Recombinant lipase mutant and application thereof in preparation of vitamin E succinate

Abstract

The invention provides a recombinant lipase mutant and application thereof in preparation of vitamin E succinate. The recombinant lipase mutant is obtained by performing single mutation on the 104th site, the 106th site, the 154th site, the 190th site, the 278th site, the 281th site or the 282th site of an amino acid sequence as shown in SEQ ID NO.1. The invention further provides a preparation method of the recombinant lipase mutant. The method has the beneficial effects that the recombinant lipase mutant for efficiently expressing the lipase CALB is used as a catalyst, so that the vitamin E succinate can be synthesized in an organic reaction system, the conversion rate of the vitamin E succinate can reach 90% or above, the relative enzyme activity of the recombinant lipase mutant is improved by 15% compared with that of a wild type, and the yield of the recombinant lipase mutant is improved. And the vitamin E succinate is synthesized by using the recombinant lipase mutant, so that the reaction condition is mild, the process operation is simple, the method is environment-friendly, the requirement on equipment is reduced, the production cost is greatly reduced, and the method has a wide application prospect in industrial application.

Description

Technical Field

[0001] The invention belongs to the technical field of bio-enzyme catalysis and relates to a recombinant lipase mutant and application thereof in the preparation of vitamin E succinate, in particular to a recombinant lipase mutant that efficiently expresses lipase CALB and application thereof in the preparation of vitamin E succinate. Background Art

[0002] Natural vitamin E, also known as tocopherol, is an indispensable fat-soluble vitamin and natural antioxidant in the human body. It is widely found in vegetable oils, oily seeds and malt in nature. The structural characteristics of vitamin E give it significant antioxidant properties, which can effectively eliminate free radicals in the body, thereby maintaining the normal structure and function of cells, and plays a key role in human health, growth and development. With the deepening of the understanding of vitamin E, its application value in the fields of medicine, health care, food, etc. has become increasingly prominent. However, the phenolic hydroxyl group of vitamin E is easily oxidized by air and heat, which brings challenges to its production, transportation and storage; at the same time, fat-soluble vitamin E is almost insoluble in water, which limits its effective absorption in the body. Vitamin E esterified derivatives are more stable than vitamin E. The esterified vitamin E not only significantly improves the absorption and utilization rate, but also overcomes the shortcomings of traditional oily vitamin E that is difficult to make tablets and hard capsules. Therefore, commercial vitamin E is mostly provided to the market in the form of esters.

[0003] Among vitamin E ester derivatives, vitamin E succinate has attracted much attention due to its unique pharmacological function and high melting point. Currently, there are two main production processes for vitamin E succinate, chemical synthesis and bio-enzyme catalytic synthesis. The traditional chemical method for synthesizing vitamin E succinate mainly uses triethylamine, pyridine or acid and base as catalysts, which has problems such as environmental unfriendliness, the use of volatile organic solvents, and chemical catalyst residues. In contrast, the bio-enzyme method has the advantages of mild reaction conditions, high catalytic efficiency, and strong catalytic specificity. It is a green and efficient synthesis route that has been widely studied in recent years. Xia et al. (Lipase nanogel catalyzed synthesis of vitamin E succinate in non-aqueous phase [J]. Journal of the Science of Food and Agriculture, 2021, 101 (8): 3186-3192.) used Candida rugosa lipase (CRL) as a catalyst in an intermittent reactor to prepare vitamin E succinate by catalyzing the esterification reaction of vitamin E and succinic anhydride. The reaction was carried out in dimethyl sulfoxide (DMSO) with a molar ratio of vitamin E to succinic anhydride of 1:4, an enzyme concentration of 6 mg / L, a speed of 150 r / min, and a temperature of 55 ° C for 15 h, and the optimal yield reached 62.58%. However, the reaction time of this method is too long, the catalytic efficiency of enzymatic synthesis of vitamin E succinate in organic solvents is not high, the product yield is low, and it is difficult to achieve industrial continuous production. Therefore, although enzymatic synthesis avoids the shortcomings of chemical synthesis, which is toxic and harmful and has many by-products, it is still a huge challenge to synthesize vitamin E succinate efficiently, at low cost, and with high yield.

