Candida antarctica lipase b mutants and uses thereof

Abstract

The present application relates to the field of genetic engineering and bioengineering technology, in particular to a Candida antarctica lipase B mutant and application thereof. The Candida antarctica lipase B mutant provided by the present application is obtained by joint mutation of the 143th and 284th positions in the amino acid sequence shown in the wild type lipase B. The Candida antarctica lipase B mutant constructed by the present application has 12.5 times higher enzyme activity than the wild type enzyme, and the protein expression amount is as high as 1.8-1.9 g / L under the scale of 5 L fermenter, which can significantly improve the yield and reduce the production cost. In addition, the Candida antarctica lipase B mutant can be used for synthesis of high value-added, non-natural substrates such as vitamin C fatty acid ester and vitamin A oleate, which lays a foundation for the application of lipase in the field of fine chemical industry.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and bioengineering technology, specifically to a Candida antarcticis lipase B mutant and its applications. Background Technology

[0002] Lipase (EC 3.1.1.3) is an important industrial biocatalyst, widely used in food processing, pharmaceutical synthesis, biofuel production, and fine chemical manufacturing due to its advantages such as high substrate selectivity, mild reaction conditions, and environmental friendliness. Among these, lipases derived from *Candida antarctica* (…) are… Candida antarctica CALB (carbohydrate lipase B) has become one of the most widely used commercial lipases due to its excellent catalytic performance and high stability.

[0003] However, despite the commercial availability of CALB, its large-scale industrial application still faces several challenges. First, the low expression levels of wild-type CALB in its natural host lead to high costs for downstream separation and purification. Second, free wild-type CALB exhibits poor stability in non-aqueous reaction systems and has limited catalytic activity and conversion efficiency for non-natural substrates, making it difficult to meet the synthesis requirements of high-value-added products.

[0004] Currently, existing technologies have attempted to modify CALB using protein engineering techniques to improve its performance. Patent document CN119709695A discloses a mutant of *Candida antarcticus* lipase B with high transesterification activity and its applications. This mutant is obtained by mutating the wild-type *Candida antarcticus* lipase B gene sequence at positions 11, 13, 15, 18, 24, 25, 58, 74, 88, 96, 98, 99, 126, 139, 141, 144, 163, 168, 189, 206, 208, 242, 243, 244, 249, 250, 263, 278, 281, 282, 285, 286, 301, 305, and 307. This highly transesterification-active Candida antarcticis lipase B mutant exhibits higher lipolysis and transesterification activities than the original enzyme, making it suitable for enzymatic preparation of biodiesel and possessing potential industrial application value.

[0005] Patent document CN120442595A discloses a mutant of Candida antarcticus lipase B and its applications. This mutant is based on the wild-type Candida antarcticus lipase B gene sequence, with alanine at position 146 replaced by glycine, glycine at position 207 replaced by alanine, aspartic acid at position 223 replaced by glycine, and leucine at position 278 replaced by methionine. Compared to the wild type, this Candida antarcticus lipase B mutant exhibits higher enzyme activity and thermostability, and can be used for the synthesis of chlorogenic acid derivatives, showing promising industrial application prospects.

[0006] However, current mutant designs for Candida antarctic lipase B mostly target mutations in the "lid" region of the active pocket, relying primarily on high-throughput screening. The resulting increases in catalytic activity are typically limited and insufficient to meet current needs. Therefore, developing a lipase mutant with significantly higher catalytic activity than the wild-type, while also being compatible with highly efficient expression systems, remains a pressing challenge. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a *Candida antarcticis* lipase B mutant with improved enzyme activity and high-efficiency expression, solving the problems of poor catalytic activity and low catalytic efficiency in the preparation of wild-type *Candida antarcticis* lipase B. Furthermore, the *Candida antarcticis* lipase B mutant provided by this invention can also be applied to the preparation of non-natural substrates such as vitamin C derivatives and vitamin A derivatives, meeting the synthesis needs of high-value-added products.

[0008] To achieve the above objectives, the present invention discloses the following technical solutions:

[0009] The present invention is derived from Candida antarctica ( Candida antarctica The amino acid sequence of wild-type Candida antarcticis lipase B (CALB) is shown in SEQ ID NO.2.

[0010] The present invention provides a Candida antarcticis lipase B mutant, which is obtained by jointly mutating positions 143 and 284 of the amino acid sequence shown in SEQ ID NO.2.

