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Cyclodextrin glycosyltransferase mutants and application thereof

A glucose-based, mutant technology, applied in the field of genetic engineering and enzyme engineering, to achieve the effect of improving the conversion rate

Active Publication Date: 2019-03-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few reports on the affinity of CGTase for maltose receptors in domestic and foreign literature, only Bacillus circulans and Paenibacillus sp. have related reports, and the CGTase derived from Bacillus circulans has a much higher affinity for maltose receptors than Paenibacillus sp. .

Method used

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  • Cyclodextrin glycosyltransferase mutants and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Expression of wild-type cyclodextrin glucosyltransferase

[0032] Cgt / pET20b(+) / BL21(DE3) (Yang Yulu, Wang Lei, Chen Sheng, etc.) was inoculated from glycerol tubes stored in the laboratory in the early stage. The production process of β-cyclodextrin glucosyltransferase Condition optimization [J]. Biotechnology Bulletin, 2014, 8:175-181.) Grow in LB liquid medium (containing 100mg / L ampicillin) for 8h, insert the seeds into TB liquid fermentation medium ( Contains 100mg / L ampicillin). After Escherichia coli was cultured and fermented on a shaking table at 25°C for 48 hours, a certain volume of fermentation broth was centrifuged at 4°C and 12,000 rpm for 15 minutes, and the fermentation supernatant was taken, which was the fermented crude enzyme liquid.

Embodiment 2

[0033] Example 2: Preparation and expression of cyclodextrin glucosyltransferase single mutant

[0034] (1) Preparation of mutants

[0035]According to the gene sequence of Bacillus circulans cyclodextrin glucosyltransferase, primers for introducing M234I, M234A, M234L, and M234V mutations were designed and synthesized, and the cyclodextrin glucosyltransferase gene Cgt was subjected to site-directed mutation, and the cyclodextrins were confirmed by sequencing. Whether the coding gene of the glucosyltransferase mutant is correct; the vector carrying the mutant gene is introduced into Escherichia coli for expression, and a single mutant cyclodextrin glucosyltransferase is obtained.

[0036] PCR amplification of the gene encoding the site-directed mutant: using the rapid PCR technique, the expression vector Cgt / pET-20b(+) carrying the gene encoding wild-type cyclodextrin glucosyltransferase was used as a template.

[0037] The site-directed mutagenesis primers for introducing th...

Embodiment 3

[0062] Example 3: Analysis of dismutation activity of cyclodextrin glucosyltransferase and its affinity for maltose receptor

[0063] The fermented supernatant crude enzyme liquid obtained in Example 1 and Example 2 was subjected to enzyme activity determination. Enzyme activity and kinetic parameter K of wild-type cyclodextrin glucosyltransferase (WT) and mutants m Listed in Table 1, the results showed that all mutants had higher receptor affinity to maltose than the wild type.

[0064] Table 1 Shake Flask Enzyme Dismutation Activity of Wild-type and Mutant Cyclodextrin Glucosyltransferase and Its Affinity with Maltose as Acceptor

[0065]

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Abstract

The invention discloses cyclodextrin glycosyltransferase mutants and application thereof and belongs to the technical field of enzyme engineering. Methionine from site 234 of cyclodextrin glycosyltransferase of Bacillus circulans is subjected to site-specific mutagenesis; affinities of mutants M234I, M234A, M234L and M234V to a maltose receptor are increased by 45.6%, 34.7%, 30.3% and 36.9% as compared to wild types. The cyclodextrin glycosyltransferase mutants are applicable to the preparation of trehalose; trehalose conversion rate is further increased.

Description

technical field [0001] The invention relates to a cyclodextrin glucosyltransferase mutant and its application, especially a cyclodextrin glucosyltransferase mutant with improved receptor affinity to maltose in disproportionation reaction and its application, which belongs to genetic engineering and enzyme engineering technology. Background technique [0002] Trehalose (Trehalose) is composed of two glucopyranose molecules connected by α,α-1,1-glycosidic bonds. It is a stable non-reducing disaccharide with unique high moisture retention, high safety and It has good stability and is widely used in the fields of medicine, food, cosmetics and agriculture. [0003] Since the 1980s, countries have successively carried out research on the effect of trehalose on physiological functions, and now it has become one of the main oligosaccharides developed and researched internationally. [0004] The synthesis of trehalose mainly includes three methods: phosphorylase method, trehalose s...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/12C12R1/19
CPCC12N9/1074C12N15/70C12P19/12C12Y204/01019
Inventor 吴敬宿玲恰杜立
Owner JIANGNAN UNIV
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