Lipase mutant and application thereof in preparation of dihydrooat alkaloid through biological catalysis

Abstract

The invention discloses a lipase mutant and application of the lipase mutant to preparation of dihydrooat alkaloid through biological catalysis, and relates to the technical field of biological engineering. The invention provides a lipase mutant. The amino acid sequence of the lipase mutant is as shown in SEQ ID NO: 2. The invention also provides a method for synthesizing the dihydroavena alkaloid D by using the lipase mutant. According to the invention, the biosynthesis of the dihydroavena alkaloid D is realized, one-step synthesis is realized by adopting an enzymic method, the steps of group protection and deprotection are not needed, the production process is green and environment-friendly, the emission of industrial wastewater is reduced on a large scale, and the lipase mutant has important application value.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a lipase mutant and use thereof in biocatalytic preparation of dihydroavenate alkaloids. Background Art

[0002] Dihydroavenate alkaloid D is a nitrogen-containing phenolic acid derivative designed based on natural oat alkaloids. It has rapid anti-inflammatory and antipruritic activity and is an important active ingredient in many anti-allergic skin care products. Its chemical structure is as follows:

[0003]

[0004] Dihydroavenous alkaloid D is an organic compound formed by connecting p-hydroxycinnamic acid and anthranilic acid with an amide bond (-HNCO-). It has strong antioxidant, lipid-lowering, anti-irritation, antipruritic and anti-inflammatory effects, and is therefore widely used in beauty, nursing and medicine. Since dihydroavenous alkaloid D has a low concentration in oats, it requires a high cost to purify it on an industrial scale. Therefore, the current research focus on this type of substance is on developing a synthetic process that can be applied to industrial production. At present, the synthesis method for dihydroavenous alkaloid D is mainly prepared by chemical methods. In the research report of Chinese patent CN 104418764B, p-hydroxyphenylpropionic acid and anthranilic acid are used as raw materials, and N, N-carbonyldiimidazole is used as a condensation agent. A condensation reaction is carried out in an environment where pyridine is a reaction medium to obtain dihydroavenous alkaloid D. However, this method has the disadvantages of low yield, generation of a large amount of polluted wastewater and difficulty in product separation. In view of the above problems, Chinese patent CN 112939803 B uses p-hydroxyphenylpropionic acid and acetic anhydride as raw materials and adopts a three-step process in a chlorinated solvent to prepare dihydroavenate alkaloid D. This process effectively reduces the discharge of wastewater, reduces environmental pollution, improves the product yield and reduces the production cost. However, the method has complicated synthesis steps, relies on the phenolic hydroxyl protection step, and requires strict control of the reaction conditions of each step. The three-step reaction yield is low, which is not conducive to large-scale industrial synthesis.

[0005] Compared with traditional chemical synthesis, the use of biological enzymes with high efficiency, high stereoselectivity and specificity to synthesize dihydroavenate alkaloid D can effectively solve many of the above problems, so biological methods are more ideal industrial alternatives. It is worth noting that in the enzyme-catalyzed synthesis system, enzymes are subject to many limitations of aqueous reaction media, including the difficulty of many compounds to dissolve in water, the occurrence of side reactions in water, the degradation of products, and the fact that products in water are not conducive to the purification of subsequent products. The above problems largely limit the further industrial application of enzymes. In order to improve the enzyme catalytic process, biocatalytic systems with organic solvents as reaction media have been favored by many researchers. Compared with enzyme catalysis in traditional aqueous phase, non-aqueous phase catalysis can effectively inhibit adverse side reactions and make the reaction tend to proceed in the direction of synthesis. In addition, since natural enzymes often have low biocatalytic activity in the process of catalyzing non-natural substrates, this seriously limits the industrial application of enzymes. With the continuous breakthroughs and developments in the field of biocatalysis, structure-oriented protein engineering can effectively transform enzyme molecules to obtain efficient biocatalysts.

[0006] According to the reaction mechanism, the amidation reaction of p-hydroxycinnamic acid and its derivatives with anthranilic acid can realize the synthesis of the corresponding dihydroavenous alkaloid D. There are many types of enzymes that can realize this catalytic process, including lipase, acyltransferase, amide synthetase, carboxylic acid reductase, penicillin acylase and aminoacylase, etc. However, there are no reports on the enzymatic research and production of dihydroavenous alkaloid D. Summary of the invention

[0007] The purpose of the present invention is to provide a lipase mutant and its application and method in the biocatalytic preparation of dihydroavenate alkaloids.

