Cyclodextrin glycosyltransferase mutant, coding gene and application of cyclodextrin glycosyltransferase mutant

Abstract

The invention relates to a cyclodextrin glycosyl transferase mutant, a coding gene and application thereof. The novel cyclodextrin glycosyl transferase mutant is obtained by mutating wild type cyclodextrin glycosyl transferase from bacillus alcalophilus (Bacillus sp.), the amino acid sequence of the wild type cyclodextrin glycosyl transferase is as shown in SEQ ID NO.2, the mutant is obtained by performing single-point or two-point mutation on the 217th site and the 283th site of the wild type cyclodextrin glycosyl transferase, and the mutant has a nucleotide sequence as shown in SEQ ID NO.1. The novel cyclodextrin glycosyl transferase mutant has a nucleotide sequence as shown in SEQ ID NO.2. The novel cyclodextrin glycosyl transferase mutant has a nucleotide sequence as shown in SEQ ID NO.2. The amino acid sequence of the mutant is preferably as shown in SEQ ID NO. 4. The novel cyclodextrin glycosyl transferase mutant is obtained through a site-directed mutagenesis technology, the mutant with the amino acid sequence shown as SEQ ID NO.4 has the highest enzyme activity, the highest conversion rate of a substrate can reach 94.8%, and the novel cyclodextrin glycosyl transferase mutant has the advantages that the novel cyclodextrin glycosyl transferase mutant is a novel cyclodextrin glycosyl transferase mutant for improving a biological catalytic synthesis technology of alpha-glucosyl hesperidin; the method has important significance in realizing high-yield and high-purity alpha-glucosyl hesperidin.

Description

technical field [0001] The invention relates to a cyclodextrin glycosyltransferase mutant and its coding gene, and its application in the biocatalytic preparation of α-glucosyl hesperidin. Background technique [0002] Among the main functional components of citrus peel, hesperidin is one of the functional components with relatively high added value. It is a dihydroflavone with a chemical structure shown in formula (I). Dihydroflavonoids are one of the 60 kinds of flavonoids in citrus. Hesperidin mainly exists in the peel, juice and seeds of Rutaceae citrus plants, especially in the peel, with a content of about 3%. Hesperidin is weakly acidic, insoluble in water, natural and non-toxic. It has the functions of maintaining normal osmotic pressure of blood vessels, enhancing capillary toughness, shortening bleeding time, and reducing vascular fragility. It has also been found recently that it can lower blood pressure. , anti-allergic, lowering low-density cholesterol so as t...

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Problems solved by technology

However, cyclodextrin glycosyltransferase cannot achieve efficient conversion of hesperidin. The inventor's research group submitted an application number 201911420161.5 in the early stage, and the name is "An alkaline cyclodextrin glucosyltransferase in the production of α-glucose Application of Hesperidin in Hesperidin", the application provides a cyclodextrin glycosyltransferase derived from Bacillus alkalophilus, the amino acid sequence is shown in SEQ ID NO.2, the enzyme in 5% orange peel 88% of the substrate conversion rate can be achieved when the glycoside dosage is 15%, and the conversion rate is reduced to 58% when the 15% hesperidin dosage is used. The remaining hesperidin is difficult to separate, which seriously affects the water solubility of the final product, which leads to Product yield and purity are difficult to balance

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