[0011] Furthermore, the Antarctic Candida lipase B mutant is specifically obtained by mutating proline at position 143 to leucine and alanine at position 284 to glutamic acid in the amino acid sequence of wild-type Antarctic Candida lipase B.

[0012] Furthermore, the amino acid sequence of the Candida antarcticis lipase B mutant is shown in SEQ ID NO.4.

[0013] Furthermore, the present invention also provides a nucleic acid molecule that encodes the above-mentioned Candida antarcticis lipase B mutant.

[0014] Furthermore, the present invention also provides a recombinant vector comprising the aforementioned nucleic acid molecules. Preferably, the recombinant vector is the pPIC9k recombinant vector comprising the aforementioned 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] Furthermore, the recombinant cells are preferably Pichia pastoris containing the above-mentioned nucleic acid molecules or the above-mentioned recombinant vector. Pichia pastoris GS115.

[0017] Furthermore, the present invention also provides a method for preparing the *Candida antarcticis* lipase B (CALB) mutant, as detailed below:

[0018] S1 and CALB full gene were synthesized, and the CALB gene was linked into the vector pPIC9K to obtain the recombinant plasmid pPIC9K-CALB.

[0019] S2. Using pPIC9K-CALB as a template, prepare the CALB mutant P143L / A284E recombinant plasmid to obtain the recombinant plasmid pPIC9K-CALB-M;

[0020] Construction and induction of S3 and CALB mutant expression strains, followed by transformation of the pPIC9K-CALB-M recombinant plasmid into Pichia pastoris. Pichia pastoris GS115 was induced to express, and a culture was obtained. The CALB lipase mutant was then isolated from the culture.

[0021] Furthermore, this invention provides a method for solidifying the obtained *Candida antarcticis* lipase B mutant for use in the preparation of non-natural substrates such as vitamin C derivatives and vitamin A derivatives. Specifically, this invention provides the application of the aforementioned *Candida antarcticis* lipase B mutant, the aforementioned nucleic acid molecule, the aforementioned recombinant vector, and the aforementioned recombinant cells in the preparation of vitamin C fatty acid esters. Simultaneously, this invention also provides the application of the aforementioned *Candida antarcticis* lipase B mutant, the aforementioned nucleic acid molecule, the aforementioned recombinant vector, and the aforementioned recombinant cells in the preparation of vitamin A oleate.

[0022] This invention, through in-depth structural biological analysis of *Candida antarcticus* lipase B, precisely identified two key non-conserved regulatory sites in the lipase B molecule: P143 located in the β-turn region and A284 located at the edge of the active pocket. The inventors creatively mutated these two sites co-mutated (P143L / A284E) to construct a novel *Candida antarcticus* lipase B (CALB) mutant (EXP01). The CALB mutant obtained by this invention exhibits significantly enhanced catalytic activity compared to wild-type CALB. Experiments confirmed that the catalytic activity of the CALB mutant provided by this invention is 12.5 times higher than that of the wild-type enzyme. Simultaneously, the protein expression level reaches 1.8–1.9 g / L in a 5L fermenter, which can significantly improve the application value of the *Candida antarcticus* lipase B mutant.

[0023] Compared with the prior art, the Antarctic Candida lipase B mutant provided by the present invention has the following advantages:

[0024] (1) The Antarctic Candida lipase B mutant provided by the present invention has significantly improved catalytic activity compared with wild-type lipase, with enzyme activity increased by 12.5 times, which can significantly improve production efficiency and reduce the amount of industrial enzyme used.

[0025] (2) The Antarctic Candida lipase B mutant provided by the present invention has a protein expression level of up to 1.8~1.9 g / L in a 5L fermenter, which can significantly reduce production costs and is very suitable for large-scale industrial production.

[0026] (3) The Antarctic Candida lipase B mutant provided by the present invention can be used for the synthesis of high-value-added, non-natural substrates such as vitamin C fatty acid esters and vitamin A oleate. The conversion rates in non-aqueous phase reactions are 95.6% and 97.3%, respectively, which can meet the synthesis requirements of high-value-added products and lay the foundation for the application of lipase in the field of fine chemicals. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the construction of the recombinant plasmid pPIC9K-CALB.

[0028] Figure 2 This is a schematic diagram illustrating the construction of the recombinant plasmid pPIC9K-CALB-M.