[0008] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0009] In one aspect, the present invention provides a lipase mutant, wherein the amino acid sequence of the lipase mutant is shown in SEQ ID NO:2.

[0010] Specifically, the lipase mutant is a derivative protein formed by replacing the 286th proline in the amino acid sequence shown in SEQ ID NO: 1 with alanine.

[0011] SEQ ID NO: 1:

[0012] MRRRPGLDTASRCFRGIRYATAERFQPAELLGFEGLDGDRGRGPVSPQDPSRLDMIMGPAAQLAQSEHCQVLSVFTPSLDGRRPVMVWLHGGAFVSGGGELPW YDAARLSAEQDVVVVTVTYRLGAFGFLQLADTAGPSPGTSDQIAALQWVHRHIGQFGGDPDNVTLFGHSAGGASIEAILQWGHASLVRRAILQSGNARTHRRTR EEADKVAQMFVGLTGTDPRLLAEDEILPIQRELSRGRHFAFDWWPTSPDVPAEFAVDLMAGWTRDDGLPFLMLADQARPGPDTLSSYSDRIGPANAMFSVGSHAAAQAAVAAGRRAWLYRFDWEAPASHLGAPHCIELPFLLGEPEAWTAAPMLAGASWHEIDSLGAGLRAAWASFARGDGPGDAWRACSDADQPLNILPMRDKGRNP

[0013] SEQ ID NO:2:

[0014] MRRRPGLDTASRCFRGIRYATAERFQPAELLGFEGLDGDRGRGPVSPQDPSRLDMIMGPAAQLAQSEHCQVLSVFTPSLDGRRPVMVWLHGGAFVSGGGELPW YDAARLSAEQDVVVVTVTYRLGAFGFLQLADTAGPSPGTSDQIAALQWVHRHIGQFGGDPDNVTLFGHSAGGASIEAILQWGHASLVRRAILQSGNARTHRRTR EEADKVAQMFVGLTGTDPRLLAEDEILPIQRELSRGRHFAFDWWPTSPDVPAEFAVDLMAGWTRDDGLPFLMLADQARPGPDTLSSYSDRIGPANAMFSVGSHAAAQAAVAAGRRAWLYRFDWEAPASHLGAPHCIELPFLLGEPEAWTAAAMLAGASWHEIDSLGAGLRAAWASFARGDGPGDAWRACSDADQPLNILPMRDKGRNP

[0015] In yet another aspect, the present invention provides a gene encoding the above-mentioned lipase mutant, the nucleotide sequence of the gene is shown in SEQ ID NO:3.

[0016] SEQ ID NO:3:

[0017]

[0018] In yet another aspect, the present invention provides an expression vector comprising the above-mentioned gene.

[0019] Specifically, the expression vector includes but is not limited to a recombinant expression vector and a recombinant expression transformant.

[0020] Specifically, the present invention provides a host cell comprising the above-mentioned recombinant expression vector.

[0021] In another aspect, the present invention provides a method for enzymatically synthesizing dihydroavenate alkaloid D in a non-aqueous medium, using an organic solvent as a reaction medium, adding methyl p-hydroxybenzoate and anthranilic acid, and using the recombinant lipase mutant shown in SEQ ID NO: 2 as a catalyst to synthesize dihydroavenate alkaloid D.

[0022] Specifically, the organic solvent is selected from one or more of n-pentane, n-hexane, cyclohexane, isooctane, dichloromethane, toluene, o-xylene, ethyl ether, dibenzyl ether, and methyl tert-butyl ether.

[0023] Furthermore, the organic solvent is methyl tert-butyl ether.

[0024] Specifically, the reaction concentration of methyl parahydroxybenzoate is 1-50mmol / L;

[0025] Further, the reaction concentration of methyl parahydroxybenzoate is 10-30mmol / L;

[0026] Furthermore, the reaction concentration of methyl parahydroxybenzoate is 20 mmol / L.

[0027] Specifically, the reaction concentration of anthranilic acid is 1-80 mmol / L;

[0028] Furthermore, the reaction concentration of anthranilic acid is 50-70 mmol / L;

[0029] Furthermore, the reaction concentration of the anthranilic acid is 60 mmol / L.

[0030] Specifically, the molar ratio of the anthranilic acid and methyl parahydroxybenzoate is 1-5:1;

[0031] Furthermore, the molar ratio of the anthranilic acid and methyl parahydroxybenzoate input is 3:1.

