A lipase mutant and its application in synthesis of vitamin a palmitate

Abstract

The application belongs to the technical field of genetic engineering of enzymes, and particularly relates to a lipase and preparation and application thereof. Tilletia walkeri A wild-type lipase TWG5L coding gene is obtained from Tilletia wirti (syn. twg5l. The wild-type lipase enzyme gene is reformed by using a key site amino acid residue site-directed mutation technology to obtain a lipase mutant TWG5L N87Y and a coding gene thereof twg5l2 , a recombinant plasmid is constructed, and high-efficiency expression of the lipase in Pichia pastoris is realized, and the relative enzyme activity of the mutant is 299% of that of the wild type. The yield of the lipase mutant in catalyzing vitamin A acetate and palmitic acid to generate vitamin A palmitate is 190% of that of the wild type, and the lipase mutant has important industrial value.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to the cloning and expression of lipase mutant genes and their application in catalyzing the synthesis of vitamin A esters, and particularly to the efficient production of lipase mutants with specific transesterification activities using a Pichia pastoris expression system. Background Technology

[0002] Lipase (EC 3.1.1.3) is a serine hydrolase belonging to the α / β hydrolase family. It catalyzes the hydrolysis of triglycerides (triacylglycerols) into glycerol and fatty acids, and participates in transesterification and ester synthesis reactions. Its unique catalytic properties and wide industrial applications make it an important tool in biochemistry and industry. Lipases are widely found in plants, animals, and microorganisms, with microbial enzymes being the mainstream for industrial applications due to their high stability and broad substrate specificity. Lipase is a key enzyme in the synthesis pathway of vitamin A derivatives, achieving directed hydrolysis or transesterification of ester bonds at the oil-water interface, catalyzing the formation of vitamin A palmitate from vitamin A acetate and palmitic acid. Vitamin A palmitate is a fat-soluble vitamin A derivative. As a stable form of vitamin A, it links retinol and palmitic acid through an ester bond, significantly improving storage stability and bioavailability. As a multifunctional nutrient, vitamin A palmitate offers multiple benefits in the health field, including vision protection, immune regulation, antioxidation, and skin repair. However, there has been very little research on its production method using bio-enzymatic synthesis to date.

[0003] Currently, the main methods for producing vitamin A palmitate include chemical synthesis and enzymatic catalysis. While chemical synthesis is a mature process, it has significant drawbacks. For example, when using retinol and palmitoyl chloride as raw materials, the reaction requires strongly alkaline conditions, resulting in severe byproduct pollution. This poses significant risks to human health and the environment, and also presents challenges such as numerous side reactions and difficulties in product separation. Enzymatic catalysis offers advantages such as being environmentally friendly, operating under mild conditions, and achieving high yields, making it the future mainstream technology for vitamin A palmitate production. It particularly aligns with the demands of high-purity, low-residue products in high-end fields such as pharmaceuticals and cosmetics. However, it faces challenges such as high enzyme costs and poor stability. Therefore, developing a novel, green, safe, and efficient lipase has significant application value for vitamin A palmitate production.

[0004] Lipases are influenced by their protein chemical properties, and their catalytic activity, substrate specificity, optimal temperature and pH, acid-base stability, and solvent tolerance are all major factors limiting their application. Furthermore, the preparation and purification processes of lipases are relatively complex and costly, and the enzymes themselves exhibit poor stability and solvent compatibility, thus restricting the lipase-catalyzed synthesis of vitamin A esters. Therefore, obtaining lipases with high activity and good performance is of great significance for the industrial production of vitamin A palmitate.

[0005] The Pichia pastoris expression system is a highly efficient and powerful eukaryotic protein expression platform. Its core advantage lies in combining the ease of operation of prokaryotic systems with the complex post-translational modification capabilities of eukaryotes. Utilizing the potent AOX1 (alcohol oxidase 1) promoter, this system achieves highly efficient expression of exogenous genes under methanol induction, significantly outperforming traditional prokaryotic systems (such as *E. coli*) and some eukaryotic systems (such as *Saccharomyces cerevisiae*). Through its unique methanol induction mechanism, highly efficient eukaryotic modification capabilities, and mature large-scale production process, the Pichia pastoris expression system has become the preferred platform for exogenous protein production. Its advantages of low cost, high yield, and near-natural protein functionality make it crucial in the development of recombinant protein drugs, industrial enzyme preparations, and vaccines. Summary of the Invention

[0006] The purpose of this invention is to: 1) provide a novel lipase mutant with enhanced activity and its encoding gene; 2) provide a recombinant vector or host bacterium expressing the lipase mutant; and 3) provide the application of the lipase mutant in the catalytic synthesis of vitamin A palmitate.

