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

A technology of glucosidase and mutant, applied in the direction of glycosylase, recombinant DNA technology, introduction of foreign genetic material using vectors, etc., can solve the problems of reducing conformational flexibility, enzyme inactivation, organic solvent intolerance, etc.

Active Publication Date: 2015-06-24
ANHUI UNIVERSITY
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  • Abstract
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  • Application Information

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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.

Method used

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

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Embodiment Construction

[0028] The implementation methods in the following examples are conventional methods unless otherwise specified.

[0029] (1), the construction of the expression bacterial strain that contains β-glucosidase mutant gene of the present invention

[0030] 1. Selection of β-glucosidase gene mutation sites

[0031] Based on sequence alignment, Bgl1A is most similar to β-glucosidase BglB (PDB code: 2O9R) from Paenibacillus polymyxa, with an amino acid sequence identity of 43%. Using the structure of BglB as a template, using the Swiss-Model (http: / / swissmodel.expasy.org / ; Kiefer F, Arnold K, Künzli M, Bordoli L, Schwede T. The SWISS-MODEL Repository and associated resources. Nucleic Acids Research .2009, 37, D387-392.) Modeling the structure of β-glucosidase (Bgl1A) of marine uncultured microbial origin.

[0032] According to the modeled structural information, it is determined that the site-directed mutations are alanine A at position 24 and phenylalanine F at position 297, and t...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/70C12N1/21C12R1/19
CPCC12Y302/01021
Inventor 房伟肖亚中杨旸方泽民张学成何超
Owner ANHUI UNIVERSITY
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