[0004] Therefore, there is an urgent need to provide a recombinant lipase mutant and its application in the preparation of vitamin E succinate, wherein the recombinant lipase mutant can produce a lipase mutant having high catalytic activity in traditional organic solvents, which is used to improve the catalytic efficiency, increase the product yield, and prepare high-purity vitamin E succinate. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a recombinant lipase mutant and its use in the preparation of vitamin E succinate, so as to solve the problems of low catalytic efficiency, low product yield and difficulty in achieving industrial continuous production in the current enzymatic synthesis of vitamin E succinate in organic solvents.

[0006] In order to achieve the above-mentioned purpose and other related purposes, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides a recombinant lipase mutant, characterized in that: the recombinant lipase mutant is obtained by single mutation of the 104th, 106th, 154th, 190th, 278th, 281st or 282nd position of the amino acid sequence shown in SEQ ID NO.1. The amino acid sequence shown in SEQ ID NO.1 of the present invention is derived from a recombinant esterase mutant of wild-type Candida antarctica lipase B (hereinafter referred to as wild-type lipase CALB), and its nucleotide sequence is shown in SEQ ID NO.2. The amino acid sequence corresponding to the wild-type lipase CALB is 317aa long, and the protein molecular weight is about 33.02kDa. By performing site-directed mutagenesis on the recombinant esterase mutant, a recombinant lipase mutant that can efficiently express Candida antarctica lipase B (hereinafter referred to as lipase CALB) and has high catalytic performance is obtained.

[0008] Preferably, the recombinant lipase mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.1 is mutated into one of the following:

[0009] (1) Tryptophan at position 104 was mutated to alanine (W104A);

[0010] (2) glutamine at position 106 was mutated to alanine (Q106A);

[0011] (3) valine at position 154 mutated to alanine (V154A);

[0012] (4) valine at position 190 mutated to alanine (V190A);

[0013] (5) leucine at position 278 mutated to alanine (L278A);

[0014] (6) alanine at position 281 was mutated to glycine (A281G);

[0015] (7) Alanine at position 282 was mutated to glycine (A282G).

[0016] The recombinant lipase mutant is one of the following: W104A, Q106A, V154A, V190A, L278A, A281G, A282G.

[0017] Conservative substitution forms of other amino acid sites of the above-mentioned recombinant lipase mutants, forms of adding or deleting one or several amino acids, forms of amino-terminal truncation, forms of carboxyl-terminal truncation, these mutant forms are also included in the scope of the present invention.

[0018] The present invention also provides a gene encoding the recombinant lipase mutant, and its nucleotide sequence is shown as SEQ ID NO.2.

[0019] Due to the particularity of the nucleotide sequence, any variant of the polynucleotide shown in the present invention, as long as it has more than 90% homology with the aforementioned polynucleotide, belongs to the scope of protection of the present invention. The variant of the polynucleotide refers to a polynucleotide sequence with one or more nucleotide changes. The variant of this polynucleotide can be a natural variant or a non-natural variant, including a substitution variant, a deletion variant and an insertion variant. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of a polynucleotide, but will not substantially change the function of the peptide protein encoded by it.

[0020] The invention also provides a recombinant expression plasmid comprising the encoding gene.

[0021] The present invention also provides a recombinant genetic engineering bacterium comprising the coding gene or the recombinant expression plasmid.

[0022] The present invention also provides a method for preparing the recombinant lipase mutant, characterized in that it comprises the following steps:

[0023] Step 1, connecting the coding gene with the nucleotide sequence as shown in SEQ ID NO.2 to an expression vector and then transferring it into a host bacterium to construct a recombinant genetic engineering bacterium containing the coding gene;

[0024] Step 2: ferment and culture the recombinant genetically engineered bacteria, induce expression, centrifuge, resuspend, and centrifuge at low temperature, then collect the supernatant to obtain a crude enzyme solution containing a recombinant lipase mutant that efficiently expresses lipase CALB.

[0025] Preferably, the expression vector is pET28b(+). The host cell may be any conventional host cell in the art, and preferably the host bacterium is E. coli BL21(DE3).