[0007] This invention uses wheat smut ( Tilletia walkeri Using wild-type lipase TWG5L (whose amino acid and gene sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively) as the parent, a high-activity lipase was obtained by mutating key amino acid residue sites. This enzyme catalyzes the synthesis of vitamin A palmitate from vitamin A acetate and palmitic acid. This lipase provides a new route for the enzymatic synthesis of vitamin A palmitate and has significant application prospects.

[0008] One of the technical solutions provided by this invention is a high-activity lipase mutant, which is obtained by mutating the amino acid residue at position 87 of the wild-type lipase TWG5L shown in SEQ ID NO.1 from asparagine (Asn) to tyrosine (Tyr), and is named lipase mutant TWG5L. N87Y The lipase mutant TWG5L N87Y The amino acid sequence is shown in SEQ ID NO.3.

[0009] The second technical solution provided by this invention is the lipase mutant TWG5L described in the first technical solution. N87Y The encoding gene; Furthermore, the lipase mutant TWG5L N87Y Encoding genes twg5l2 The nucleotide sequence is shown in SEQ ID NO.4.

[0010] The third technical solution provided by the present invention is a recombinant vector or recombinant strain containing the encoding gene described in the second technical solution; Furthermore, the expression vector used for the recombinant vector includes, but is not limited to: pET-22b, pET-28a(+), and pPIC9K; preferably pPIC9K. Furthermore, the host cells used in the recombinant strain include, but are not limited to: Escherichia coli, Pichia pastoris, and Bacillus subtilis; Pichia pastoris is preferred. Preferably, the expression vector used is pPIC9K, and the host cell is Pichia pastoris GS115.

[0011] The fourth technical solution provided by the present invention is the application of the recombinant vector or recombinant strain described in the third technical solution, particularly its application in the preparation of the lipase mutant described in the first technical solution.

[0012] The fifth technical solution provided by the present invention is the application of the lipase mutant described in the first technical solution; particularly its application in transesterification reaction; even more particularly its application in the synthesis of vitamin A derivatives, preferably, the vitamin A derivative is vitamin A palmitate; even more particularly its application in catalyzing the reaction of vitamin A acetate and palmitic acid to produce vitamin A palmitate.

[0013] Beneficial effects: 1. This invention involves site-directed mutagenesis of wild-type TWG5L to obtain a mutant TWG5L with enhanced activity. N87Y The activity was increased by 199% compared to wild-type TWG5L.

[0014] 2. This invention identifies the gene for synthesizing a lipase mutant and expresses it in a Pichia pastoris expression system.

[0015] 3. The lipase of this invention can be used to synthesize vitamin A palmitate efficiently.

[0016] 4. The present invention also relates to the application of the lipase and the vitamin A palmitate catalyzed by the lipase in food, feed, chemical, and pharmaceutical preparation. Attached Figure Description

[0017] Figure 1 The mutant TWG5L of this invention N87YRecombinant plasmid pPIC9K- twg5l2 Agarose gel electrophoresis verification image Where: M represents DNA Marker, and 1 represents pPIC9K- twg5l2 Recombinant plasmid, 2 is pPIC9K- twg5l2 Linearized recombinant plasmids; Figure 2 The mutant TWG5L of this invention N87Y Recombinant plasmid pPIC9K- twg5l2 Colony PCR validation diagram Where: M represents DNA Marker, and 1 represents pPIC9K- twg5l2 Image of colony PCR products; Figure 3 The mutant TWG5L of this invention N87Y SDS-PAGE image of purified sample Where: M represents protein marker, and 1 represents TWG5L. N87Y Purify the sample. Detailed Implementation

[0018] The technical content of the present invention will be further described below with reference to the embodiments. However, the present invention is not limited to these embodiments, and the scope of protection of the present invention cannot be limited by the following embodiments.

