CN101565713B - Candida antarctica lipase B gene and its application in yeast display - Google Patents

Abstract

The invention relates to candida Antarctica lipase B gene and applications thereof in yeast display. Coded protein of improved candida Antarctica B and wild type candida Antarctica lipase protein havethe same function on the amino acid level; the heat resistance capacity of the enzyme is 50-80 DEG C, and the half-lift is 3-24 hours; a nucleotide sequence is hybridized with SEQ.ID.NO2 from 1st to 978th of nucleotide under the moderate precise condition; and a preservation number of colon bacillus DH5Alpha/Puc57-CALB (Escherichia coliDH5Alpha/pUC57-CALB) which carries the plasmids is CCTCC M 209081. The candida Antarctica lipase B gene is transferred into pichia pastoris host bacteria so as to realize the high-efficient display expression of the candida Antarctica lipase B in pichia pastoris; and the provided pichia pastoris bacteria can effectively display candida Antarctica lipase B, can be widely applied to the synthesis of ethyl caproate, has different melting points and does not contain triglyceride of various fatty acids, a plurality of structured lipids, and the like.

Description

Candida antarctica lipase B gene and its application in yeast display

technical field

The invention relates to an improved gene sequence capable of highly expressing Candida antarctica lipase B in Pichia pastoris, a yeast showing high-activity lipase, and a genetically engineered lipase obtained from the recombinant yeast .

Background technique

Candida antarctica lipase B (CALB) from Antarctica (Candida antarctica) is an important class of lipase. Better catalytic performance. However, the stability of free lipase to temperature and organic solvents is poor, the separation of reaction products is difficult, and it is difficult to adapt to the needs of different industrial production conditions. Using yeast surface display technology, exogenous lipase can be immobilized on the surface of yeast cells with the help of carrier proteins anchored on the cell wall, similar to the immobilization of enzymes, maintaining the relatively independent spatial conformation and original biological activity of lipase, showing Excellent characteristics such as temperature resistance, organic solvent resistance and good thermal stability, such as natural Rhizopus oryzae lipase (Rhizopus oryzae lipase, ROL) can not catalyze enantiomer resolution in organic solvents, but the displayed ROL can Catalytic enantiomeric resolution.

The lipase protein yeast surface display technology can realize the immobilization (display) of enzymes on the surface of yeast cells during the standard fermentation process, which not only retains the advantages of convenient recovery and reusability of immobilized enzymes, but also saves the separation and purification of enzymes and Immobilization and other steps are expected to become one of the alternative methods for enzyme immobilization.

Based on the excellent characteristics of CALB, people have carried out commercial development of it, but the high cost still limits the wide application of CALB in practical production. Recently, researchers in Japan tried to display wild-type CALB on the surface of Saccharomyces cerevisiae cells, but the catalytic efficiency of the enzyme displayed on the surface of Saccharomyces cerevisiae cells was low, which became a bottleneck restricting its development.

Pichia pastoris has a strong promoter of AOX1 gene regulated by methanol, which can stably display modified eukaryotic proteins such as glycosylation and disulfide bond isomerization. Compared with Saccharomyces cerevisiae, it has a higher expression level and extracellular expression Advantages such as less bottom protein. Pichia display Compared with Saccharomyces cerevisiae display, Pichia pastoris is easier to achieve high-density culture, so generally higher enzyme production than Saccharomyces cerevisiae can be obtained. It is currently reported that there are few lipases displayed on the surface of Pichia pastoris, which is related to the late development of the surface display system of Pichia pastoris, immature vector system and unstable display ability. Specific to the display of CALB, the inventor (Shuang-yan Han, et al. Highly efficient synthesis of ethyl hexanoate catalyzed by CALB-displaying Saccharomyces cerevisiae whole-cells in non-aqueous phase, Journal of Molecular Catalysis B: 1095, 9:09matic -172) has displayed the unmodified original CALB gene on the surface of Saccharomyces cerevisiae, the enzymatic hydrolysis activity is 17U/g dry cells, and the yield is low, which greatly limits its application as a whole-cell catalyst and the development of CALB lipase enzyme preparation. Therefore, there is an urgent need to improve the research on increasing the amount of enzyme display, finding a more suitable display host, and improving the catalytic properties of the displayed enzyme, so as to lay the foundation for its application.

