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Preparation and application of cyclodextrin glycosyltransferase mutant

A glucosyl and cyclodextrin technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low AA-2G conversion rate and low enzyme yield, and achieve the effects of high yield and simple purification

Active Publication Date: 2018-05-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, there are many studies on the genetic modification and high-efficiency expression of cyclodextrin glucosyltransferase, but there are still big problems in the industrial production of cyclodextrin glucosyltransferase, such as low enzyme yield, catalytic preparation of AA -2G conversion rate is low, etc.

Method used

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  • Preparation and application of cyclodextrin glycosyltransferase mutant
  • Preparation and application of cyclodextrin glycosyltransferase mutant
  • Preparation and application of cyclodextrin glycosyltransferase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Preparation of Cyclodextrin Glucosyltransferase Mutants

[0038] (1) Preparation of mutants

[0039] According to the gene sequence of cyclodextrin glucosyltransferase shown in the amino acid sequence of SEQ ID NO.1, primers for introducing K124E, G450S, I465F, I641T, K647E, and I631T mutations were designed and synthesized, respectively, for cyclodextrin glucosyltransferase Carry out site-directed mutation of the enzyme gene, determine the DNA coding sequence, change the codon of the 124th Lys to the codon of Glu, mutate the codon of the 450th Gly to the codon of Ser, and mutate the codon of the 465th Ile to Phe The 641st Ile codon was changed to a Thr codon, the 647th Lys codon was changed to a Glu codon, and the 631st Ile codon was changed to a Thr codon. The mutant gene is placed in an appropriate expression vector and introduced into Escherichia coli for expression to obtain the cyclodextrin glucosyltransferase.

[0040] Site-directed mutagenesis of mu...

Embodiment 2

[0062] Example 2 Single mutant cyclodextrin glucosyltransferase activity and soluble expression analysis

[0063] (1) Expression of mutant enzymes

[0064] Pick the positive clones transferred into the expression host Escherichia coli BL21 (DE3) and grow them in LB liquid medium (containing 100 μg / mL ampicillin) for 8-10 h, and connect the seed fermentation liquid to TB medium (containing 100 μg / mL ampicillin) according to 5% inoculum / mL ampicillin), cultured in a shaker at 25°C for 60h, centrifuged at 4°C, 8000rpm for 10min to remove bacteria, and collected the centrifuged supernatant. The supernatant fermented with wild cgt / pET20b(+) / E.coli BL21(DE3) was used as wild enzyme control.

[0065] The enzyme activities of wild-type cyclodextrin glucosyltransferase and mutant enzymes after 60 h culture in shake flasks are listed in the table, among which I641T and K647E have the most significant effects, and the enzyme activities are 2.5 times and 2.4 times of wild enzymes respec...

Embodiment 3

[0070] Example 3: Preparation and expression of cyclodextrin glucosyltransferase double mutants

[0071] (1) Preparation of mutants

[0072] The above three sites were mutated and combined, and double mutations were designed to obtain mutants I641T / K647E, I641T / I631T, and I631T / K647E. The gene sequence is used as a template, primers are designed and synthesized to introduce double mutations, the sequence is determined, the mutant gene is placed in an appropriate expression vector and introduced into Escherichia coli for expression to obtain cyclodextrin glucosyltransferase. Site-directed mutagenesis of mutants I641T / K647E, I641T / I631T, and I631T / K647E: using rapid PCR technology and using the expression vector cgt / pET20b(+) as a template.

[0073] The site-directed mutagenesis primers for introducing the K647E mutation are:

[0074] Forward primer: 5'-CGAGTTTAAATTCATC GAA AAAGACTTCTCAGGGC-3' (the underline is the mutated base)

[0075] Reverse primer: 5'-GCCCTGAGAGTCTTT ...

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Abstract

The invention relates to preparation and application of a cyclodextrin glycosyltransferase mutant, and belongs to the field of gene engineering and enzyme engineering. The amino acid of cyclodextrin glycosyltransferase is subjected to mutation; the enzyme activity of the obtained mutant can reach 2.5 times of that of wild enzyme; the purification of the obtained cyclodextrin glycosyltransferase mutant is simple; the industrial production can be realized.

Description

technical field [0001] The invention relates to the preparation and application of a cyclodextrin glucosyltransferase mutant, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Vitamin C (VC) is a water-soluble vitamin that cannot be synthesized by the human body itself. It participates in many physiological activities in the body, such as promoting the conversion of cholesterol into bile acids, promoting the synthesis of adrenal cortex hormones, participating in the metabolism of aromatic amino acids, promoting iron absorption and Participate in various redox reactions in the body and play an important role in maintaining and promoting human health. In addition, vitamin C can also promote the synthesis of collagen, restore the formed melanin, and play a certain role in maintaining skin elasticity, whitening, and wrinkle removal. Widely used in food, medicine, cosmetics and other fields. However, the hydroxyl group of the 2...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/70C12P19/60
CPCC12N9/1074C12N15/70C12P19/60C12Y204/01019
Inventor 吴敬宿玲恰陶秀梅王昱升董俊辰
Owner JIANGNAN UNIV
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