is known that, using the lipase mutant provided by this invention in a non-aqueous system, the catalytic synthesis of aspirin can achieve a salicylic acid conversion rate of 98%, an aspirin yield of 95%, a purity of over 99%, no byproducts, and high product purity.

[0055] Example 7: The reaction of aspirin synthesized by lipase ASP01 in a solvent-free system. 1. Experimental Method: The immobilized lipase ASP01 obtained in Example 5 was used for a catalytic reaction. 10 g of salicylic acid, 15 mL of acetic anhydride, and 1 g of immobilized lipase ASP01 were mixed and reacted at 40–60 °C and 220 rpm. The reaction formula for the synthesis of aspirin catalyzed by the lipase mutant is as follows. Figure 5 As shown. Samples were taken after 30 min of reaction and analyzed by HPLC.

[0056] HPLC detection conditions: Column: octadecylsilane-bonded silica gel (C18, 4.6 mm × 250 mm, 5 μm); Mobile phase A: acetonitrile-tetrahydrofuran-glacial acetic acid-water = 20:5:5:70; Mobile phase B: acetonitrile; Flow rate: 1 mL / min; Elution method: gradient elution, as shown in Table 4; Column temperature: 30 ℃; Injection volume: 10 uL; Detector: UV detector, aspirin detection wavelength 276 nm, salicylic acid detection wavelength 303 nm.

[0057] Table 4 Gradient elution settings for the mobile phase Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 60 20 80 2. Experimental Results: Experimental results are as follows Figure 4 As shown.

[0058] Figure 4 This is an HPLC chromatogram of the reaction sample in the solvent-free lipase mutant-catalyzed synthesis of aspirin. Figure 4 It can be seen that the conversion rate of salicylic acid can reach 99.9%, and salicylic acid was not detected. The yield of aspirin is 98.3%, and the purity is greater than 99%. At the same time, 50 μL of the above reaction sample was added dropwise to 200 μL of 1% ferric chloride test solution. The solution color did not change, which also proves that the salicylic acid has been consumed.

[0059] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A lipase mutant, characterized in that, The lipase mutant was obtained by jointly mutating positions 125 and 198 of the amino acid sequence shown in SEQ ID NO.

2.

2. The lipase mutant according to claim 1, wherein The amino acid sequence of the lipase mutant is shown in SEQ ID NO.

4.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule is encoding the lipase mutant described in claim 1 or 2.

4. The nucleic acid molecule as described in claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

3.

5. A recombinant cell, characterized in that, The recombinant cells comprise the nucleic acid molecules as described in claim 3 or 4.

6. The use of the lipase mutant as described in claim 1 or 2, the nucleic acid molecule as described in claim 3 or 4, or the recombinant cell as described in claim 5 in the synthesis of aspirin.

7. A method for preparing aspirin in a non-aqueous phase system, characterized in that, Includes the following steps: Using salicylic acid and acetic anhydride as raw materials and n-hexane as solvent, an acetylation reaction is carried out under the catalysis of the lipase mutant described in claim 1 or 2 at a temperature of 40-60 °C to produce aspirin.

8. The method for preparing aspirin in a non-aqueous phase system as described in claim 7, characterized in that, The ratio of salicylic acid to acetic anhydride in the liquid is 1 g: 1.2 mL, the ratio of salicylic acid to n-hexane in the liquid is 1 g: 2 mL, and the mass ratio of salicylic acid to lipase mutant is 10:

1.

9. A method for preparing aspirin in a solvent-free system, characterized in that, Includes the following steps: Aspirin is produced by acetylation of salicylic acid and acetic anhydride under the catalysis of the lipase mutant described in claim 1 or 2 at a temperature of 40-60 °C.

10. The method for preparing aspirin in a solvent-free system as described in claim 9, characterized in that, The ratio of salicylic acid to acetic anhydride in the liquid solution is 1 g: 1.5 mL, and the mass ratio of salicylic acid to lipase mutant is 10:1.

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