[0026] Preferably, step 1 of constructing a recombinant genetically engineered bacterium containing the encoding gene comprises the following steps:

[0027] (1) The coding gene of wild-type Candida antarctica lipase B with a nucleotide sequence as shown in SEQ ID NO.2 was connected to the expression vector pET28b(+) to construct a recombinant plasmid containing the coding gene of lipase CALB;

[0028] (2) Designing site-directed mutagenesis primers, using a recombinant plasmid carrying a gene fragment having a nucleotide sequence such as SEQ ID NO. 2 as a template, and performing gene modification by site-directed mutagenesis technology to obtain a recombinant expression plasmid containing the coding gene;

[0029] (3) The successfully constructed recombinant expression plasmid was transformed into the host bacterium E. coli BL21 (DE3) to obtain a recombinant engineered bacterium containing the lipase mutant gene.

[0030] Preferably, step 2 is specifically:

[0031] The obtained recombinant engineered bacteria are fermented and cultured to induce expression, and the fermentation broth is centrifuged to obtain wet bacterial cells containing the recombinant lipase mutant;

[0032] The wet cells were resuspended in phosphate buffer, ultrasonically disrupted in an ice bath, and the supernatant was collected by low-temperature centrifugation to obtain a crude enzyme solution containing the recombinant lipase mutant.

[0033] More preferably, in step (2), the cell concentration of the wet cells is 40 g / L to 140 g / L.

[0034] Preferably, the induction agent in step 2 is IPTG.

[0035] More preferably, the concentration of IPTG is 0.1-1 mM, and the induction temperature is 16-37° C. Most preferably, the concentration of IPTG is 0.5 mM, and the induction temperature is 28° C.

[0036] Preferably, the method for preparing the recombinant lipase mutant further comprises:

[0037] Step 3: The crude enzyme solution containing the recombinant lipase mutant is subjected to cell disruption, and the protease solution obtained after separation and purification is subjected to immobilization treatment to obtain immobilized lipase. After immobilization, immobilized lipase is obtained. The immobilization treatment can further improve the enzymatic activity of the lipase.

[0038] Preferably, the immobilization treatment is carried out according to the following steps:

[0039] 1) taking the clear liquid after cell wall breaking and diluting it to a bacterial concentration of 20-60 g / L to obtain a lipase dilution solution;

[0040] 2) The lipase dilution was filtered through a 0.45 μm filter membrane and then subjected to Ni column affinity chromatography. The eluted fractions were collected into a 10 kDa (Millipore) ultrafiltration tube using an elution buffer (containing 300 mM imidazole). The buffer was replaced by centrifugal concentration, and finally concentrated to obtain a lipase CALB protease solution;

[0041] 3) Weighing macroporous adsorption resin AB-8 and lipase CALB protease solution, mixing them, and continuously stirring at 150 rpm for 4 hours to fully mix the lipase CALB and the macroporous adsorption resin to obtain a reaction solution;

[0042] 4) The reaction solution is filtered off, the supernatant is discarded, the immobilized lipase is rinsed with a buffer solution, and the immobilized lipase is dried at room temperature to obtain the immobilized lipase.

[0043] More preferably, the added amount (g) of the macroporous adsorption resin AB-8 is 20 w / v% to 40 w / v% of the volume (L) of the lipase CALB protease solution.

[0044] The present invention also provides an application of the recombinant lipase mutant that efficiently expresses lipase CALB or the recombinant lipase mutant prepared by the preparation method in the preparation of vitamin E succinate.

[0045] The present invention also provides a method for preparing vitamin E succinate, which is characterized in that it comprises the following steps: using vitamin E and succinic anhydride as substrates and an organic solvent as a reaction medium, carrying out an esterification reaction under the catalytic action of the recombinant lipase mutant or the recombinant lipase mutant prepared by the preparation method of the recombinant lipase mutant, to obtain vitamin E succinate.

[0046] Preferably, the final concentration of vitamin E added is 0.01-0.1 mol / L.

[0047] Preferably, the final molar ratio of the vitamin E to the succinic anhydride is 1:(4-12).

[0048] More preferably, the final molar ratio of the vitamin E to the succinic anhydride is 1:4.

[0049] Preferably, the recombinant lipase mutant is added in the form of immobilized lipase.

[0050] More preferably, the added amount of the immobilized lipase is 20-40 g / L.

[0051] Preferably, the esterification reaction time is 4 to 6 hours, and the esterification reaction temperature is 0°C to 60°C.

[0052] More preferably, the reaction temperature is 20°C to 60°C.

[0053] More preferably, the reaction temperature is 45°C to 50°C.

[0054] Preferably, the organic solvent includes at least one of tert-butyl alcohol, tert-amyl alcohol, acetone, petroleum ether, n-hexane, methanol and acetonitrile.