[0019] 1. The technical route for achieving the objective of this invention is summarized as follows: According to the sequence shown in SEQ ID NO.2, wheat smut fungus was obtained through gene synthesis. Tilletia walkeri The wild-type TWG5L gene from which this is derived. twg5l ,Will twg5l Genes through EcoR I and Not I was cloned into the vector pPIC9k, and sequencing yielded samples containing the wild-type... twg5l Recombinant plasmid pPIC9K- sequence twg5l Using plasmid pPIC9K- twg5l Using a template, primers for site-directed mutagenesis were designed for PCR amplification. The reaction product was then circularized and ligated to construct a mutant expression vector. High-activity lipase mutant TWG5L was obtained through fermentation and purification techniques using Pichia pastoris for efficient expression. N87Y Using the aforementioned lipase as a biocatalyst, and vitamin A acetate and palmitic acid as substrates, vitamin A palmitate was synthesized.

[0020] 2. Nomenclature of amino acids and DNA nucleic acid sequences (1) Use the recognized IUPAC nomenclature for amino acid residues, in three-letter / single-letter code form. DNA nucleic acid sequences use the recognized IUPAC nomenclature.

[0021] (2) Identification (naming) principles of TWG5L mutants The mutated amino acid in the TWG5L mutant is represented by "original amino acid residue + residue position + substituted amino acid residue". For example, Asn87Tyr (N87Y) indicates that the amino acid at position 87 is replaced by tyrosine (Tyr) in wild-type TWG5L with asparagine (Asn). The position number corresponds to the amino acid sequence number of wild-type TWG5L in SEQ ID NO.1.

[0022] In this invention, lowercase italics twg5l Indicates the gene encoding wild-type TWG5L, in lowercase italics. twg5l2 This indicates the mutant TWG5L N87Y The encoding genes are shown in the table below.

[0023]

[0024] In this invention, the wild-type TWG5L is derived from wheat smut (… Tilletia walkeri The amino acid sequence is shown in SEQ ID NO.1: MPPTSDPAFVTPQATLDANIKCPGTKGGYAAVSNPILLVPGTGNTGSESFDSTYVILTRNLGYQPCYISPPPFMLNDSQINAEYVVNAISRLNQAAGKKIPVLGWSQGNLIIQWALTFFPSTVQKTDRFVSFAGDFRGTFLAYLLDAQPLGIAPSVWQQSTLSAYLTALRNAGGLTAKV PTTSIYSVTDEIVQPQIGGPALESSYLFGDMAMNVKVQDYCPLLIVEHSQQLFNIFTYSVAKAALQSPTGKAEAGSFSSAKCSLAFPPGLGLGDQLVAPTIITQAAVHIVAGPRVACEPPLLPYAAKYYPFAKQACNPLTQVVDGFVPQTPEQLNSFEAITVYILTRSLRNILGTAGK.

[0025] In this invention, the mutant TWG5L N87Y The amino acid sequence is shown in SEQ ID NO.3: MPPTSDPAFVTPQATLDANIKCPGTKGGYAAVSNPILLVPGTGNTGSESFDSTYVILTRNLGYQPCYISPPPFMLNDSQINAEYVVYAISRLNQAAGKKIPVLGWSQGNLIIQWALTFFPSTVQKTDRFVSFAGDFRGTFLAYLLDAQPLGIAPSVWQQSTLSAYLTALRNAGGLTAKV PTTSIYSVTDEIVQPQIGGPALESSYLFGDMAMNVKVQDYCPLLIVEHSQQLFNIFTYSVAKAALQSPTGKAEAGSFSSAKCSLAFPPGLGLGDQLVAPTIITQAAVHIVAGPRVACEPPLLPYAAKYYPFAKQACNPLTQVVDGFVPQTPEQLNSFEAITVYILTRSLRNILGTAGK.

[0026] 3. Some of the culture media involved in the embodiments of the present invention are as follows: LB medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, the remainder being water.

[0027] MD medium: YNB 13.4 g / L, glucose 20.0 g / L, 4×10⁻⁶ -5 % Biotin, the remainder is water.

[0028] YPD medium: peptone 20.0 g / L, yeast extract 10.0 g / L, glucose 20.0 g / L, the remainder being water.

[0029] BMGY medium: peptone 20.0 g / L, yeast extract 10.0 g / L, YNB 13.4 g / L, 4×10 -5 % (v / v) Biotin, 10% (v / v) 1 mmol / L potassium phosphate buffer (pH 6.0), 0.5% (v / v) Glycerol, and the remainder is water.