Contents of the invention

The purpose of the present invention is to overcome the low catalytic efficiency of existing lipase display, and provide a gene sequence of Candida antarctica lipase B (CALB) that can be efficiently displayed on the surface of Pichia pastoris.

The second object of the present invention is to provide a vector for cloning the above gene sequence.

The third object of the present invention is to provide a recombinant plasmid vector for directing the display and expression of CALB on the cell surface of Pichia pastoris.

The fourth object of the present invention is to provide Pichia pastoris engineered bacteria capable of displaying high-activity CALB with the above-mentioned gene sequence.

The nucleotide sequence of Candida antarctica lipase provided by the present invention uses genetic engineering methods to realize the efficient display of the gene on the cell surface of Pichia pastoris, so as to increase the display amount and thermal stability of lipase on the cell surface, Thereby providing a high-efficiency, heat-resistant lipase and reducing the production cost of lipase.

The purpose of the present invention is to realize through the following technical solutions:

(1) Improved CALB gene highly expressed in Pichia pastoris: According to the reported three-dimensional structure of Candida antarctica lipase B, combined with bioinformatics calculations, multiple amino acid mutations and combinations of different mutation sites were designed to establish The mutant library of wild-type CALB was screened by thermal stability and enzyme activity, and it was found that the amino acid sequence of wild-type CALB (Genbank: Z30645, derived from Candida antarctica LF058 strain) was designed with the following mutations, Pro226Asn, Leu227Lys, Phe228Thr, Val229Ser , Leu285Gly, Ala286Met, Ala288Ile, the specific amino acid sequence of the modified CALB is SEQ.ID.NO1, the heat resistance of the mutated CALB is significantly improved, and the activity is reduced by 50% when incubated at 75°C for 16h, and the half-life is 8h compared with the wild type at 75°C Increased by 1 times. Further, the rare codons in the wild-type CALB gene were replaced by the preferred codons of Pichia pastoris, and the sequence had 85% homology with SEQ.ID.NO2 from nucleotide 1-978. The above-mentioned improved CALB gene can be synthesized by a chemical method (beta-acetonitrile phosphorous acid amine chemical synthesis method) using an automatic synthesizer. This DNA molecule synthesized by using an automatic nucleic acid synthesizer in vitro encodes the same protein as the wild-type CALB protein at the amino acid level. There are 7 nucleotide sequences with different positions but the same function and different catalytic abilities.

In the present invention, the work of artificially synthesizing and improving the lipase gene can be completed by a specialized biotechnology company or institution (such as Shenzhen Huada Gene Technology Co., Ltd., Shanghai Sangong Bioengineering Co., Ltd.) using an automatic nucleic acid synthesizer and PCR splicing method.

(2) Construction of vectors with above-mentioned gene sequence cloning

A non-template-dependent A can be added to the 3' end of the PCR product using Taq enzyme, and the T vector is a vector with a 3'T overhang. Under the effect of in vitro ligation, transform Escherichia coli competent, smear and culture overnight, carry out blue-white screening, and select positive clones to extract plasmids and verify them by sequencing. The T vector used for cloning the lipase gene can be any commercially available T vector, such as pUC57, pGEM-T, PMD18-T, PMD19-T, etc. The present invention selects pUC-57, and obtains the plasmid vector pUC57-CALB carrying the improved CALB gene.

The recombinant carrier CCTCC M 209081 of gene of the present invention, wherein RML refers to lipase gene sequence; The bacterial strain Escherichia coli DH5a/pUC57-CALB (Escherichia coli DH5a/pUC57-CALB) carrying this plasmid was on April 22, 2009 in Preserved by China Center for Type Culture Collection, the preservation number is: CCTCC NO: M 209081. The preservation address is Wuhan University (430072), Wuhan City, Hubei Province.

