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Candida Antarctica lipase B gene and applications thereof in yeast display

A technology of Candida Antarctica and lipase, applied in the field of yeast, can solve the problems of limited application and development, low yield, etc., and achieve the effects of increasing display amount, high catalytic performance and high efficiency

Active Publication Date: 2009-10-28
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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: Enzymatic, 2009, 59: 168 -172) The unmodified original CALB gene has been displayed 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

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: the synthesis of improved Rhizomucor miehei lipase gene

[0028] 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.

[0029] 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 Pich...

Embodiment 2

[0030] Embodiment 2: Construction of pUC-CALB plasmid

[0031] 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 carrier, 3 μL of fully synthetic gene product, 1 μL of 10×Buffer containing ATP, 1 μL of T4 DNA ligase, and ddH 2 O to make up to 10 μL. After a little centrifugation, connect to a water bath at 16°C overnight. The ligation product was transformed into E.coli DH5α, and then spread on the indicator plate containing 0.5mM IPTG, 40μg / ml X-Gal, cultured overnight, and the recombinant plasmid PMD18-T-RML was sent to Shanghai Sheng Sequencing by Gong Biological Engineering Co., Ltd., the sequencing showed that the cloned gene was consistent with the fully synthetic gene we described.

Embodiment 3

[0032] Embodiment 3: Construction of recombinant plasmid pKFS-CALB

[0033] 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+ ); 2.5mmol / L dNTP 4μL; 20μM mol / L primers 1μL each; Taq DNA polymerase 0.75μL, add sterile water to a total volume of 50μL. 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.

[0034] Both the PCR product of the synthetic gene and the pKFS plasmid were digested with EcoRI and Not I, 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 lig...

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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 have the 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

technical field [0001] 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 [0002] 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 chara...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/63C12N15/81C12N1/19C12P7/64C12P7/62C12P19/00C12R1/72C12R1/84
CPCY02E50/13Y02E50/10
Inventor 林影韩双艳苏国栋郑穗平黄登峰
Owner SOUTH CHINA UNIV OF TECH
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