[0029] Figure 3 Nucleic acid gel images of linearized recombinant plasmids pPIC9K-CALB and pPIC9K-CALB-M.

[0030] Figure 4SDS-PAGE electrophoresis images of the expression supernatant of wild-type CB-CK strain and EXP01 mutant strain.

[0031] Figure 5 This is the HPLC chromatogram of vitamin C fatty acid esters.

[0032] Figure 6 This is the HPLC chromatogram of vitamin A oleate. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. 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.

[0034] Example 1: Construction of pPIC9K-CALB recombinant plasmid

[0035] 1. Genes synthesized from the entire genome:

[0036] The company commissioned Qingke Biotechnology to obtain Candida antarcticis (Candida) using whole genome synthesis techniques. Candida antarctica The complete gene of CALB lipase (GenBank: CAA83122.1) is shown in SEQ ID NO.2. The nucleic acid sequence of this gene has been codon-optimized.

[0037] 2. Construct the pPIC9K-CALB recombinant plasmid:

[0038] The CALB gene was ligated between the EcoRI and NotI restriction sites of the vector pPIC9K to form the recombinant plasmid pPIC9K-CALB. A schematic diagram of the recombinant plasmid pPIC9K-CALB constructed in this invention is shown below. Figure 1 As shown.

[0039] Example 2: Construction of the CALB mutant P143L / A284E recombinant plasmid

[0040] 1. Gene synthesis of CALB mutant P143L / A284E:

[0041] The P143L / A284E Candida antarctic lipase B (CALB) mutant was obtained by jointly mutating positions 143 and 284 of the amino acid sequence shown in SEQ ID NO.2. Specifically, proline at position 143 of the CALB gene was mutated to leucine, and alanine at position 284 was mutated to glutamic acid. The amino acid sequence of the P143L / A284E Candida antarctic lipase B (CALB) mutant obtained by this invention is shown in SEQ ID NO.4.

[0042] 2. Construct the CALB mutant P143L / A284E recombinant plasmid:

[0043] (1) Using pPIC9K-CALB from Example 1 as a template, reverse PCR was performed using the primers in Table 1. The PCR system is shown in Table 2, and the PCR conditions are shown in Table 3.

[0044] Table 1. Primers required for PCR

[0045] sequence Primer name Primer sequence SEQ ID NO.5 P143L-F TCTGGCTGGTCTGCTGGATG SEQ ID NO.6 P143L-R AAAGCATCCAGCAGACCAGC SEQ ID NO.7 A284E-F CCAGCTGCTGCTGAAATTGTTGC SEQ ID NO.8 A284E-R AGCAACAATTTCAGCAGCAGCT

[0046] Table 2. PCR reaction system

[0047] reaction system volume P143L-F 0.5 µL P143L-R 0.5 µL A284E-F 0.5 µL A284E-R 0.5 µL Template plasmid 1 µL 2*PCR polymerase 12.5µL <![CDATA[ddH2O]]> 9.5µL Total volume 25 µL

[0048] The PCR polymerase was purchased from TaKaRa Company in Dalian.

[0049] Table 3. PCR reaction conditions

[0050] PCR steps PCR conditions Pre-variation 98 ℃, 3 min transsexual 98 ℃，30 s annealing 58 ℃, 30 s (denaturation, annealing; 30 batches cycled) extend 72 ℃, 1 min 30 s Low temperature preservation Store at 4 ℃ for long-term storage

[0051] (2) The above PCR products were examined by gel electrophoresis. Then, 1 μL of DpnI restriction endonuclease (DpnI restriction endonuclease was purchased from Dalian TaKaRa Company) was added to 20 μL of PCR products to digest the template plasmid, and the product was incubated at 37 °C for 1 h.

[0052] (3) Take 5 μL of the product from step (2) and convert it into Escherichia coli DH5α (the Escherichia coli ( Escherichia coli (The DH5α strain and gene were purchased and synthesized by Beijing Qingke Biotechnology Co., Ltd.) to obtain the corresponding recombinant Escherichia coli, which was plated on LB agar plates containing kanamycin (100 mg / L) and cultured overnight at 37 °C.

[0053] (4) Randomly selected clones were subjected to colony PCR identification and sequencing verification. The results showed that the recombinant expression vector containing the gene encoding the CALB mutant was successfully transformed into the expression host *Escherichia coli* DH5α. The plasmid that successfully mutated, as verified by sequencing by Beijing Qingke Biotechnology Co., Ltd., was the recombinant plasmid containing the mutant, named pPIC9K-CALB-M. A schematic diagram of the recombinant plasmid pPIC9K-CALB-M constructed in this invention is shown below. Figure 2 As shown.