[0055] More preferably, the organic solvent is at least one of acetone and n-hexane. Acetone and n-hexane play a solubilizing role in the reaction, which can increase the solubility of vitamin E and succinic anhydride in the reaction solution, and is beneficial to improving the reaction efficiency.

[0056] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0057] (1) The recombinant lipase mutant provided by the present invention is mutated at position 104, 106, 154, 190, 278, 281 or 282 of the amino acid sequence of the wild-type lipase, among which the best effect is achieved by mutation of valine at position 154 to alanine and alanine at position 281 to glycine.

[0058] (2) Using the recombinant lipase mutant provided by the present invention as a catalyst, not only can vitamin E succinate be synthesized in an organic reaction system, but the conversion rate of vitamin E succinate can reach more than 90%, and the relative enzyme activity of the recombinant lipase mutant is increased by 15% compared with the wild type.

[0059] (3) The recombinant lipase mutant provided by the present invention is used to synthesize vitamin E succinate. The conditions are mild, the process operation is simple, the pollution is small, it is green and safe, and the product has high purity, which is in line with the "green and safe" production concept. The product is suitable for use in pharmaceutical raw materials and cosmetic active ingredient additions. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is the SDS-PAGE gel image of the lipase CALB and its mutant proteins in Example 4.

[0061] Figure 2 Schematic diagram of HPLC detection of vitamin E standard, vitamin E succinate standard and reaction solution in implementation case 6.

[0062] Figure 3 This is the vitamin E standard curve in implementation case 6.

[0063] Figure 4 This is the standard curve of vitamin E succinate in Example 6.

[0064] Figure 5 Schematic diagram of the relative enzyme activities of lipase CALB and recombinant lipase mutants in Example 7.

[0065] Figure 6 Schematic diagram of substrate conversion rate of lipase CALB and recombinant lipase mutant in implementation case 7. DETAILED DESCRIPTION

[0066] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0067] It should be noted that the process equipment or devices not specifically specified in the following embodiments are all conventional equipment or devices in the art.

[0068] In addition, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise specified; it should also be understood that the combination connection relationship between one or more devices / apparatuses mentioned in the present invention does not exclude the existence of other devices / apparatuses before or after the combination device / apparatus or the insertion of other devices / apparatuses between these explicitly mentioned two devices / apparatuses, unless otherwise specified. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or the scope of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the present invention without substantially changing the technical content.

[0069] In the present invention:

[0070] LB plate: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl and 2 g / L agar powder.

[0071] LB liquid medium: 10 g / L peptone, 5 g / L yeast powder and 10 g / L NaCl.

[0072] Example 1

[0073] Construction of genetically engineered bacteria E. coli BL21 (DE3) / CALB for efficient expression of lipase:

[0074] The gene sequence of wild-type Candida antarctica lipase B (abbreviated as wild-type lipase CALB, whose amino acid sequence is shown in SEQ ID NO.1 and nucleotide sequence is shown in SEQ ID NO.2) in the gene library was synthesized by whole gene and transferred into the expression vector pET28b(+) to obtain the recombinant plasmid pET28b(+)-CALB, and the recombinant plasmid pET28b(+)-CALB was transferred into the host bacteria E. coli BL21(DE3) to obtain the genetically engineered bacteria E. coli BL21(DE3) / CALB expressing the wild-type lipase CALB, and the corresponding lipase was recorded as wild-type lipase CALB.

[0075] SEQ ID NO:1 (CALB)

[0076] LPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMVNAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAGPLDALAVSAPSVWQQT TGSALTTALRNAGGLTQIVPTTNLYSATDEIVQPQVSNSPLDSSYLFNGKNVQAQAVCGPLFVIDHAGSLTSQFSYVVGRSALRSTTGQARSADYGITDCNPLPANDLTPEQKVAAAALLAPAAAAIVAGPKQNCEPDLMPYARPFAVGKRTCSGIVTP

[0077] SEQ ID NO:2 (CALB)