(3) Construction of lipase display recombinant plasmid vector expressed outside yeast cells

In order to realize the display expression of improved CALB in Pichia pastoris, adopt pKFS (application number 200810028631.9, this display vector utilizes restriction endonuclease EcoRI and NotI on the basis of pPIC9K (Invitrogen company's eukaryotic expression vector) The part of the signal peptide gene on the vector was excised, and on this basis, it was replaced with the floculin gene FS from ^S. Elements such as Kna ⁺ , and multiple cloning sites that can be used to clone lipase genes, including restriction endonuclease sites MluI, ApaI, SacII, EcoRI, AvrII, NotI) are cloned expression vectors. The sequence corresponding to 24 amino acids encoding its own signal peptide upstream of RML was removed to display foreign proteins using the flocculin gene FS of pKFS. According to the Candida antarctica lipase B sequence reported in GenBank (GenBank: Z30645.1), when cloning the CALB gene, the 18 amino acids encoding its own signal peptide upstream of CALB and the nucleotide sequence corresponding to the 7 amino acids of the leader peptide were removed , to display foreign proteins using the flocculin gene FS of pKFS. According to the above ideas, design primer P1 as 5'TAGAATTCGCCACTCCTTTGGTGAAGCGTC (inside the frame is divided into EcoRI restriction site), primer P2 5'TATGCGGCCGCTCAGGGGGTGACGATG (inside the frame is divided into NotI restriction site)

The plasmid pUC57-CALB was used as a template, and P1 and P2 were used as primers for PCR amplification. Both the PCR product and the pKFS plasmid were digested with EcoRI and NotI, and ligated in vitro to construct the recombinant plasmid pKFS-CALB.

(4) Provide Pichia engineered bacteria with high expression of Candida antarctica lipase B having the above gene sequence

The SalI linearized recombinant plasmid pKFS-CALB was transformed into Pichia pastoris host strain GS115 or KM71 by the LiCl method, and the transformed product was spread on an MD plate and cultured for 2-3 days. The transformants on the MD plate were respectively inoculated on the G418-resistant YPD plate containing different concentrations, and cultured for 3-5 days. Pick the corresponding single clone of the transformants appearing on the high-concentration G418-YPD plate, extract the yeast genomic DNA as a template according to the Invitrogen operating instructions, and carry out yeast genome PCR identification to obtain recombinant transformants.

Recombinant transformants were first inoculated in 200mL of YPD medium, cultured to OD ₆₀₀ to 8-12 as seed solution. Cultivate 8-10 bottles of seed liquid, then transfer to culture in the 50L fermenter containing 20-35LBSM medium with 5-10% inoculum size (volume ratio), and then add 5-8L 50% ( Glycerol with a volume ratio of 1:1) until the secondary glycerol is consumed, and the OD ₆₀₀ reaches about 280-320. After starvation for 0.5-1h, methanol is fed, and the flow rate of methanol is 30g/L·h-60g/L·h , Dissolved oxygen is controlled at 5-10%, ammonia water is added automatically, and the fermentation ends after 120-140h, showing that CALB hydrolyzes p-nitrophenol butyrate on the surface of Pichia pastoris up to 130U/g yeast stem cells.

Pichia pastoris transformed with the expression vector pKFS-CALB can be used for catalytic synthesis of short-chain aromatic esters, biodiesel and glycosides.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

The present invention adopts the yeast surface display system to express the enzyme, while maintaining the high activity and high catalytic performance of the enzyme, compared with the free enzyme or immobilized enzyme, omitting the cumbersome steps of purification and separation, it has the advantages of immobilized enzyme and can be used repeatedly. Strong operational stability. When lipase is used in production practice, in addition to the requirement for high enzyme activity, the heat resistance of the enzyme is also the content that is emphasized. The present invention uses bioinformatics and combined with directed evolution means to obtain the mutated CALB activity that can withstand 75°C High temperature, the optimum temperature is 55°C, which is 10°C higher than that of the wild type. At the same time, taking into account the problems of low yield and low expression ability when expressing foreign genes of different species and genus in yeast, the RML gene suitable for its expression was synthesized according to the Pichia pastoris expression system, which effectively solved the above-mentioned problems. The problem is that the high expression of the enzyme has been achieved, and the expression activity is as high as 130U/g yeast stem cells, which is the highest level reported.