[0054] Example 3: Construction and Induction of CALB Mutant Expression Strains

[0055] 1. Constructing CALB mutant expression strains:

[0056] After linearizing the recombinant plasmid pPIC9K-CALB constructed in Example 1 and the recombinant plasmid pPIC9K-CALB-M constructed in Example 2 using SacI, the nucleic acid gel images of the linearized recombinant plasmids pPIC9K-CALB and pPIC9K-CALB-M are as follows: Figure 3 As shown. Figure 3 Nucleic acid gel images of linearized recombinant plasmids pPIC9K-CALB and pPIC9K-CALB-M (M: Marker; 1: pPIC9K-CALB linearization; 2: pPIC9K-CALB-M linearization). After linearization, recombinant plasmids pPIC9K-CALB and pPIC9K-CALB-M were electroporated into Pichia pastoris. Pichia pastoris GS115, the strain constructed by pPIC9K-CALB was named CB-CK, and the strain constructed by pPIC9K-CALB-M was named EXP01. Then, screening was performed using MD solid medium (MD solid medium consisted of: 20 g / L glucose, 20 g / L agar powder, and 6.7 g / L amino acid-free yeast nitrogen source (YNB)) at 30 ℃ for 2-3 days until clearly visible colonies appeared.

[0057] 2. Validation of CALB mutant engineered strain expression:

[0058] Single colonies were picked and inoculated into 5 mL YPD liquid medium (YPD liquid medium consists of 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose) in test tubes and cultured at 30 ℃ and 180 rpm / min for 18–24 h. The bacterial culture was then inoculated at a 2% ratio into 50 mL BMGY liquid medium (BMGY liquid medium consists of 20 g / L peptone, 10 g / L yeast extract, 100 mmol / L potassium phosphate buffer (pH 6.0), and 4 × 10⁻⁶ biotin). -5In a culture medium containing 10 g / L glycerol and 6.7 g / L amino acid-free yeast nitrogen source (YNB), the culture was carried out at 30 °C and 180 rpm / min until the OD reached 100%. 600 =4~6, centrifuge for 10 min, and resuspend the bacterial cells in 50 mL of BMMY liquid medium (BMMY liquid medium consists of: 20 g / L peptone, 10 g / L yeast extract, 100 mmol / L potassium phosphate buffer (pH 6.0), and 4 × 10⁻⁶ biotin). -5 The culture medium was prepared with 2% methanol (20 g / L) and 6.7 g / L amino acid-free yeast nitrogen source (YNB). 2% volume of methanol was added to the culture medium every 24 h, and expression was induced at 30 ℃ for 96 h. All bacterial cultures were collected, centrifuged at 4 ℃ for 10 min, and the supernatant was used for SDS-PAGE analysis and subsequent reactions.

[0059] SDS-PAGE electrophoresis images of the expression supernatants of the wild-type CB-CK strain and the EXP01 mutant strain constructed in this invention are shown below. Figure 4 As shown. Figure 4 SDS-PAGE electrophoresis images of the expression supernatants of wild-type CB-CK strain and EXP01 mutant strain are shown (M: Marker; 1-7: recombinant proteins from CB-CK plate screening strains; 8-14: recombinant proteins from EXP01 plate screening mutant strains). The plates described in Example 1 were induced for expression according to the method described in Example 2. Different single colonies showed varying protein expression levels. Strains 5 and 14, with the highest protein expression, were selected as the production strains of wild-type CB-CK and EXP01 mutant, respectively.

[0060] Example 4: Activity analysis of CB-CK lipase and EXP01 lipase

[0061] 1. Experimental Method:

[0062] The CB-CK and EXP01 strains collected in Example 3 were used as catalysts. Principle: Lipase hydrolyzes the substrate 4-nitrophenylbutyrate to generate p-nitrophenol (pNP) under alkaline conditions. p-Nitrophenol has maximum absorption near 405 nm. Enzyme activity can be calculated by measuring the absorbance and converting it to the amount of p-nitrophenol. Enzyme activity is defined as the amount of enzyme required to release 1 μmol of p-nitrophenol per minute at 45 °C and pH = 8. Enzyme protein concentration was determined using Coomassie brilliant blue staining, with bovine serum albumin as the standard.