[0078] TTACCTAGTGGTTCCGACCCTGCTTTCTCTCAGCCTAAGAGTGTGCTGGATGCAGGTCTTACATGTCAAGGTGCATCCCCATCTTCCGTTAGTAAACCTATTTTACTGGTTCCAGGAACTGGTACTACTGGACCACAAAGTTTCGATTCTAATTGGATCCCTCTGTCCACCCAACTAGGATATACGCCATGTTGGATTTCTCCTCCACCATTCATGTTAAACGATACTCAAGTTAACACTGAGTACATGGTTAACGCTATTACCGCACTTTACGCTGGTTCAGGAAACAATAAATTGCCTGTCTTGACCTGGTCTCAGGGTGGCTTAGTCGCCCAATGGGGACTGACATTCTTCCCTTCAATCAGATCAAAGGTCGACAGACTTATGGCCTTTGCTCCTGACTACAAAGGTACCGTGTTGGCTGGTCCACTTGACGCCTTGGCAGTGTCCGCTCCTTCCGTCTGGCAACAAACCACGGGTTCAGCTTTGACGACTGCCCTGCGAAATGCTGGAGGATTGACTCAAATAGTGCCCACTACTAACCTATACTCAGCTACAGACGAAATTGTTCAGCCTCAAGTTAGTAACAGTCCACTAGATTCATCCTATCTATTTAACGGCAAGAATGTTCAAGCACAGGCAGTCTGTGGTCCTCTTTTCGTTATCGATCATGCAGGATCTTTGACATCACAGTTCTCATACGTAGTGGGTCGATCCGCCTTGAGGTCAACAACGGGTCAAGCCAGATCTGCCGACTACGGTATCACCGATTGTAACCCTCTGCCTGCAAACGATCTGACCCCTGAACAAAAGGTCGCTGCCGCAGCCCTGCTGGCTCCAGCAGCTGCCGCTATCGTTGCTGGTCCAAAACAAAATTGCGAACCTGATTTAATGCCTTACGCAAGACCTTTCGCTGTCGGAAAGAGAACCTGTTCAGGAATTGTTACTCCT

[0079] Example 2

[0080] Genetically engineered bacteria E. coli BL21(DE3) / CALB-muts for construction of recombinant esterase mutants by site-directed mutagenesis:

[0081] Based on the wild-type lipase CALB, the following mutants were designed using SnapGene as primer design software. The mutant upstream and downstream primers were synthesized by Qingke Company. The designed mutant primers were amplified by PCR (KOD One MaterMix, Shanghai Toyobo) to obtain the mutant PCR product. The PCR reaction program is as follows: 95℃5min; 95℃30s, 60℃30s, 72℃3min, repeated 30 cycles; 72℃ continued to extend for 10min. The above PCR stock solution was used to remove the DNA template in the product with DpnⅠ (ThermoScientific, FD1703), and then transformed into competent cells (DH5α) to circularize the PCR product. The transformed competent cell culture was applied to LB solid medium containing 50μg / mL Kan resistance and cultured at 37℃ for 15h. Single clones were selected and the mutant plasmid was extracted with a plasmid extraction kit. The mutant plasmid with the correct sequence was determined by DNA sequencing, which was the recombinant expression plasmid containing the coding gene of the lipase mutant. The primer sequences of lipase mutants are shown in Table 1.

[0082] The recombinant plasmid pET-28b(+)-CALB obtained in Example 1 and the mutant plasmid obtained in Example 2 were transformed into E. coli BL21 (DE3) competent cells, respectively, spread on LB solid plates containing 50 μg / mL Kan resistance, and inverted in a 37°C constant temperature incubator for overnight culture; a single colony was picked from the plate and inoculated into a test tube containing LB liquid culture medium, cultured at 37°C for 8-12 hours, and the strain was preserved in a -80°C refrigerator with 30% glycerol to obtain a genetically engineered bacterium E. coli BL21 (DE3) / CALB-muts containing a gene encoding a lipase mutant.

[0083] Table 1 Mutant sites and primer sequences

[0084]

[0085] Example 3

[0086] Preparation of wet cells of genetically engineered bacteria containing lipase mutants:

[0087] The genetically engineered bacteria E. coli BL21 (DE3) / CALB-muts obtained in Example 2 were inoculated into a test tube containing 10 mL of LB liquid medium (containing ampicillin resistance at a final concentration of 50 μg / mL), and cultured at 37 ° C, 180 rpm, for 10-12 hours to obtain seed liquid. The seed liquid was then inoculated into a shake flask containing 100 mL of LB liquid medium (containing ampicillin resistance at a final concentration of 50 μg / mL) at an inoculum volume of 2% (v / v), and cultured at 37 ° C, 180 rpm until the bacterial OD 600nm When the pH reaches 0.6-0.8, add IPTG (Sigma, 367-93-1) with a final concentration of 0.1-1 mM to induce expression, and the induction temperature is 16-37°C. The preferred IPTG concentration is 0.5 mM, and the induction temperature is 28°C. After induction for 15 hours under the preferred conditions, centrifuge at 4°C, 8000 rpm, and centrifuge for 10 minutes, discard the supernatant, and collect the cells to obtain the wet cells of the recombinant Escherichia coli containing the coding gene of the lipase mutant. The same method is used to prepare the genetically engineered bacteria containing the coding gene of the lipase mutant, that is, the recombinant E. coli BL21 (DE3) / CALB wet cells.

[0088] Example 4

[0089] Preparation of crude enzyme solution of lipase CALB and lipase mutants:

[0090] Preparation of the crude enzyme solution of the recombinant lipase CALB of the present invention: add 10 mL of lysis buffer solution (50 mM Tris-HCl pH=7.4, 150 mM NaCl, 20 mM imidazole, 10% (v / v) glycerol) to 1 g of the wet bacteria collected by the method of Example 3 and resuspend, add phenylmethylsulfonyl fluoride (PMSF, final concentration of 1 mM) before disruption, perform ultrasonic disruption under ice bath conditions (60 W power, continuous 2 s, intermittent 4 s, continuous disruption for 10 min), and then obtain cell disruption solution. The cell disruption solution obtained after ultrasonic disruption is centrifuged at 8000 rpm and 4°C for 10 min, and the supernatant obtained is the desired crude enzyme solution of lipase CALB. The SDS-PAGE image of the cell disruption supernatant and precipitate of E. coli BL21 (DE3) / CALB is as shown in FIG. Figure 1 As shown in the figure, it can be found that lipase CALB exists in a soluble form and the protein molecular weight is consistent with the expected value.

[0091] Example 5

[0092] Preparation of immobilized lipase:

[0093] (1) Preparation of lipase: The crude CALB lipase solution prepared in Example 4 and Ni 2+-NET medium was rotated and mixed at 4°C for 1 hour to allow the protein with 6×His to bind to the medium. The binding solution was added to the gravity column and naturally flowed out under the action of gravity. The column was washed with 10 volumes of lysis buffer, and then elution buffer (50mM Tris-HCl pH=7.4, 150mM NaCl, 300mM imidazole, 10% (v / v) glycerol) was used to collect the eluted components into a 10kDa (Millipore) ultrafiltration tube, and the imidazole and salt were removed by centrifugal concentration and replacement of the buffer to obtain a lipase solution.

[0094] (2) Enzyme immobilization: Weigh the macroporous adsorption resin AB-8 and mix it with the lipase solution, and stir it continuously at 150 rpm for 4 hours to fully mix the lipase and the resin. Finally, remove the supernatant by suction, wash it with water three times to remove the bacteria on the surface and the enzyme molecules that are not firmly bound, dry it in an environment below 30°C, and place it in a refrigerator at 4°C for use.

[0095] Example 6

[0096] Lipase CALB enzyme activity detection method and HPLC identification:

[0097] (1) Enzyme activity assay method: In a 50 mL reactor, weigh 0.5 g of vitamin E and 0.5 g of succinic anhydride, add 10 mL of acetone and mix well, add immobilized lipase, place at 50°C, shake on a 200 rpm shaker for 6 h. After the reaction is completed, evaporate the acetone, add methanol to the system, mix well, filter, and take the filtrate for HPLC detection.

[0098] (2) HPLC identification of substrates and products: Vitamin E and vitamin E succinate were quantitatively analyzed by high performance liquid chromatography. A high performance liquid chromatograph produced by Thermo Fisher Scientific was used, the chromatographic column was a C18 reverse column (250 mm × 4.6 mm, 5 μm), the reactants and products were detected at 285 nm by a UV-Vis detector, the mobile phase was methanol-glacial acetic acid (500:3.2), the flow rate was 1.0 mL / min, the column temperature was 30°C, and the injection volume was 10 μL. The measurement results are shown in Figures 2 to 4 As shown, Figure 2 HPLC analysis chart of the substrate standard vitamin E in this example (A), HPLC analysis chart of the product standard vitamin E succinate (B) and HPLC analysis chart of the lipase-catalyzed product generation (C). Figure 3 Schematic diagram of the standard curve of the substrate standard vitamin E in this example. Figure 4 Schematic diagram of the standard curve of the product standard vitamin E succinate in this example. Figures 2 to 4It can be determined that the peak time of the product generated under the catalysis of lipase CALB is consistent with the peak time of vitamin E succinate.