[0030] BMMY medium: peptone 20.0 g / L, yeast extract 10.0 g / L, YNB 13.4, 4×10 -5 % (v / v) Biotin, 10% (v / v) 1 mmol / L potassium phosphate buffer (pH 6.0), 0.5% (v / v) methanol, and the remainder water.

[0031] All of the above-mentioned solid culture media were supplemented with 20.0 g / L agar.

[0032] The present invention will be further explained and illustrated below through specific embodiments.

[0033] Example 1: Obtaining the wild-type lipase TWG5L encoding gene The wild-type lipase TWG5L encoding gene was synthesized. Codon optimization was performed based on Pichia pastoris codon preference (shown in SEQ ID NO.2), with Pichia pastoris codon usage frequencies referenced from the Codon Usage Database. Optimal codons were used for all codons without altering the amino acid sequence. The designed gene was synthesized in its entirety by Jiutian Gene Technology (Tianjin) Co., Ltd., and inserted into the pPIC9K cloning vector, named pPIC9K- twg5l Example 2: Obtaining the TWG5L mutant N87Y 1. Recombinant plasmid pPIC9K- obtained using the wild-type lipase TWG5L encoding gene twg5l Using TWG5L as a template, site-directed mutagenesis was performed at the N87Y site. The mutation primers are shown below: Upstream F N87Y :AATATGTGGTGTATGCCATTAGCCG Downstream R N87Y :CGGCTAATGGCATACACCACATATT The PCR amplification reaction system consists of 50 μL units. The components of the reaction system are listed in the table below.

[0034] Note: The reagents required above are from Takara Bio Inc.

[0035] The PCR reaction program was as follows: 98 °C, 30 s (pre-denaturation); 98 °C, 10 s (denaturation); 54 °C, 20 s (annealing); 72 °C, 2 min (extension); 72 °C, 10 min (extension); 16 °C, 10 s (restoring to room temperature), for 30 cycles.

[0036] After the reaction, 2 μL of the PCR product was taken for agarose gel electrophoresis to verify the bands (see [link]). Figure 1 The corresponding target band is approximately 10,000 bp, which is considered to contain [the target band]. twg5l2 (Linear recombinant plasmid of the mutant gene). Add 2 μL to the PCR product with the correct band size. DpnI. A 5 μL buffer was added to the culture medium, and the enzyme digestion reaction was carried out at 37°C for 3 h. After the enzyme digestion reaction, the digested gene fragment was purified and recovered. The purified and recovered product was transformed into *E. coli* JM109 competent cells. The cells were plated on LB solid medium containing Amp (100 μg / mL) and incubated statically at 37°C for 12 h. Plasmid extraction and DNA sequencing were then performed to obtain the mutant expression vector pPIC9K- twg5l2 The expression vector contains the mutant TWG5L shown in SEQ ID NO.4. N87Y Encoding genes twg5l2 .

[0037] 2. Mutant TWG5L N87Y Recombinant plasmid pPIC9K- twg5l2 linearization To achieve a high recombination frequency in the yeast genome, recombinant expression vectors need to be linearized before transformation into yeast cells. This is achieved using restriction endonucleases. Sal I (Enzyme brand: TaKaRa Q cut) Sal I) Regarding the recombinant plasmid pPIC9K- twg5l2 Linearization was performed by enzyme digestion. The system was digested at 37 ℃ for 3 h. 5 μL of the product was subjected to agarose gel electrophoresis. After successful single-enzyme digestion, the linearized plasmid DNA was purified and recovered.

[0038]

[0039] Preparation of Pichia pastoris electrocompetent cells: A single colony of Pichia pastoris GS115 was transferred to 5 mL of YPD medium and incubated overnight at 30°C and 200 rpm. The next day, 1 mL of the culture was inoculated into a shake flask containing 100 mL of fresh YPD medium and allowed to grow overnight to OD. 600 Centrifuge at approximately 1.3-1.5 °C, 4,000 rpm for 5 min to collect cells. Resuspend cells in 20 mL of pre-chilled ddH2O and centrifuge as above, repeating once. Resuspend cells in 20 mL of pre-chilled 1 mol / L sorbitol and centrifuge as above, repeating once. Resuspend cells in 2 mL of pre-chilled 0.1 mol / L sorbitol (containing 10% glycerol), aliquot into 100 µL containers, and store on ice for later use.

