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RECOMBINANT VECTOR CONSTRUCTED FROM AN ENCODING GENE OF A NITRILASE MUTANT, A RECOMBINANT GENETIC ENGINEERED STRAIN AND APPLICATION THEREOF
RECOMBINANT VECTOR CONSTRUCTED FROM AN ENCODING GENE OF A NITRILASE MUTANT, A RECOMBINANT GENETIC ENGINEERED STRAIN AND APPLICATION THEREOF
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机译:由硝基酶突变体的编码基因构成的重组载体,重组遗传工程化菌株及其应用
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摘要
The present invention discloses a recombinant vector constructed from an encoding gene of a nitrilase mutant, a recombinant genetic engineered strain and application thereof. the nucleotide sequence of the gene is shown in SEQ ID No.5, and the amino acid sequence of the mutant is shown in SEQ ID No.6. In the present invention, by the protein molecular modification, thermostability of the purified nitrilase LNIT5 is increased by up to 4.5 folds; and by utilizing recombinant E. coli containing the nitrilase mutant to hydrolyze 1-cyanocyclohexylacetonitrile at a high temperature (45° C.), product tolerance is increased, activity of NIT5-L201F is increased by 20%, and the mutant NITLNIT5-AcN can completely hydrolyze 750 mM 1-cyanocyclohexylacetonitrile within 8 hours and achieve an doubled conversion rate. Therefore, the mutants obtained by the present invention have a good application prospect in efficiently catalyzing 1-cyanocyclohexylacetonitrile to synthesize gabapentin intermediate, 1-cyanocyclohexyl acetic acid.
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机译:本发明公开了一种由硝基酶突变体的编码基因构成的重组载体,其重组遗传工程化菌株及其应用。基因的核苷酸序列在SEQ ID No.5中示出,突变体的氨基酸序列示于SEQ ID No.6中。在本发明中,通过蛋白质分子修饰,纯化的硝基酶LNIT5的热稳定性增加到4.5倍;通过利用重组剂e。 Coli i>在高温(45℃)下含有硝酸酶突变体以水解1-氰基己酰乙二乙腈,增加产物耐受性,NIT5-L201F的活性增加了20%,突变体NITLNIT5-ACN可以完全水解在8小时内750mM 1-氰基碘酸己基腈,达到加倍的转化率。因此,本发明获得的突变体具有有效催化1-氰基己酰乙二腈的良好应用前景,以合成加巴邦中间体,1-氰基己基乙酸。
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