Mutant of cyclodextrin glucosyl transferase having highly alpha-cyclodextrin yielding property and mutation method

Abstract

The invention relates to a mutant of a cyclodextrin glucosyltransferase with the capability of highly yielding alpha-cyclodextrin and a mutation method, which belong to the fields of gene engineering and enzyme engineering. The invention improves the specificity of products of the cyclodextrin glucosyltransferase (CGT enzyme for short), provides a mutant proposal for improving the capability of CGT enzyme from Peanibacillus macerans JFB05-01 (CCTCC NO: M 208063) for producing the alpha-cyclodextrin, and substitutes Asp on the 372 position of the CGT enzyme for Lys, and Tyr on the 89 position as Asp and Arg to respectively obtain single mutant enzyme D372K, Y89D and Y89R; the alpha-cyclodextrin production capacity of the obtained mutant enzyme is improved compared with wild type CGT enzymes; genetic fragments of the CGT enzyme with Lys 372 are substituted by corresponding genetic fragments of Y89R so as to obtain double mutant enzyme D372K / Y89R; and the yield of the alpha-cyclodextrin of the ouble mutant enzyme D372K / Y89R is improved by 1.5 times compared with the wild type CGT enzyme, while the yield of the beta-cyclodextrin is reduced by 57 percent. The mutants are more favorable for industrial production of the beta-cyclodextrin than the wild type CGT enzymes.

Description

Technical field [0001] The present invention belongs to the field of genetic engineering and enzyme engineering with a mutant of cyclodextrin glucosyltransferase with high alpha-cyclodextrin ability and a mutation method. Specifically, the present invention is a technique for improving the product specificity of cyclodextrin glucosyltransferase (CGTase) by using the site-directed mutagenesis method of protein engineering. Background technique [0002] Cyclodextrin is a series of oligosaccharide non-reducing compounds with cyclic hydrophobic conical structure formed by connecting more than six glucoses. Among them, the most commonly used are α-, β-, and γ-cyclodextrins, which are composed of It is composed of 6, 7, 8 glucose units. At present, the industrial production of cyclodextrin is synthesized by enzymatic method, that is, cyclodextrin is synthesized by converting starch and related substrates through cyclization reaction under the catalysis of CGT enzyme. Since cyclodextrin...

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Problems solved by technology

Decyl alcohol is generally used for α-cyclodextrin, but this compound is difficult to remove from aqueous solution because its boiling point is as high as 229°C; cyclododexone is generally used for complexation and selective precipitation of γ-cyclodextrin, but this solvent Too expensive for commercial use, and the disadvantages of using organic solvents include their toxicity and flammability and the need for solvent recovery processes, so the utilization of α-, γ-cyclodextrins is largely limited at present

Even the production process of β-cyclodextrin is not ideal, because the current process makes β-cyclodextrin production cost too high for most applications

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