[0032] Specifically, the concentration of the recombinant lipase mutant is 12-24 g / L;

[0033] According to some embodiments of the present invention, the concentration of the recombinant lipase mutant is 12 g / L, 16 g / L, 20 g / L, or 24 g / L.

[0034] Further, the concentration of the recombinant lipase mutant is 16-24 g / L;

[0035] Furthermore, the concentration of the recombinant lipase mutant is 16 g / L.

[0036] Specifically, the recombinant lipase mutant is derived from a recombinant Escherichia coli lipase expression strain.

[0037] Specifically, the reaction conditions are constant temperature, and the reaction temperature is 25-40°C;

[0038] According to some embodiments of the present invention, the reaction temperature is 25°C, 30°C, 37°C, or 40°C.

[0039] Furthermore, the reaction temperature is 37°C.

[0040] Specifically, the shaking condition is 100-300 rpm;

[0041] According to some embodiments of the present invention, the oscillation conditions are 100 rpm, 175 rpm, 200 rpm, 250 rpm, and 300 rpm.

[0042] Further, the shaking condition was 250 rpm.

[0043] Specifically, the reaction time is 6-24h;

[0044] According to some embodiments of the present invention, the reaction time is 6 h, 8 h, 10 h, 12 h, or 24 h.

[0045] Furthermore, the reaction time is 12 h.

[0046] According to some embodiments of the present invention, the technical route for synthesizing dihydroavenate alkaloid D is as follows:

[0047]

[0048] Specifically, high performance liquid chromatography was used for analysis after the reaction was completed.

[0049] Furthermore, the detection conditions of the high performance liquid chromatography are:

[0050] (1) Chromatographic column: XDB-C18;

[0051] (2) Mobile phase: acetonitrile: water (1‰ trifluoroacetic acid) = 45:55;

[0052] (3) Column temperature: 30°C;

[0053] (4) Detection wavelength: 254 nm;

[0054] (5) Injection volume: 20 μL.

[0055] In another aspect, the present invention provides dihydroavenate alkaloid D prepared by the above method.

[0056] Specifically, the dihydroavenate alkaloid D has an amide bond structure of methyl parahydroxybenzoate and anthranilic acid.

[0057] The beneficial effects of the present invention are:

[0058] The invention relates to engineering design of a wild-type recombinant lipase from Rhizorhabdus wittichii, transforming the active center of the wild-type recombinant lipase by means of molecular docking and "alanine scanning", replacing the 286th proline of the amino acid sequence shown in SEQ ID NO:1 with alanine to form a derivative protein, whose new amino acid sequence is shown in SEQ ID NO:2, obtaining a recombinant lipase mutant P286A, realizing the biosynthesis of dihydroavenate alkaloid D for the first time, and compared with the traditional chemical method for synthesizing dihydroavenate alkaloid D, the method adopts an enzymatic one-step synthesis, does not require group protection and deprotection steps, has a green and environmentally friendly production process, reduces the discharge of industrial wastewater on a large scale, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is a high performance liquid chromatography analysis chart of methyl p-hydroxybenzoate. The retention time of the substrate peak is about 6.999.

[0060] Figure 2 This is a high performance liquid chromatography analysis chart of anthranilic acid. The retention time of the substrate peak is about 3.976.

[0061] Figure 3 This is a high performance liquid chromatography analysis chart of dihydroavenate alkaloid D. The product peak retention time is about 9.420.

[0062] Figure 4 The figure shows the screening results of recombinant lipase mutants.

[0063] Figure 5 This is a diagram of the reaction results of different proportions of anthranilic acid and methyl p-hydroxybenzoate.

[0064] Figure 6 The graph shows the reaction results of adding different amounts of engineered E. coli containing recombinant lipase.

[0065] Figure 7 The graph shows the reaction results at different reaction temperatures.

[0066] Figure 8 The graphs show the reaction results under different oscillation conditions. DETAILED DESCRIPTION

[0067] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0068] Example 1 Preparation of reaction catalyst

[0069] The recombinant Escherichia coli lipase expression strain (host bacteria BL21 DE3, Qingke Biotechnology) was activated, induced to express, and freeze-dried bacterial powder and crude enzyme solution were prepared;

[0070] Strain activation: The recombinant Escherichia coli lipase expression strain was inoculated into 5 mL of LB medium (containing 50 μg / mL kanamycin), and cultured at 37°C and 200 rpm for 12 hours. Then, 0.5 mL was aspirated and inoculated into 50 mL of LB medium (containing 50 μg / mL kanamycin), and cultured at 37°C and 200 rpm for about 2.5 hours.