[0063] 2. Experimental Results:

[0064] The results are shown in Table 4.

[0065] Table 4. Activity analysis results of CB-CK lipase and EXP01 lipase

[0066] Lipase Enzyme activity (U / mL) The increase was several times compared to wild-type lipase. Wild-type CB-CK lipase 56.2 - mutant EXP01 lipase 702.5 12.5

[0067] As shown in Table 4, the EXP01 mutant constructed in this invention can significantly improve the catalytic activity of lipase, with an enzyme activity increase of 12.5 times, which can significantly improve production efficiency and reduce the amount of industrial enzyme used.

[0068] The specific sequences of the wild-type lipase and mutant lipase provided by this invention are as follows:

[0069] (1) Wild-type CB-CK amino acid sequence (SEQ ID NO.2):

[0070] LPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMVNAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAGPLDALAVSAPSVWQQTTGSA LTTALRNAGGLTQIVPTTNLYSATDEIVQPQVSNSPLDSSYLFNGKNVQAQAVCGPLFVIDHAGSLTSQFSYVVGRSALRSTTGQARSADYGITDCNPLPANDLTPEQKVAAAALLAPAAAAIVAGPKQNCEPDLMPYARPFAVGKRTCSGIVTPHHHHHH.

[0071] (2) Amino acid sequence of EXP01 mutant (SEQ ID NO.4):

[0072] LPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMVNAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAGLLDALAVSAPSVWQQTTGSA LTTALRNAGGLTQIVPTTNLYSATDEIVQPQVSNSPLDSSYLFNGKNVQAQAVCGPLFVIDHAGSLTSQFSYVVGRSALRSTTGQARSADYGITDCNPLPANDLTPEQKVAAAALLAPAAAEIVAGPKQNCEPDLMPYARPFAVGKRTCSGIVTPHHHHHH.

[0073] Example 5: Enzyme production process on a 5 L fermenter scale

[0074] 1. Seed culture:

[0075] (1) Primary seed culture: Pick one single colony of EXP01 constructed in Example 3 from the plate and put it into 5 mL of YPD liquid culture medium (the YPD liquid culture is: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose), and incubate overnight at 30 ℃ and 180 rpm.

[0076] (2) Secondary seed culture: The EXP01 single bacteria constructed in Example 3 were inoculated into 200 mL YPD liquid culture medium (the YPD liquid culture medium consists of 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose) at a ratio of 2‰ (v / v) and cultured at 30 °C and 200 rpm for 20 h.

[0077] 2. Fermentation culture:

[0078] 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.

[0079] 3. Detect protein expression levels:

[0080] The supernatant collected by centrifugation of the fermentation broth was determined by Coomassie brilliant blue staining. The results showed that the protein expression level of the EXP01 single strain constructed in this invention was in the range of 1.8~1.9 g / L.

[0081] Example 6: Immobilization method of lipase

[0082] 1. Screening and immobilization conditions:

[0083] After decolorizing the supernatant collected in Example 5 with activated carbon, the collected supernatant enzyme solution was concentrated 5-10 times and used for immobilization experiments. Optimization was performed on the following parameters: resin type, resin-enzyme ratio, immobilization temperature, immobilization pH, immobilization time, immobilization rotation speed, drying temperature, and drying time.

[0084] 2. The optimal immobilization conditions for the experiment are:

[0085] (1) The resin type is: Acrylonitrile-Butadiene-Acrylate Copolymer (ABA resin).

[0086] (2) The ratio of resin to enzyme: ABA resin: enzyme solution = 1:3 (m / V);

[0087] (3) The immobilization temperature is 30 ℃;

[0088] (4) The immobilization pH was: pH 7.0;

[0089] (5) The immobilization time is 4 hours;

[0090] (6) The fixed rotational speed is 150 rpm;

[0091] (7) The drying temperature is 30 ℃;

[0092] (8) Drying time: 1 h.

[0093] Therefore, the optimal immobilization conditions for the Antarctic Candida lipase B mutant constructed in this invention are: ABA resin: enzyme solution = 1:3 (m / V), pH 7.0, 30 ℃, shaking at 150 rpm for 4 h, and drying at 30 ℃ for 1 h after washing.

