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Mutant of trehalose synthetase from corynebacterium glutamicum and application thereof

A technology of trehalose synthase and Corynebacterium glutamicum, applied in the field of modern enzyme engineering, can solve the problems of high cost, limited trehalose, low content, etc., and achieve the effect of reducing production cost and improving conversion efficiency

Inactive Publication Date: 2010-01-20
南宁中诺生物工程有限责任公司
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Problems solved by technology

Due to the low content, the extraction process is more complicated and the cost is extremely high
Such expensive trehalose can only be limited to some special fields such as the application of activity maintenance of medical biological products

Method used

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  • Mutant of trehalose synthetase from corynebacterium glutamicum and application thereof

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Embodiment 1

[0020] 1. Site-directed saturation mutation of trehalose synthase gene

[0021] The energy optimization of the protein molecular structure of the trehalose synthase of the present invention is carried out, combined with the analysis of its amino acid sequence with high homology, the 263rd amino acid residue in the conserved region is selected for site-specific saturation mutation, and the wild-type benzene Alanine residues were mutated to other 19 amino acid residues to obtain 19 new mutant enzymes. Using the constructed expression plasmid of the trehalose synthase gene as a template, the Stratagene company's gene mutation kit was used to perform site-directed saturation mutation of the gene, and the obtained mutant gene was sequenced and analyzed to confirm that the obtained mutant was consistent with the expected result, and then The mutated gene is introduced into Escherichia coli for expression experiments, further purified to obtain the mutated trehalose synthase, and the...

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Abstract

The invention relates to a mutant of trehalose synthetase from corynebacterium glutamicum. A method for obtaining the mutant performs site-directed saturation mutation on a phenylalanine residue at the 263rd site of an amino acid sequence of trehalose synthetase from corynebacterium glutamicum by using a site-directed mutation technology, and the method for obtaining the mutant also comprises the following step: the saturation mutation is performed on at least one amino acid residue in at least 80 percent of homologous trehalose synthetase sequences of an amino acid sequence including SEQ ID NO.1, SEQID NO.2, SEQ ID NO.3 and SEQ ID NO.4. The reaction kinetics of the trehalose synthetase expressed by a mutated trehalose synthetase gene changes, various enzymology characteristics such as specific activity of enzyme, optimum reaction temperature, optimum pH value, and the like also change, and the enzyme can improve the transformation efficiency of the trehalose synthetase and further reduce the production cost of the trehalose.

Description

technical field [0001] The invention relates to the technical field of modern enzyme engineering, in particular to a mutant of Corynebacterium glutamicum trehalose synthase and application thereof. Background technique [0002] Trehalose, more precisely α,α-1,1-trehalose is composed of two molecules of glucose connected by α,α-1,1-glycosidic bonds. Trehalose is a natural disaccharide widely present in microorganisms, shrimps, Saccharomyces cerevisiae, mushrooms and various fungi, insects, plants, etc. But the content is very small. In the past, it was mainly extracted from dry yeast. Due to the low content, the extraction process is complicated and the cost is extremely high. Such expensive trehalose can only be limited to some special fields such as the application of activity maintenance of medical biological products. With the development and progress of human society, it is becoming more and more obvious to apply trehalose to maintain the umami taste and improve the ...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N15/63C12R1/15
Inventor 黄日波韦宇拓韦航朱绮霞杜丽琴罗兆飞黄福宝周志强
Owner 南宁中诺生物工程有限责任公司
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