[0071] Induce expression: When the OD600 value is 0.6-0.8, add the inducer IPTG (final concentration is 0.1mM) and culture in a shaker at 20°C and 200rpm for 18-22 hours. After the culture is completed, transfer the bacterial solution to a centrifuge tube, balance it and centrifuge it (8000rpm, 10min) to collect the bacteria, and discard the supernatant. Add physiological saline to resuspend and wash twice, and then discard the supernatant.

[0072] Preparation of freeze-dried bacterial powder: After being refrigerated in a -40°C refrigerator for at least 6 hours, the bacteria were prepared into freeze-dried bacterial powder using a freeze dryer.

[0073] Preparation of crude enzyme solution: according to the mass of wet bacteria, add phosphate buffer (100mM, pH7.0) in proportion to resuspend, put into ice box for crushing. The program of ultrasonic cell crusher is set to work for 3 seconds and 5 seconds interval, crushing time is 15 minutes, and the power is set to 250-300W. After crushing, centrifuge at 8000rpm for 15 minutes, add 4mL 75% glycerol to each 8mL supernatant, mix well, and then divide into EP tubes, place in -40℃ refrigerator for storage, and precipitate is resuspended and mixed with an equal volume of sodium phosphate buffer for use.

[0074] Example 2 Screening of Catalyst

[0075] The reaction was carried out in a shaking table at 37°C and 150 rpm. A 1 mL buffer screening system was established. A 2 mL EP tube was used as a reaction container. 10 mmol / L methyl p-hydroxybenzoate, 50 mmol / L anthranilic acid, 10% dimethyl sulfoxide as a reaction cosolvent and 1 mL crude enzyme solution were added to the container. After sufficient vortexing, the reaction was carried out in a shaking table for 12 hours. After the reaction was completed, the sample was allowed to stand in a 90°C water bath for 10 minutes, and two times the equivalent of dimethyl sulfoxide was added to the sample. After the reaction was completed, the supernatant was taken by centrifugation (8000 rpm, 10 min) and the sample was prepared for subsequent high performance liquid chromatography analysis.

[0076] The detection conditions of HPLC are:

[0077] (1) Chromatographic column: XDB-C18;

[0078] (2) Mobile phase: acetonitrile: water (1‰ trifluoroacetic acid) = 45:55;

[0079] (3) Column temperature: 30°C;

[0080] (4) Detection wavelength: 254 nm;

[0081] (5) Injection volume: 20 μL.

[0082] A wild-type recombinant lipase was screened from a library of 69 lipases. The wild-type lipase (amino acid sequence shown in SEQ ID NO: 1) catalyzed the reaction of methyl p-hydroxybenzoate and anthranilic acid. After 12 hours of reaction, the conversion rate was 35%, and it had the biological activity of preliminarily synthesizing dihydroavenous alkaloid D. The HPLC analysis of methyl p-hydroxybenzoate is shown in FIG. Figure 1 As shown, the HPLC analysis of anthranilic acid is as follows Figure 2 As shown, the HPLC analysis of dihydroavenate alkaloid D is as shown in Figure 3 shown.

[0083] Example 3 Screening of organic solvents

[0084] The raw materials of the reaction, methyl parahydroxybenzoate and anthranilic acid, are difficult to dissolve in water, and the lipase-mediated enzymatic reaction may decompose the product in the aqueous phase. Using an organic solvent as a reaction medium can effectively inhibit adverse side reactions, making the reaction tend to proceed in the direction of synthesis. Considering that polar solvents may cause enzyme denaturation and inactivation, in order to find a suitable organic solvent, ten non-polar solvents, including n-pentane, n-hexane, cyclohexane, isooctane, dichloromethane, toluene, o-xylene, ether, dibenzyl ether and methyl tert-butyl ether, were selected as reaction media, and a screening system was established.

[0085] In a 1mL reaction screening system, a 2mL EP tube was used as a reaction container, 10mmol / L methyl p-hydroxybenzoate and 50mmol / L anthranilic acid were added to the tube, an organic solvent was used to fill the 1mL reaction system, and 24g / L wild-type recombinant lipase engineered Escherichia coli was used as a reaction catalyst, and the reaction temperature and time were 37°C and 12 hours, respectively. After the reaction was completed, the results were analyzed using high performance liquid chromatography (high performance liquid chromatography conditions were the same as in Example 2), and the results showed that the wild-type recombinant lipase was only in a catalytic system with methyl tert-butyl ether as the reaction medium, and after 12 hours of reaction, the reaction conversion rate reached 43%, and no obvious catalytic activity was shown in other organic solvents, so methyl tert-butyl ether was selected to establish a non-aqueous phase catalytic system to synthesize dihydroavenous alkaloid D.

