Beta-glucosaccharase mutant, recombinant expression plasmid thereof and transformed engineering strain

Abstract

The invention provides a beta-glucosaccharase gene, mutant plasmid and engineering bacteria. The beta-glucosaccharase gene of which the ethyl alcohol concentration is improved is obtained by a gene site-directed mutation method. After the engineering bacteria are subjected to inducible expression, the beta-glucosaccharase of which the ethyl alcohol concentration is improved is obtained. On the basis of the beta-glucosaccharase which is from uncultured organisms of the sea, a mutant gene is obtained through PCR (polymerase chain reaction) site-specific mutagenesis. Under the condition that the temperature is 35 DEG C, the ethyl alcohol concentration IC50 of obtained mutant protein is increased 1.8 times relative to wild type beta-glucosaccharase protein, the glucose tolerance concentration is increased twice, pH (potential of hydrogen) and temperature stability are improved, and the affinity for a substrate and the catalytic efficiency are also improved. Soybean isoflavone glycoside can be efficiently hydrolyzed by the mutant within a short time so as to prepare aglycone, and the potential application value is high.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to obtaining a β-glucosidase gene, a mutant plasmid, an engineering bacterium and a mutant enzyme with an increased ethanol tolerance concentration through a gene-directed mutation method. Background technique [0002] β-glucosidase (EC 3.1.2.21, β-glucosidase) belongs to fiber hydrolytic enzymes, mainly hydrolyzing β-1,4-glucosidic bonds in glycosides or oligosaccharides, and releasing terminal non-reducing glucose and aglycone It has important application value in modern industrial biotechnology such as food, medicine, feed processing, energy refining and other fields. The main process involved in the preparation of soybean isoflavone aglycones is as follows: ① Prepare soybean isoflavone glycoside extract with soybean isoflavone powder or defatted soybean meal as raw materials; ② Add β-glucosidase to the glycoside conversion system, ℃ for a certain period of time to carry out...

AI Technical Summary

Problems solved by technology

However, it is known that β-glucosidase generally has the weakness of intolerance to organic solvents. Organic solvents cause enzyme inactivation by destroying the conformation of enzyme molecules, reducing conformational flexibility, and depriving the active center of essential water.

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