[0040] 3. Linearized expression vector electroporation transformation of Pichia pastoris GS115 10 µL of linearized pPIC9K- twg5l2Add 80 µL of competent cells to a pre-chilled 0.2 cm electroporation cuvette and incubate on ice for 5 min. After a 1.5 kV electroporation, immediately add 1 mL of pre-chilled 1 mol / L sorbitol to the cuvette, then transfer it to a sterile centrifuge tube and incubate at 30°C for 1 h to allow the cells to recover. Centrifuge at 4,000 r / min for 5 min, discard most of the supernatant, resuspend the cells in a small amount of liquid at the bottom, plate the resuspended cells on MD plates, and incubate at 30°C for 3 days.

[0041] 4. Screening of high-copy transformants of Pichia pastoris All transformants from the MD plates were transferred to YPD solid plates with a final concentration of 0.5 mg / mL G418. Single colonies (larger in diameter) from the 0.5 mg / mL G418 YPD solid plates were then transferred to YPD solid plates with a final concentration of 2 mg / mL G418. Single colonies (larger in diameter) from the 2 mg / mL G418 YPD solid plates were then extracted for PCR verification.

[0042] Extraction of yeast genome (1) Collect the yeast cells after overnight culture by centrifugation, add 300 µL of genomic lysis buffer to them, and repeatedly pipette to suspend the cells; (2) Add a certain amount of quartz sand and shake on a shaker for 25 minutes to fully break the cell walls of the yeast; (3) Add 400 µL of genome lysis buffer and centrifuge at 12,000 r / min for 10 min; (4) Add an equal volume of Tris-saturated phenol / chloroform (1:1) mixture to the obtained supernatant, mix thoroughly, centrifuge at 12,000 r / min for 15 min, and transfer the supernatant to another EP tube; (5) After two repeated extractions, extract once with an equal volume of chloroform to remove residual phenol; (6) After centrifuging at 12,000 r / min for 15 min, transfer the supernatant to another EP tube and add 0.6 times the volume of isopropanol. After inverting the tube several times, place it at -80℃ for 20 min and centrifuge at 12,000 r / min for 8 min to recover the genomic DNA precipitate. (7) Wash the precipitate 2-3 times with 70% ethanol; (8) Dry the EP tube in the air for 20-30 min until there is no alcohol smell, then add 40 µL of sterile water to dissolve the precipitate.

[0043] PCR verification of positive transformants: PCR amplification was performed using genomic DNA as a template and universal primers AOX1-3' and AOX1-5' as primers. The PCR verification was correct (see...). Figure 2 ), to obtain pPIC9K- twg5l2 Pichia pastoris recombinant strain GS115 / pPIC9K- twg5l2 The PCR reaction conditions are as follows:

[0044] Similarly, using the same method described above, the recombinant plasmid pPIC9K- twg5l The strain was transferred into Pichia pastoris GS115 to obtain the recombinant strain GS115 / pPIC9K- twg5l.

[0045] Example 3: Lipase TWG5L and its mutant TWG5L N87Y Induction and expression in recombinant Pichia pastoris strains Lipase TWG5L and its mutant TWG5L N87Y The steps for inducing expression in recombinant Pichia pastoris strains are as follows: (1) Wild-type strain GS115 / pPIC9K- twg5l and mutant strain GS115 / pPIC9K- twg5l2 Activate the bacteria, pick a single colony and inoculate it into a YPD tube, and incubate overnight at 30°C and 220 r / min.

[0046] (2) Take YPD seed culture and inoculate it into 50 mL of BMGY medium to make the initial OD 600 The value was set to 0.1, and the yeast transformants were pre-cultured in BMGY medium at 30°C and 220 r / min for 16 h.

[0047] (3) The culture was then centrifuged at 6000 r / min for 10 min and then resuspended in 50 mL of BMMY medium containing 0.5% methanol. The culture was then incubated at 30°C and 220 r / min. Methanol was added to the medium every 12 h until the final concentration was 0.5%, and the culture was induced for 120 h.

[0048] Example 4: Lipase TWG5L and its mutant TWG5L N87Y Purification and preparation of recombinant Pichia pastoris strains After induction as described in Example 3, the fermentation broth was centrifuged at 6,000 r / min for 10 min at 4°C, and the fermentation supernatant was collected (the Pichia pastoris expression of TWG5L is a secretory expression and can be directly extracted and purified from the fermentation broth), thus obtaining TWG5L and TWG5L2. N87YCrude enzyme solution. The crude enzyme solution was subjected to nickel column affinity chromatography. The resin was washed with solution A (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole); the target protein was eluted with solution B (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 200 mM imidazole). The eluted protein was transferred to Tris-HCl buffer and stored at 4°C. The purified enzyme solution was analyzed by SDS-PAGE, and the results are shown below. Figure 3 As shown, a TWG5L with a size of 35 kDa was obtained. N87Y A single band was observed. After purification, the enzyme solution was replenished with 20% glycerol and stored at -80°C for later use.

