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Lipase with improved catalytic performance and application thereof

A technology of lipase and parent enzyme, applied in the field of genetic engineering, to achieve the effects of good thermal stability and catalytic activity, improved catalytic activity, and improved optimal reaction temperature

Active Publication Date: 2020-06-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, compared with the wild type, the specific activity of the mutant A251E was reduced by half (references: Park, H.J., Park, K., Kim, Y.H., Yoo, Y.J., 2014. Computational approach for designing thermostable Candida antarctica lipase B by molecular dynamics simulation. Journal of Biotechnology. 192, 66-70.)

Method used

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  • Lipase with improved catalytic performance and application thereof
  • Lipase with improved catalytic performance and application thereof
  • Lipase with improved catalytic performance and application thereof

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Experimental program
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Effect test

Embodiment 1

[0047] The prediction of embodiment 1 lipase mutant mutation site

[0048] Based on the crystal structure of Candida antarctica lipase B, computer-aided protein design was used, and the identified mutation sites are shown in Table 1.

[0049] Table 1 Selection of Mutant Amino Acids

[0050]

Embodiment 2

[0051] Construction of the recombinant plasmid of embodiment 2 lipase mutant

[0052] Carry out primer synthesis according to mutation site, take the gene (nucleotide sequence as shown in SEQ ID NO.1) of the lipase B of coding Candida antarctica origin as template, carry out site-directed mutagenesis by overlap extension PCR (site-directed mutagenesis primer see Table 2). After the obtained point mutant gene was amplified by PCR, the point mutant gene was connected to the expression vector pPICZαA by T4 ligase. Subsequently, the ligation product was transformed into Escherichia coli JM109 competent by chemical transformation method, and spread to contain 25 μg·mL -1 Bleomycin LLB Screening Plate. The transformants were picked to extract plasmids, sequenced and identified, and finally recombinant plasmids pPICZαA-A130C, pPICZαA-A146G, pPICZαA-N181V, pPICZαA-N264P, pPICZαA-L278M, pPICZαA-S50R, pPICZαA-S56M, pPICZαA- Q112L, pPICZαA-A151P, pPICZαA-G226R.

[0053] Table 2 Prime...

Embodiment 3

[0056] Transformation and verification of the recombinant plasmid of embodiment 3 lipase mutant

[0057] The above-mentioned recombinant plasmids were linearized respectively using restriction endonuclease PmeI. Take 200ng of linearized fragments respectively, add them to Pichia pastoris GS115 competent cells, mix well, transfer to the electroporation cup, and use electroporation (Eppendorf) for 5 minutes after electroporation, add 1mL sorbitol for recovery after electroporation, and incubate at 30℃ , 200rpm under recovery culture 2h. Then the resuscitation solution was spread to contain 100 μg·mL -1 Bleomycin YPD screening plate, cultivated for 2-3 days.

[0058]Transformants were picked and inoculated into YPD medium, and cultured overnight. Collect the bacteria to extract the genome. Using the genome as a template, the universal primers 3'AOX and 5'AOX were used for PCR verification. Positive clones can be amplified to obtain a 1500bp band.

[0059] The general primer...

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Abstract

The invention discloses lipase with improved catalytic performance and application thereof, particularly discloses lipase with improved optimal reaction temperature, activity and thermal stability andapplication thereof, and belongs to the technical field of gene engineering. The invention provides genes of lipase CALB mutants A146G-L278M and A146G-L278M-A151P with improved thermal stability, andester synthesis application. According to the invention, on the basis of a wild lipase gene sequence, reasonable design is carried out to select proper mutation sites, and finally the mutants with Tmvalues increased by 3.3 DEG C and 4.2 DEG C, the optimal reaction temperature increased by 5 DEG C, and the catalytic efficiency increased by 4.1 times are respectively obtained.

Description

technical field [0001] The invention relates to a lipase with improved catalytic performance and application thereof, in particular to a lipase with improved optimum reaction temperature, activity and thermal stability and application thereof, belonging to the technical field of genetic engineering. Background technique [0002] Lipase has good catalytic ability for hydrolysis, transesterification, esterification, transesterification and other reactions, and is widely used in food, medicine, fine chemical industry, bioenergy and other industries. Lipases of microbial origin have become the main source of lipases for industrial applications. Catalytic reactions in industry are generally carried out at higher temperatures. On the one hand, increasing the temperature can increase the enzyme reaction rate, and on the other hand, because the increase in temperature can increase the solubility of reactants and reduce the risk of microbial contamination. In addition, the use of co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/81C12N1/19C12R1/84
CPCC12N9/20C12N15/815C12Y301/01003
Inventor 喻晓蔚徐岩温露文
Owner JIANGNAN UNIV
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