[0099] Example 7

[0100] Enzyme activity assay of lipase CALB and its mutants:

[0101] Based on the methods of Examples 1 to 4, lipase CALB and its mutants were constructed and expressed, and crude enzyme solutions of WT and M1 to M10 were obtained respectively; based on the method of Example 5, immobilized lipases were prepared from the obtained WT and M1 to M10 respectively; enzyme activity was detected based on the detection method of lipase activity in Example 6, and the results were as follows: Figure 5-6 shown. Figure 5 This is a diagram showing the relative enzyme activity of the lipase CALB mutation sites in this example. Figure 6 Schematic diagram of substrate conversion rate of lipase CALB and its mutants in this example.

[0102] from Figure 5 Data analysis shows that there are 7 mutants with significantly improved relative activity compared to the original enzyme activity, including W104A (109.09%), Q106A (104.88%), V154A (113.89%), V190A (106.19%), L278A (112.22%), A281G (115.38%) and A282G (103.54%), among which the relative enzyme activity of mutant A281G increased by 15%. The yield of the catalytic product of lipase CALB and its mutants was determined by the external standard method, and the results are as follows Figure 6 As shown, the conversion rate of mutant A281G in synthesizing vitamin E succinate was as high as 90.25%.

[0103] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of methods and compositions in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A recombinant lipase mutant, characterized in that: The mutant is obtained by taking a single mutation at position 104, 106, 154, 190, 278, 281 or 282 of the amino acid sequence shown in SEQ ID NO.

1.

2. The recombinant lipase mutant according to claim 1, characterized in that: The mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.1 is mutated into one of the following: (1) Tryptophan at position 104 mutated to alanine; (2) glutamine at position 106 was mutated to alanine; (3) Valine at position 154 was mutated to alanine; (4) Valine at position 190 was mutated to alanine; (5) leucine at position 278 mutated to alanine; (6) alanine at position 281 was mutated to glycine; (7) Alanine at position 282 was mutated to glycine.

3. A gene encoding the recombinant lipase mutant according to claim 1.

4. A recombinant genetically engineered bacterium comprising the coding gene according to claim 3.

5. A method for preparing the recombinant lipase mutant according to claim 1, characterized in that: The following steps are involved: Step 1, connecting the coding gene with the nucleotide sequence as shown in SEQ ID NO.2 to an expression vector and then transferring it into a host bacterium to construct a recombinant genetic engineering bacterium containing the coding gene; Step 2: ferment and culture the recombinant genetically engineered bacteria, induce expression, centrifuge, resuspend, and centrifuge at low temperature, then collect the supernatant to obtain a crude enzyme solution containing the recombinant lipase mutant.

6. The method for preparing the recombinant lipase mutant according to claim 5, characterized in that: The method further comprises: step 3, performing cell disruption on the crude enzyme solution containing the recombinant lipase mutant, and performing immobilization treatment on the protease solution obtained after separation and purification to obtain immobilized lipase.

7. Use of the recombinant lipase mutant prepared by the recombinant lipase mutant according to claim 1 or the method for preparing the recombinant lipase mutant according to any one of claims 5 to 6 in the preparation of vitamin E succinate.

8. A method for preparing vitamin E succinate, characterized in that: The following steps are involved: Vitamin E and succinic anhydride are used as substrates and an organic solvent is used as a reaction medium. An esterification reaction is carried out under the catalysis of the recombinant lipase mutant described in claim 1 or 2 or the recombinant lipase mutant prepared by the preparation method described in any one of claims 5 to 6 to obtain vitamin E succinate.

9. The method for preparing vitamin E succinate according to claim 8, characterized in that: The final molar ratio of the vitamin E to the succinic anhydride is 1:(4-12).

10. The method for preparing vitamin E succinate according to claim 8, characterized in that: The recombinant lipase mutant is added in the form of immobilized lipase, and the added amount of the immobilized lipase is 20-40 g / L.

Images