[0086] Example 4 Engineering design of wild-type recombinant lipase

[0087] In order to obtain a biocatalyst that can be used in industrial production, a variety of protein engineering methods including molecular docking and alanine scanning were used to transform the wild-type recombinant lipase. First, the protein model of the wild-type recombinant lipase was established using an online website (https: / / swissmodel.expasy.org / ), and the model structures of two small molecules, methyl p-hydroxybenzoate and anthranilic acid, were obtained from the organic small molecule biological activity database (https: / / pubchem.ncbi.nlm.nih.gov / ); then the computational software AutoDock 4.2.6 was used for molecular docking, and the results were analyzed by visualization software to determine the 10 amino acid residues that constitute the active center of the wild-type recombinant lipase; considering that non-natural substrates may face unfavorable steric hindrance when binding to the active center of the wild-type recombinant lipase, affecting the catalytic efficiency of the substrate, the "alanine scanning" method was used to mutate the 10 amino acid residues in its active center to alanine.

[0088] Using the plasmid of wild-type recombinant lipase as the parent, primers were designed for the intended mutation sites. The upstream and downstream primer sequences are shown in Table 1 below:

[0089] Table 1

[0090] Primer Sequence (5'-3') SEQ ID NO: D54AF cgccggctgaaaggcttccgcggtcgca SEQ ID NO:4 D54AR tgcgaccgcggaagcctttcagccggcg SEQ ID NO:5 M55A F ccagatgatcttccaaacgaaaggccagaccattc SEQ ID NO:6 M55AR gaatggtctggcctttcgtttggaagatcatctgg SEQ ID NO:7 I56A F gcccaacgacgctgaaacggatcgccatctgc SEQ ID NO:8 I56A R gcagatggcgatccgtttcagcgtcgttgggc SEQ ID NO:9 M57A F gtatccaaacctgggctcctccgcatgga SEQ ID NO:10 M57A R tccatgcggaggagcccaggtttggatac SEQ ID NO:11 G58AF agcagttcgccgcccttaataattgcaacattaaagcccggacc SEQ ID NO:12 G58AR ggtccgggctttaatgttgcaattattaagggcggcgaactgct SEQ ID NO:13 P286AF tcatcaatgcgcgtaaacggccaaataccgcctgcgg SEQ ID NO:14 P286AR ccgcaggcggtatttggccgtttacgcgcattgatga SEQ ID NO:15 S335AF aatgcgtgcatccgataacggaacaaattcgccatccaca SEQ ID NO:16 S335AR tgtggatggcgaatttgttccgttatcggatgcacgcatt SEQ ID NO:17 L337AF ccccactgaaaatttttaaccgtcggatcaatcacattacgcgg SEQ ID NO:18 L337AR ccgcgtaatgtgattgatccgacggttaaaaattttcagtgggg SEQ ID NO:19 M360A F gtatgcggaccaggcttaccatcaccaataacggtatcatca SEQ ID NO:20 M360A R tgatgataccgttattggtgatggtaagcctggtccgcatac SEQ ID NO:21 L361A F taaaatggcgctatcataaccgcgatcgcctgcttcctg SEQ ID NO:22 L361A R caggaagcaggcgatcgcggttatgatagcgccatttta SEQ ID NO:23

[0091] The PCR reaction system is shown in Table 2 below:

[0092] Table 2.

[0093]

[0094]

[0095] PCR reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 98°C for 5 s, annealing at 58°C for 15 s, extension at 72°C for 5 min, this stage was cycled 30 times; final extension at 72°C for 5 min; storage at 4°C.

[0096] After the PCR amplification was completed and the agarose gel electrophoresis was verified to be positive, 0.5 μL of DpnI enzyme was added to specifically remove the methylated template DNA, and placed in a 37°C metal bath for 3 hours. Then, transformation, bacterial selection, and sequencing were performed, and the corresponding recombinant lipase mutants were prepared using the method described in Example 1.

[0097] A 1mL reaction system was set up to screen mutants. A 2mL EP tube was used as the reaction container. 10mmol / L methyl p-hydroxybenzoate and 50mmol / L o-aminobenzoic acid were added to the tube. Methyl tert-butyl ether was used to make up the 1mL reaction system. 24g / L (dry weight) of engineered Escherichia coli containing recombinant lipase was used as the reaction catalyst. The reaction temperature and time were 37°C and 12 hours, respectively.