Detailed ways

The present invention will be further described below in conjunction with the figures and examples, but the scope of protection claimed by the present invention is not limited to the scope of the examples.

Embodiment 1: the synthesis of improved Rhizomucor miehei lipase gene

The existing Candida antarctica lipase B (Genbank: Z30645, derived from Candida antarctica LF058 strain), its gene is shown in SEQ.ID.NO3, and the codon preference of Candida antarctica is similar to that of Pichia pastoris Yeast has a certain gap.

The present invention first realizes the saturation mutation of different sites and different amino acids on the wild-type Candida antarctica lipase B with the help of bioinformatics prediction, and then screens out a strain of enzyme activity and heat resistance through directed evolution and high-throughput screening All significantly improved strains, through sequencing, found that the amino acid sequence of wild-type CALB has the following mutations, Pro226Asn, Leu227Lys, Phe228Thr, Val229Ser, Leu285Gly, Ala286Met, Ala288Ile, the specific amino acid sequence of the improved CAL is SEQ.ID.NO1 On this basis, the codons preferred by Pichia pastoris were used to replace the codons with low frequency of use in Pichia pastoris by CALB, and the gene sequence with high frequency of use in Pichia pastoris was designed to form the following A specific gene sequence, such as SEQ.ID.NO2, can increase the expression of CALB in Pichia pastoris. The gene sequence provided by the present invention can be synthesized by a specialized biotechnology company or institution using an automatic synthesizer.

Embodiment 2: Construction of pUC-CALB plasmid

The fully synthetic gene obtained in the previous step can be TA cloned with pUC57 to achieve in vitro linkage, and the operation can be performed according to the instructions. The 10 μL volume reaction system is as follows: 1 μL (50 ng) of T vector, 3 μL of fully synthetic gene product, 1 μL of 10× Buffer containing ATP, 1 μL of T4 DNA ligase, made up to 10 μL with ddH ₂ O. After a little centrifugation, connect to a 16°C water bath overnight. The ligation product was transformed into E.coli DH5α, and then spread onto an indicator plate containing 0.5mM IPTG, 40μg/ml X-Gal, cultured overnight, and the recombinant plasmid PMD 18-T-RML was sent to Shanghai Sequencing by Sangon Bioengineering Co., Ltd. The sequencing showed that the cloned gene was consistent with the fully synthetic gene we described.

Embodiment 3: Construction of recombinant plasmid pKFS-CALB

The plasmid pUC57-CALB was used as a template, and P1 and P2 were used as primers for PCR amplification. The system is 1 μL of template; 5 μL of 10×Taq DNA polymerase buffer (containing Mg ²⁺ ); 4 μL of 2.5 mmol/L dNTP; 1 μL of each 20 μM mol/L primer; 0.75 μL of Taq DNA polymerase, add sterile water to a total volume 50 μL. The reaction conditions are: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 45°C for 45 s, and extension at 72°C for 2 min, a total of 30 cycles; the 30th cycle was extended at 72°C for 10 min, and the PCR product was detected by 0.8% agarose gel electrophoresis And cut the gel to recover and purify.

Both the PCR product of the synthetic gene and the pKFS plasmid were digested with EcoRI and NotI, and connected in vitro. The 20 μL volume reaction system is as follows: pKFS plasmid 2 μL (50 ng), PCR product 15 μL (50 ng), 10×Buffer 2 μL, T4 DNA ligase 1 μL. After overnight ligation in a water bath at 16°C, 10 μL of the ligation product was transformed into E.coli Top10F by CaCl ₂ transformation method, plated on the Amp ⁺ LB (50mg/mL) plate, and the positive transformant plasmid was extracted for EcoRI and Not I double enzyme digestion Identification. After the identification was correct, Shanghai Sangon Bioengineering Co., Ltd. was also entrusted to perform sequencing.

