Application of heat-resistant beta-glucosidase and mutants thereof
A technology of glucosidase and heat resistance, applied in the direction of glycosylase, enzyme, hydrolase, etc., can solve the problem of low specificity, achieve high conversion efficiency, less reaction by-products, and high purity
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Embodiment 1
[0025] According to the β-glucosidase gene sequence of family 1 of Thermotoga (Thermotiga), sequence number: X74163), design mutation primers, specifically
[0026] 5'-AGTTTTCAACAACACCTATTTCGAACCTGCGAGT-3' (SEQ ID NO. 4) and 5'-ATTCCGATCTTTCCATCTTTCACG-3' (SEQ ID NO. 5);
[0027]The recombinant plasmid pET-20b-bglA (J Ind Microbiol Biotechnol, 2009, 36:1401–1408) containing the wild-type β-glucosidase gene of Thermotoga was selected as a template for inverse PCR amplification to obtain β-glucosidase The nucleotide fragment of the enzyme mutant gene is phosphorylated, connected, transformed, and the plasmid is extracted to obtain a recombinant plasmid containing the G224T nucleotide fragment, and the host is further transformed to express it, and the β-glucoside of the present invention is obtained The amino acid sequence of the enzyme mutant G224T is shown in SEQ ID NO.1.
Embodiment 2
[0029] It is basically the same as Example 1, except that the primers used are: 5'-AGTTTTCAATAGTGGATATTTCGAACCTGCGAGT-3' (SEQ ID NO.6) and 5'-ATTCCGATCTTTCCATCTTTCACG-3' (SEQ ID NO.7), to obtain The recombinant plasmid of the nucleotide fragment is further transformed into a host to express it, thereby obtaining the β-glucosidase mutant N223S of the present invention; its amino acid sequence is shown in SEQ ID NO.2.
Embodiment 3
[0031] It is basically the same as Example 1, except that the primers used are: 5'-AGTTTTCAATAGTACCTATTTCGAACCTGCGAGT-3' (SEQ ID NO.8) and 5'-ATTCCGATCTTTCCATCTTTCACG-3' (SEQ ID NO.9), to obtain The recombinant plasmid of / G224T nucleotide fragment is further transformed into a host to express it, thereby obtaining the β-glucosidase mutant N223S / G224T of the present invention; its amino acid sequence is shown in SEQ ID NO.3.
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