[0098] The reaction results are as follows Figure 4 As shown, after 12 hours of reaction, the reaction conversion rate of the recombinant lipase mutant P286A increased from 43% to 68% compared with the wild type, indicating that the binding efficiency of the substrate and the enzyme can be effectively improved by modifying the active pocket.

[0099] Example 5 Determination of the Optimal Synthesis Conditions

[0100] Since anthranilic acid is difficult to dissolve, its reaction concentration was selected as 60 mmol / L.

[0101] In the experiment with different molar ratios of anthranilic acid and methyl paraben ( Figure 5 ), when the input molar ratio was 3:1, the reaction conversion rate reached the highest, which was 72%. Therefore, the optimal reaction concentration of methyl parahydroxybenzoate was 20mmol / L.

[0102] The amount of engineered Escherichia coli added to the recombinant lipase mutant P286A was optimized. In the experiment, 8 g / L, 12 g / L, 16 g / L, 20 g / L, and 24 g / L were selected as the single reaction variables. The experimental results are shown in Figure 6 As shown, when the addition amount of engineered Escherichia coli of recombinant lipase was increased from 8 g / L to 16 g / L, the reaction conversion rate increased from 54% to 72%. However, when the addition amount of engineered Escherichia coli of recombinant lipase was further increased, the reaction conversion rate did not increase significantly. Therefore, 16 g / L was selected as the optimal reaction condition.

[0103] Optimize the reaction temperature. In the experiment, 25℃, 30℃, 37℃, 40℃, 45℃, and 50℃ were selected as the reaction temperatures. The results are as follows Figure 7 As shown, the reaction conversion rate was the highest at 37°C, reaching 72%, so 37°C was selected as the optimal condition;

[0104] The oscillation conditions were optimized. In the experiment, 100 rpm, 175 rpm, 200 rpm, 250 rpm, and 300 rpm were selected as the oscillation conditions. The results are as follows: Figure 8 As shown, when 250 rpm was selected, the reaction conversion rate was the highest, which was 72%.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

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

2.

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

3.

3. An expression vector, characterized in that: The expression vector comprises the gene according to claim 2.

4. A method for enzymatically synthesizing dihydroavenate alkaloid D in a non-aqueous medium, characterized in that: An organic solvent is used as a reaction medium, methyl p-hydroxybenzoate and anthranilic acid are added, and the recombinant lipase mutant according to claim 1 is used as a catalyst to synthesize dihydroavenate alkaloid D.

5. The method according to claim 4, characterized in that The organic solvent is selected from one or more of n-pentane, n-hexane, cyclohexane, isooctane, dichloromethane, toluene, o-xylene, ether, dibenzyl ether, and methyl tert-butyl ether.

6. The method according to claim 5, characterized in that The organic solvent is methyl tert-butyl ether.

7. The method according to claim 4, characterized in that The reaction concentration of the methyl p-hydroxybenzoate is 1-50 mmol / L; the reaction concentration of the anthranilic acid is 1-80 mmol / L.

8. The method according to claim 7, characterized in that The reaction concentration of the methyl p-hydroxybenzoate is 20 mmol / L; the reaction concentration of the anthranilic acid is 60 mmol / L.

9. The method according to claim 4, characterized in that The molar ratio of the anthranilic acid and methyl parahydroxybenzoate input is 1-5:

1.

10. The method according to claim 9, characterized in that The molar ratio of the anthranilic acid and methyl parahydroxybenzoate input is 3:

1.

11. The method according to claim 4, characterized in that The concentration of the recombinant lipase mutant is 12-24 g / L.

12. The method according to claim 11, characterized in that The concentration of the recombinant lipase mutant is 16-24 g / L.

13. The method according to claim 12, characterized in that The concentration of the recombinant lipase mutant is 16 g / L.

14. The method according to claim 4, characterized in that The reaction conditions are constant temperature, the reaction temperature is 25-40°C; the shaking conditions are 100-300 rpm; and the reaction time is 6-24 hours.

15. Dihydroavenate alkaloid D prepared by the method according to any one of claims 4 to 14.

16. The dihydroavenate alkaloid D according to claim 15, characterized in that The dihydroavenate alkaloid D has an amide bond structure of methyl p-hydroxybenzoate and anthranilic acid.

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