Embodiment 4: the cultivation of the Pichia pastoris engineered bacterium expressing high activity lipase

The recombinant plasmid pKFS-CALB linearized by SalI was transformed into the host strain GS115 by the LiCl method, and the transformant was spread on the MD plate and cultured at 30°C for 2 days. Transformants on the MD plate were inoculated on YPD plates containing G4180.5mg/mL, 1.0mg/mL, 1.5mg/mL, 2.0mg/mL, and 3.0mg/mL, respectively, and cultured at 30°C for 3 days. Single clones were picked from the transformants appearing on the high-concentration G418-YPD plate (3.0 mg/mL). Extract yeast genomic DNA as a template according to the Invitrogen operation guide, use the PCR primers of the target gene sequence for yeast genome PCR identification, the total reaction volume is 20 μL, and the amount of Taq enzyme is 2U. Take 2 μL of PCR products for 0.8% agarose gel electrophoresis identification.

The correctly identified recombinant transformant GS115/pKFS-CALB was first inoculated in 200 mL of YPD medium and cultured to OD ₆₀₀ to 10 as the seed solution. Cultivate 8 bottles of seed liquid, then transfer to culture in the 50L fermentor containing 30LBSM medium with 5% inoculum size (volume ratio), and then add the glycerol of 6L 50% (mass volume ratio) after glycerol is exhausted, to After the secondary glycerin is consumed, the OD ₆₀₀ reaches 300. After half an hour of starvation, feed methanol. The flow rate of methanol is 30g/L·h-60g/L·h. Dissolved oxygen is controlled at 5-10%, and ammonia water is automatically fed. Finish after 120h of fermentation.

The fermentation broth was centrifuged at 7000rpm and 4°C for 10 minutes, the supernatant was discarded, the cells were washed three times with deionized water, and the cells were resuspended and freeze-dried in vacuum for 24 hours to obtain freeze-dried cells. Utilize p-nitrophenol butyrate (PNPB) to do substrate and carry out NaOH method titration and measure lipase hydrolysis activity, the result shows that this lipase activity is up to 130U/g yeast stem cell, compared with prior art (T.Tanino, T.Ohno, T.Aoki, H.Fukuda, A.Kondo, Development of yeast cells displaying Candida antarctica lipase B and their application to ester synt

One unit of enzyme activity is defined as the amount of enzyme needed to hydrolyze the substrate p-nitrophenol butyrate to produce 1 μmol of p-nitrophenol per minute.

Embodiment 5: the synthesis of glucose laurate

Glucose and lauric acid are used as raw materials, organic solvents tert-amyl alcohol and dimethyl sulfoxide are used, an esterification reaction occurs under the action of Candida antarctica lipase displayed on the surface of Pichia pastoris cells, and sugar ester products are obtained.

The organic solvents tert-amyl alcohol and dimethyl sulfoxide (4ml of tert-amyl alcohol, 1ml of dimethyl sulfoxide) were pre-used with Molecular sieves fully remove water. Take substrate 0.595g glucose, 1.2g lauric acid (the molar ratio of acyl donor and acyl acceptor is 2:1) in a 25ml Erlenmeyer flask with a stopper, preheat at 55°C for 20min, then add 0.3g (about 39U) Pichia pastoris engineered bacterium lyophilized powder (prepared in Example 4), the reaction temperature was 55° C., and the reaction was shaken at 200 rpm for 72 hours. After the reaction was completed, the supernatant was centrifuged, and then quantitatively analyzed by high performance liquid chromatography (liquid chromatography: waters2695; detector evaporative light dispersion detector 2424; column: C18 column), and the final yield of glucose ester was 70%.

Embodiment 6: the synthesis of fructose laurate

Fructose and lauric acid are used as raw materials, organic solvents tert-amyl alcohol and dimethyl sulfoxide are used, esterification occurs under the action of Candida antarctica lipase displayed on the surface of Pichia pastoris cells, and sugar ester products are obtained.