[0049] Example 5: Determination of enzyme activity 1. Principle of Lipase Activity Assay p-Nitrophenol method: When lipase acts on p-nitrophenol ester, it generates p-nitrophenol (p-NP) and organic acids. The absorbance of p-NP at 405 nm is proportional to its concentration. Therefore, by measuring the absorbance of generated p-NP at 405 nm, the amount of p-NP generated can be calculated, thereby calculating the enzyme activity.

[0050] 2. Definition of lipase activity Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of p-nitrophenol (p-NP) within 1 min at 45℃ and pH 8.0, which is defined as 1 enzyme activity unit (U).

[0051] 3. Enzyme activity assay methods and procedures Using the purified enzyme solution from Example 4 as the test sample, the lipase activity was measured using the aforementioned p-nitrophenol assay method. p-Nitrophenylacetic acid (p-NPA) was used as the substrate, and the lipase activity was measured on a 96-well plate using a multi-functional microplate reader. The procedure was as follows: First, 50 μL of the diluted enzyme solution was added to 900 μL of 20 mM Tris-HCl buffer (pH 8.0), mixed and incubated at 45°C for 1 min. Then, 50 μL of 20 mM p-NPA acetonitrile solution was added and reacted for 2 min. 200 μL of the reaction solution was then measured at 405 nm.

[0052] Using the purified enzyme solution from Example 4 as the test sample, the enzyme activities of wild-type lipase TWG5L and lipase N87Y mutant were measured using the aforementioned lipase activity assay method. The results are shown in the table below: Wild-type lipase TWG5L and mutant TWG5L N87Y Specific activity (U / mg) determination results

[0053] The results above show that after the N87Y mutation, the lipase activity was increased by 199% compared with the wild-type TWG5L.

[0054] Example 6: Utilizing lipase to catalyze the reaction of vitamin A acetate and palmitic acid to produce vitamin A palmitate. 1. Reaction system Using vitamin A acetate and palmitic acid as substrates, with vitamin A acetate added at 25.46 mM and palmitic acid added at 76.35 mM, the mixture was dissolved in n-hexane, and 1 mL of lipase was added. The reaction was carried out at 30 °C and 220 rpm for 6 h. The lipase used was the wild-type lipase TWG5L and the mutant TWG5L prepared and purified in Example 4 of this invention. N87Y Then, HPLC was used for liquid phase detection.

[0055] 2. HPLC high-performance liquid chromatography determination of vitamin A palmitate One mL of the reaction solution was diluted with 1 mL of n-hexane, and 20 μL of the sample was injected for high-performance liquid chromatography (HPLC) analysis. The chromatographic column was an Agilent ZORBAX Eclipse Plus C18 (4.6 x 250 mm, 5 μm), the mobile phase was 100% methanol, the detection wavelength was 327 nm, and the flow rate was 1 mL / min. The content of vitamin A palmitate was calculated based on the standard curve.

[0056] The results showed that, compared with wild-type TWG5L, the TWG5L prepared using this invention... N87Y The mutant prepared vitamin A palmitate using vitamin A acetate and palmitic acid as substrates. The yield of vitamin A palmitate obtained was 190% of that of wild-type lipase, which is beneficial for industrial production.

[0057] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims

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

3.

2. The encoding gene of the lipase mutant according to claim 1.

3. The encoding gene as described in claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

4.

4. A recombinant vector or recombinant strain containing the encoding gene of claim 2.

5. The recombinant vector as described in claim 4, characterized in that, The recombinant vectors used include the expression vectors pET-22b, pET-28a(+), and pPIC9K.

6. The recombinant strain according to claim 4, characterized in that, The host cells used in the recombinant strains include: Escherichia coli, Pichia pastoris, and Bacillus subtilis.

7. The use of the recombinant vector or recombinant strain of claim 4 in the preparation of the lipase mutant of claim 1.

8. The use of the lipase mutant of claim 1 in catalyzing the formation of vitamin A palmitate from vitamin A acetate and palmitic acid.

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