[0094] Example 7: Synthesis of Vitamin C Fatty Acid Esters

[0095] 1. Experimental Method:

[0096] Following the lipase immobilization method described in Example 6, immobilized wild-type CB-CK lipase and immobilized mutant EXP01 lipase were prepared. Vitamin C fatty acid esters were synthesized using both immobilized wild-type CB-CK lipase and immobilized mutant EXP01 lipase as catalysts.

[0097] The reaction system consisted of: 5 mL tert-butanol, 0.1 g vitamin C, 2 mL feed oil (purchased from Chengdu Yuanda A03 model), 0.1 g immobilized wild-type CB-CK lipase / immobilized mutant EXP01 lipase, and 1.2 g molecular sieve. The reaction was carried out at 40–50 °C and 160 rpm for 10 h, followed by sampling and chromatographic analysis.

[0098] 2. Experimental Results:

[0099] The experimental results are shown in Table 5 and Figure 5 As shown.

[0100] 2.1 The conversion rate of vitamin C fatty acid esters is shown in Table 5.

[0101] Table 5. Conversion rate of vitamin C fatty acid esters

[0102] Lipase Conversion rate (%) Compared with wild-type lipase, the conversion rate is improved. Wild-type CB-CK lipase 72.3% - mutant EXP01 lipase 95.6% 23.3%

[0103] As shown in Table 5, the EXP01 mutant lipase constructed in this invention has a conversion rate of up to 95.6% for vitamin C fatty acid esters, which is 23.3% higher than that of wild-type lipase.

[0104] 2.2 HPLC chromatogram of vitamin C fatty acid esters is shown below. Figure 5 As shown. The vitamin C fatty acid ester obtained by this invention is a complex, including C. 16 Fatty acid esters and C 18 Fatty acid esters. Figure 5 The results of liquid phase analysis were vitamin C palmitate and vitamin C oleate.

[0105] Example 8: Synthesis of Vitamin A Oleate

[0106] 1. Experimental Method:

[0107] Following the lipase immobilization method described in Example 6, immobilized wild-type CB-CK lipase and immobilized mutant EXP01 lipase were prepared. Vitamin A oleate was synthesized using both immobilized wild-type CB-CK lipase and immobilized mutant EXP01 lipase as catalysts.

[0108] The reaction system consisted of 10 mL n-hexane, 0.1 g vitamin A acetate, 0.26 g oleic acid, and 0.1 g immobilized wild-type CB-CK lipase / immobilized mutant EXP01 lipase, under nitrogen protection. The reaction was carried out at 30–40 °C and 160 rpm for 8 h, followed by sampling and chromatographic analysis.

[0109] 2. Experimental Results:

[0110] The experimental results are shown in Table 6 and Figure 6As shown.

[0111] 2.1 The conversion rate of vitamin A oleate is shown in Table 6.

[0112] Table 6. Conversion rate of vitamin A oleate

[0113] Lipase Conversion rate (%) Compared with wild-type lipase, the conversion rate is improved. Wild-type CB-CK lipase 67.4% - mutant EXP01 lipase 97.3% 29.9%

[0114] As shown in Table 6, the EXP01 mutant lipase constructed in this invention has a conversion rate of up to 97.3% for vitamin A oleate, which is 29.9% higher than that of wild-type lipase.

[0115] 2.2. The HPLC chromatogram of vitamin A oleate is shown below. Figure 6 As shown. The molecular formula of the vitamin A oleate obtained by this invention is C. 38 H 62 O2; molecular weight 550.89, CAS number 631-88-9. Figure 6 The liquid phase analysis result was vitamin A oleate.

[0116] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

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

4.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule is encoding the Candida antarcticis lipase B mutant as described in claim 1.

3. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule as described in claim 2.

4. A recombinant cell, characterized in that, The recombinant cells comprise the nucleic acid molecule of claim 2 or the recombinant vector of claim 3.

5. The recombinant cell as described in claim 4, characterized in that, The recombinant cells were Pichiapastoris GS115.

6. The use of the Antarctic Candida lipase B mutant as described in claim 1, the nucleic acid molecule as described in claim 2, the recombinant vector as described in claim 3, or the recombinant cell as described in claim 4 or 5 in the preparation of vitamin C fatty acid esters.

7. The use of the Antarctic Candida lipase B mutant as described in claim 1, the nucleic acid molecule as described in claim 2, the recombinant vector as described in claim 3, or the recombinant cell as described in claim 4 or 5 in the preparation of vitamin A oleate.

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