分子筛充分除水。取底物：0.54g果糖，1.2g月桂酸(酰基供体和酰基受体摩尔比为2∶1)于25ml具塞三角瓶中，在55℃下预热20min，然后加入0.2g(约26U)毕赤酵母工程菌菌体冻干粉，反应温度55℃，然后于200rpm下振荡反应72h。反应完成后离心取上清，然后上高效液相色谱定量分析(液相色谱：waters2695；检测器蒸发光散色检测器2424；柱子：C18柱)，最终果糖的产率为80％。The organic solvents tert-amyl alcohol and dimethyl sulfoxide (4ml of tert-amyl alcohol, 1ml of dimethyl sulfoxide) were pre-used with

Molecular sieves fully remove water. Take substrate: 0.54g fructose, 1.2g lauric acid (the molar ratio of acyl donor and acyl acceptor is 2:1) in a 25ml Erlenmeyer flask with a stopper, preheat at 55°C for 20min, then add 0.2g (about 26U ) Pichia pastoris engineering bacterium lyophilized powder, the reaction temperature is 55° C., and then shaken and reacted at 200 rpm for 72 hours. After the reaction was completed, the supernatant was taken by centrifugation, and then quantitatively analyzed by high performance liquid chromatography (liquid chromatography: waters2695; detector evaporative light dispersion detector 2424; column: C18 column), and the final yield of fructose was 80%.

Embodiment 7: the synthesis of methyl oleate

Using soybean oil and methanol as raw materials, using an organic solvent isooctane (2ml), transesterification occurs under the action of Candida antarctica lipase displayed on the cell surface of Pichia pastoris to obtain a fatty acid methyl ester product.

分子筛充分除水。向25ml具塞锥形瓶中加入0.965g大豆油和0.0525g甲醇(摩尔比为1)，放于40℃的恒温空气摇床(200rpm)中预热20min，然后加入0.2g(约26U，实施例4制备)实施例4的毕赤酵母工程菌菌体冻干粉开始反应。反应温度55℃，然后于200rpm下振荡反应，在反应24h和48h后分别加入0.0525g甲醇(最终醇油摩尔比为3∶1)，反应72h，反应完成后离心取上清，然后上气相色谱分析(气相色谱：安捷伦7890C；检测器：氢离子检测器；柱子：DB-FFAP毛细管柱)，最终脂肪酸甲酯产率为65％。Isooctane (2ml) pre-prepared with

Molecular sieves fully remove water. Add 0.965g soybean oil and 0.0525g methanol (molar ratio is 1) to a 25ml Erlenmeyer flask with a stopper, preheat it in a constant temperature air shaker (200rpm) at 40°C for 20min, then add 0.2g (about 26U, implement Example 4 Preparation) The Pichia pastoris engineering bacterium thalline lyophilized powder of embodiment 4 starts to react. The reaction temperature is 55°C, then shake the reaction at 200rpm, add 0.0525g methanol (final alcohol-oil molar ratio is 3:1) respectively after 24h and 48h of reaction, react for 72h, centrifuge after the completion of the reaction to get the supernatant, and then go to the gas chromatography Analysis (gas chromatography: Agilent 7890C; detector: hydrogen ion detector; column: DB-FFAP capillary column), the final yield of fatty acid methyl ester was 65%.

Embodiment 8: Preparation of ethyl hexanoate by non-aqueous phase esterification

分子筛充分除水。在25ml具塞三角瓶中加入4.604ml的正庚烷，375μl的正己酸(终浓度0.6M)和实施例4的毕赤酵母工程菌菌体冻干粉(0.1g，约13U)放于40度的恒温摇床中预热10min，然后加入219μl无水乙醇(反应体系中，正己酸与乙醇的摩尔比为1∶1.25)，在40度摇床中反应，摇床转速为200rpm。当反应时间为0.5h和5h时，分别加入0.6g和0.3g分子筛，反应6h，己酸的转化率能达到95％。在上述条件下反应后，离心回收菌体，经溶剂正庚烷洗涤，去除产物和残余的微量底物，再加入到含有新鲜底物的反应体系中催化酯化反应，经过10批次的连续使用，毕赤酵母展示CALB仍然在每批次中使己酸的转化率保持在95％以上。Reagents are pre-used

Molecular sieves fully remove water. Add 4.604ml of n-heptane in a 25ml conical flask with a stopper, 375 μl of n-hexanoic acid (final concentration 0.6M) and the Pichia engineering bacterium lyophilized powder (0.1g, about 13U) of embodiment 4 are placed in 40 Preheat in a constant temperature shaker at 40°C for 10 minutes, then add 219 μl of absolute ethanol (the molar ratio of n-hexanoic acid to ethanol in the reaction system is 1:1.25), and react in a shaker at 40°C with a shaker speed of 200 rpm. When the reaction time is 0.5h and 5h, add 0.6g and 0.3g molecular sieve respectively, and react for 6h, the conversion rate of hexanoic acid can reach 95%. After reacting under the above conditions, the bacteria were recovered by centrifugation, washed with the solvent n-heptane to remove the product and residual trace substrates, and then added to the reaction system containing fresh substrates to catalyze the esterification reaction. After 10 batches of continuous Using, Pichia demonstrated that CALB still maintained caproic acid conversion above 95% in each batch.

