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Cyclodextrin glucosyltransferase mutant with improved cyclization activity

A glucose-based, mutant technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low cyclization activity and high production cost of cyclodextrin industry, and achieve the effect of improving cyclization activity

Active Publication Date: 2014-08-06
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 of wild CGTase on starch to produce cyclodextrin, the industrial production cost of cyclodextrin is high

Method used

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  • Cyclodextrin glucosyltransferase mutant with improved cyclization activity
  • Cyclodextrin glucosyltransferase mutant with improved cyclization activity
  • Cyclodextrin glucosyltransferase mutant with improved cyclization activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Determination of Mutation Sites

[0036] 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. The 32nd amino acid residue asparagine (Asn) of the CGTase derived from Bacillus circulans STB01 is located at the calcium ion binding site CaI, and the spatial structure of the amino acid residues at this site is complex: the distance from Asn32 The polar hydrophilic amino acid residues within are Asp27, Asn29, Asn33, Gly51, Asp53, Tyr112, Gly113, Asp115; The non-polar hydrophobic amino acid residues within are Pro30, 34, 110, Ala31, 111. This diverse and complex conformation suggests that the introduction of longer side chains or more electronegative polar hydrophilic amino acid residues at this site may affect the activity of the enzyme.

Embodiment 2

[0037] Preparation of embodiment 2 mutants N32R, N32K, N32H, N32E, N32D and N32Q

[0038] (1) Site-directed mutation

[0039] According to the wild CGTase gene sequence shown in SEQ ID NO.1, primers for introducing Arg32, Lys32, His32, Glu32 and Asp32 codon mutations were designed and synthesized respectively.

[0040] Using rapid PCR technology, site-directed mutagenesis was performed using the expression vector cgt / pST containing the wild CGTase gene as a template.

[0041] Primers for introducing the Asn32Arg mutation:

[0042] Forward primer: 5'-GCAATCCCGCC AGA AATCCGACC-3', the underline is the mutant base,

[0043] Reverse primer: 5'-GGTCGGATT TCT GGCGGGATTGC-3', the underline is the mutant base;

[0044] Primers for introducing the Asn32Lys mutation:

[0045] Forward primer: 5'-GCAATCCCGCC AAA AATCCGACC-3', the underline is the mutant base,

[0046] Reverse primer: 5'-GGTCGGATT TTT GGCGGGATTGC-3', the underline is the mutant base;

[0047] Primers for introdu...

Embodiment 3

[0065] Embodiment 3 Enzyme assay analysis

[0066] (1) Determination of enzyme activity

[0067] 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.

[0068] Determination of β-cyclization activity: Take 0.1 mL of appropriately diluted enzyme solution, add 0.9 mL of 1% (w / v) maltodextrin (DE...

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Abstract

The invention discloses a cyclodextrin glucosyltransferase mutant with improved cyclization activity, and belongs to the fields of gene engineering and enzyme engineering. According to the invention, through the adoption of a site-specific mutagenesis method, the 32nd aspartic acid (Asp) derived from beta-CGT enzyme of Bacillus circulans STB01 is mutated into alanine (Ala), glycine (Gly), glutamic acid (Glu), arginine (Arg), lysine (Lys) or glutamine (Gln) respectively. Compared with the cyclization activity of wild CGT enzyme, the cyclization activity of the mutant is obviously improved, so that the mutant is more suitable for industrial production of cyclodextrin.

Description

technical field [0001] The invention relates to a cyclodextrin glucosyltransferase mutant with improved 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/28C12N15/56C12N15/75C12P19/18
CPCC12N9/1074C12P19/04C12P19/18C12Y204/01019
Inventor 顾正彪李兆丰黄敏李才明洪雁程力
Owner JIANGNAN UNIV
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