Cyclodextrin glucosyltransferase mutants with high β-cyclization activity

A technology of glucosyl and cyclization activity, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of poor specificity, low cyclodextrin cyclization activity, and high cost of industrial production of cyclodextrin, and achieves the improvement of β-cyclization activity. Effect

Active Publication Date: 2019-01-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the low cyclization activity and poor specificity of wild CGTase acting on starch to produce cyclodextrin, the industrial production cost of cyclodextrin is relatively high

Method used

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  • Cyclodextrin glucosyltransferase mutants with high β-cyclization activity
  • Cyclodextrin glucosyltransferase mutants with high β-cyclization activity
  • Cyclodextrin glucosyltransferase mutants with high β-cyclization activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1 Determination of mutation sites

[0013] Calcium ion binding sites widely exist in the α-amylase family, and as a member of the α-amylase family (family 13), CGT enzymes also have similar calcium ion binding sites. Aspartic acid (Asp) at the 577th amino acid residue of the CGTase derived from Bacillus circulans STB01 is located at the calcium ion binding site CaIII. After Asp577 is mutated to arginine (Arg), the wild Compared with β-CGTase, the β-cyclization activity of mutant D577R was increased by 30.7%, but the product specificity did not change. In order to further improve the β-cyclization activity of β-CGTase derived from B.circulans STB01, we considered designing double mutations in order to obtain mutants with high enzyme activity and high β-cyclodextrin specificity. Amino acid residue 89 is one of the main residues located near subsite-3. We constructed and obtained double mutant recombinases Y89G / D577R, Y89D / D577R and Y89N / D577R, and also constructe...

Embodiment 2

[0014] Example 2 Preparation of mutants Y89G, Y89D, Y89N, Y89G / D577R, Y89D / D577R and Y89N / D577R

[0015] (1) Site-directed mutation

[0016] According to the wild CGTase gene sequence shown in SEQ ID NO.1, primers were designed and synthesized to introduce single mutations of Gly89, Asp89, Asn89 codons; according to the mutant D577R gene sequence shown in SEQ ID NO.15, respectively designed and synthesized Primers for introducing double mutations of Asp577 / Gly89, Asp577 / Asp89 and Asp577 / Asn89 were synthesized. The single mutant is to mutate Tyr at position 89 to Gly, Asp and Asn respectively, and the double mutant is to mutate Tyr at position 89 to Gly, Asp and Asn on the basis of mutating Asp at position 577 to Arg. . The six mutants obtained were named in turn: Y89G, Y89D, Y89N, Y89G / D577R, Y89D / D577R and Y89N / D577R

[0017] Using rapid PCR technology, site-directed mutagenesis was carried out using the expression vector cgt / pST containing the wild CGTase gene as a templa...

Embodiment 3

[0043] Example 3 Enzyme Activity Determination Analysis

[0044] (1) Determination of enzyme activity

[0045] Determination of α-cyclization activity: Take 0.1 mL of appropriately diluted enzyme solution, add 0.9 mL of 1% (w / v) maltodextrin (DE=5) prepared in advance with 10 mM phosphate buffer (pH 6.5) In the test tube of the solution, after reacting at 50°C for 10min, add 1.0mL of 1.0N hydrochloric acid to stop the reaction, then add 1.0mL of 0.1mM methyl orange solution prepared with 10mM phosphate buffer, keep warm at 20°C for 15min, and measure at 505nm Absorbance. Using the inactivated enzyme as a blank, the content of α-cyclodextrin was determined corresponding to the α-cyclodextrin standard curve. One enzyme activity unit is defined as the amount of enzyme required to generate 1 μmol of cyclodextrin per minute under the above conditions.

[0046] Determination of β-cyclization activity: Take 0.1 mL of appropriately diluted enzyme solution, add 0.9 mL of 1% (w / v) ma...

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Abstract

The invention discloses a cyclodextrin glucosyltransferase mutant with high beta-cyclizing activity, and belongs to the field of gene engineering and enzyme engineering. According to the cyclodextrin glucosyltransferase mutant with high beta-cyclizing activity, the 89th tyrosine of beta-CGT enzyme from Bacillus circulans STB01 is mutated into glycine, aspartic acid and asparaginate respectively; double mutant means mutating the 89th tyrosine into the glycine, aspartic acid and asparaginate on the basis that the 577th aspartic acid of the CGT enzyme is mutated into arginine, and the obtained mutant is significantly improved in beta-cyclizing activity than wild CGT enzyme, thus being more applicable to the industrial production of cyclodextrin.

Description

technical field [0001] The invention relates to a cyclodextrin glucosyltransferase mutant with high β-cyclization activity, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Cyclodextrin is a cyclic compound composed of D-glucopyranose connected by α-1,4-glycosidic bonds, among which α-, β- and γ-cyclodextrins composed of 6, 7 and 8 glucose units Essence is most common. Because of its hollow cylindrical structure, it has the characteristics of being hydrophilic on the outside and hydrophobic on the inside. Cyclodextrin can form inclusion complexes with many hydrophobic guest molecules, thereby changing the physical and chemical properties of the guest molecules. Therefore, it is widely used in food, medicine and other industrial fields. has a wide range of applications. [0003] The industrial production of cyclodextrin adopts enzymatic process, that is, it is synthesized by converting starch through cyclization reaction u...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/75C12P19/18
CPCC12N9/10C12N9/1074C12P19/18C12Y204/01019
Inventor 李兆丰顾正彪李才明黄敏洪雁程力
Owner JIANGNAN UNIV
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