From Examples 5, 6, 7, and 8, it can be seen that the lyophilized powder of Pichia pastoris engineering bacteria thallus prepared in Example 4 can effectively catalyze the synthesis of glycolipids, methyl oleate and ethyl caproate. Compared with free enzymes and immobilized enzymes, the lyophilized powder of the body as a catalyst saves the tedious steps of separation and purification, is easy to prepare, has a short production cycle, and has strong operational stability, which is conducive to reducing production costs and realizing large-scale applications.

sequence list

SEQ.ID.NO1: (the amino acid sequence of the improved Candida antarctica, the underlined part is the inconsistency with Genank (Z30645))

MATPLVKRLP SGSDFAFSQP KSVLDAGLTC QGASPSSVSK PILLVPGTGT TGPQSFDSNW

IPLSAQLGYT PCWISPPPFM LNDTQVNTEY MVNAITTLYA GSGNNKLPVL TWSQGGLVAQ

WGLTFFPSIR SKVDRLMAFA PDYKGTVLAG PLDALAVSAP SVWQQTTGSA LTTALRNAGG

LTQIVPTTNL YSATDEIVQP QVSNSPLDSS YLFNGKNVQA QAVCG NKTS I DHAGSLTSQF

SYVVGRSALR STTGQARSAD YGITDCNPLP ANDLTPEQKV AAAAL GMPI A AAIVAGPKQN

CEPDLMPYAR PFAVGKRTCS GIVTP*

SEQ.IDNO2：(改良后的南极假丝酵母DNA序列，包括了AA突变和密码子偏好)ATGGCTACTCCATTGGTTAAGAGATTGCCATCTGGTTCTGATCCAGCTTTTTCTCAACCAAAGTCTGTTTTGGATGCTGGTTTGACTTGTCAAGGTGCTTCTCCATCTTCTGTTTCTAAGCCAATTTTGTTGGTTCCAGGTACTGGTACTACTGGTCCACAATCTTTTGATTCTAACTGGATTCCATTGTCTGCTCAATTGGGTTACACTCCATGTTGGATTTCTCCACCACCATTTATGTTGAACGATACTCAAGTTAACACTGAATACATGGTTAACGCTATACTACTTTGTACGCTGGTTCTGGTAACAACAAGTTGCCAGTTTTGACTTGGTCTCAAGGTGGTTTGGTTGCTCAATGGGGTTTGACTTTTTTTCCATCTATTAGATCTAAGGTTGATAGATTGATGGCTTTTGCTCCAGATTACAAGGGTACTGTTTTGGCTGGTCCATTGGATGCTTTGGCTGTTTCTGCTCCATCTGTTTGGCAACAAACTACTGGTTCTGCTTTGACTACTGCTTTGAGAAACGCTGGTGGTTTGACTCAAATTGTTCCAACTACTAACTTGTACTCTGCTACTGATGAAATTGTTCAACCACAAGTTTCTAACTCTCCATTGGATTCTTCTTACTTGTTTAACGGTAAGAACGTTCAAGCTCAAGCTGTTTGTGGTAACAAGACTAGTATTGATCATGCTGGTTCTTTGACTTCTCAATTTTCTTACGTTGTTGGTAGATCTGCTTTGAGATCTACTACTGGTCAAGCTAGATCTGCTGATTACGGTATTACTGATTGTAACCCATTGCCAGCTAACGATTTGACTCCAGAACAAAAGGTTGCTGCTGCTGCTTTGGGTATGCCAATTGCTGCTGCTATTGTTGCTGGTCCAAAGCAAAACTGTGAACCAGATTTGATGCCATACGCTAGACCATTTGCTGTTGGTAAGAGAACTTGTTCT GGTATTGTTACTCCATAA//

SEQ.ID.NO3: DNA sequence of wild-type Candida antarctica:

ATGGCCACTCCTTTGGTGAAGCGTCTGCCTTCCGGTTCGGACCCTGCCTTTTCGCAGCCCAAGTCGGTGCTCGATGCGGGTCTGACCTGCCAGGGTGCTTCGCCATCCTCGGTCTCCAAACCCATCCTTCTCGTCCCCGGAACCGGCACCACAGGTCCACAGTCGTTCGACTCGAACTGGATCCCCCTCTCTGCGCAGCTGGGTTACACACCCTGCTGGATCTCACCCCCGCCGTTCATGCTCAACGACACCCAGGTCAACACGGAGTACATGGTCAACGCCATCACCACGCTCTACGCTGGTTCGGGCAACAACAAGCTTCCCGTGCTCACCTGGTCCCAGGGTGGTCTGGTTGCACAGTGGGGTCTGACCTTCTTCCCCAGTATCAGGTCCAAGGTCGATCGACTTATGGCCTTTGCGCCCGACTACAAGGGCACCGTCCTCGCCGGCCCTCTCGATGCACTCGCGGTTAGTGCACCCTCCGTATGGCAGCAAACCACCGGTTCGGCACTCACTACCGCACTCCGAAACGCAGGTGGTCTGACCCAGATCGTGCCCACCACCAACCTCTACTCGGCGACCGACGAGATCGTTCAGCCTCAGGTGTCCAACTCGCCACTCGACTCATCCTACCTCTTCAACGGAAAGAACGTCCAGGCACAGGCTGTGTGTGGGCCGCTGTTCGTCATCGACCATGCAGGCTCGCTCACCTCGCAGTTCTCCTACGTCGTCGGTCGATCCGCCCTGCGCTCCACCACGGGCCAGGCTCGTAGTGCAGACTATGGCATTACGGACTGCAACCCTCTTCCCGCCAATGATCTGACTCCCGAGCAAAAGGTCGCCGCGGCTGCGCTCCTGGCGCCGGCGGCTGCAGCCATCGTGGCGGGTCCAAAGCAGAACTGCGAGCCCGACCTCATGCCCTACGCCCGCCCCTTTGCAGTAGGCAAAAGGACCTGCTCCGGCATCGTCACCCCCTGA

Claims (7)

1. A Candida antarctica lipase B gene, the amino acid sequence of the enzyme protein encoded by it is SEQ.ID.NO1, and the enzyme protein has 7 sites at the amino acid level with the wild-type Candida antarctica lipase B protein They are different, but have the same function and different catalytic ability; when the enzyme protein is incubated at 75°C for 16 hours, its activity remains 50%. 2. a kind of candida antarctica lipase B gene according to claim 1, is characterized in that, according to pichia pastoris preferred codon design nucleotide sequence, obtains the candida antarctica that expression level improves in pichia pastoris Yeast lipase B gene, the sequence is SEQ.ID.NO2. 3. A recombinant vector pUC57-CALB containing the Candida antarctica lipase B gene according to claim 1, wherein CALB refers to the lipase B gene sequence according to claim 1. 4. an Escherichia coli DH5α/pUC57-CALB (Escherichia coli DH5α/pUC57-CALB) carrying the recombinant vector pUC57-CALB according to claim 3, its preservation number is: CCTCC NO: M 209081. 5. A recombinant plasmid pKFS-CALB containing the gene of claim 1, wherein CALB refers to the lipase B gene described in claim 1, with the recombinant vector pUC57-CALB as a template, design primers, after PCR amplification of CALB The recombinant plasmid pKFS-CALB was constructed by molecular cloning technology. 6. A Pichia bacterium transformed by the recombinant plasmid pKFS-CALB according to claim 5, characterized in that, the expression vector pKFS-CALB is transformed into Pichia bacterium. 7. The application of Pichia pastoris transformed by the plasmid pKFS-CALB according to claim 6 in the catalytic synthesis of short-chain aromatic esters, biodiesel and glycolipids.