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A cyclodextrin glucosyltransferase with improved product specificity and preparation method thereof

A glycosyltransferase and glucose-based technology, which is applied in the field of cyclodextrin glycosyltransferase and preparation, can solve problems such as low efficiency, and achieve the effects of improving glycosylation efficiency and facilitating industrial production.

Active Publication Date: 2020-08-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the glycosylation efficiency (conversion rate) of cyclodextrin glucosyltransferase (CGTase) to genistein is low, therefore, improving the glycosylation efficiency of genistein through molecular modification of CGTase technology will promote the use of dyes. The rapid development of industries related to lignin glycosyl derivatives

Method used

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  • A cyclodextrin glucosyltransferase with improved product specificity and preparation method thereof
  • A cyclodextrin glucosyltransferase with improved product specificity and preparation method thereof
  • A cyclodextrin glucosyltransferase with improved product specificity and preparation method thereof

Examples

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Effect test

Embodiment 1

[0017] Example 1: Cyclodextrin Glucosyltransferase with Improved Efficiency of Genistein Glycosylation

[0018] The cyclodextrin glycosyltransferase of the present invention is based on the gene sequence published by GenBank JX412224, and the alanine at position 156 of its mature region is replaced with other amino acids, and three kinds of mutants are specifically obtained, namely A156K, A156V, and A156N .

[0019] Amino acid substitutions can be carried out at the three sites in the mature region by chemical total synthesis or PCR.

Embodiment 2

[0020] Example 2: Preparation method of cyclodextrin glucosyltransferase with improved genistein glycosylation efficiency

[0021] This example uses the PCR method as an example for illustration, but the protection of the invention is not limited to the method of obtaining mutations only through the PCR method. The preparation method of mutant enzyme A156K, A156V, A156N is as follows:

[0022] 1) Site-directed mutation

[0023] Site-directed mutagenesis of mutant enzymes A156K, A156V and A156N to express vector cgt / pET-20b(+) 1 (1. Han, R.Z., Ge, B.B., Jiang, M.Y., Xu, G.C., Dong, J.J., and Ni, Y. (2017) High production of genistein diglucoside derivative using cyclodextrin glycosyltransferase from Paenibacillus macerans, J Ind Microbiol Biot 44, 1343- 1354.) as a template, the site-directed mutagenesis primers that introduce the A156K codon are:

[0024] Forward primer: 5'-GCTTTGCAGAAAATGGT AAA CTGTA-3', the underline is the mutant base;

[0025] Reverse primer: 5'-GAGC...

Embodiment 3

[0038] Example 3: This example illustrates the analysis of enzyme activity and the detection of glycosylation of genistein.

[0039] Enzyme activity assay method:

[0040] Method for measuring α-cyclization activity by methyl orange method: take 0.1 mL of appropriately diluted enzyme solution, add 0.9 mL of 3% soluble starch solution prepared in advance with 50 mM phosphate buffer (pH 6.5), and heat at 40 ° C After reacting for 10 min, add 1.0 mL of 1.0 M hydrochloric acid to stop the reaction, then add 1.0 mL of 0.1 mM methyl orange prepared with 50 mM phosphate buffer, incubate at 16°C for 20 min, and measure the absorbance at 505 nm. One enzyme activity unit defines the amount of enzyme required to produce 1 μmol α-cyclodextrin per minute under the conditions.

[0041] Method for measuring starch hydrolysis activity: Add appropriate amount of enzyme solution into 50mM phosphate buffer (pH 6.5) containing 1% soluble starch, react at 50°C for 10min, and then measure reducing...

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Abstract

The invention discloses a cyclodextrin glucosyltransferase and a preparation method thereof, and belongs to the field of genetic engineering and enzyme engineering. Through replacement of 156-site Alanine (Ala) of CGTase of P. Macerans strain JFB05-01 (CCTCC NO: M208063) respectively with lysine (Lys), glutamine (Gln) and valine (Val), the genistein glycosylation efficiency is increased by 23%, 44% and 32%, respectively. The mutant enzymes are more conductive to catalysis production of glycosylated genistein compared with wild-type CGTase.

Description

technical field [0001] The invention relates to a cyclodextrin glucosyltransferase with improved product specificity and a preparation method, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Genistein (also known as genistein, genistein, etc.) is considered to be a soybean isoflavone with the highest activity and function. In legumes, genistein often exists in the form of its glucoside derivative, genistin (also known as 4',5,7-trihydroxyisoflavone-7-glycoside). Genistein has a wide range of pharmacological effects in human and animal cells. The main performances are as follows: 1) It has the effect of chemical anti-cancer (breast cancer and prostate cancer, etc.). Genistein has estrogen-like and anti-hormone effects, can inhibit the activity of related enzymes in the synthesis process of tumor cells, inhibit tumor angiogenesis during the formation of tumor cells, and delay or prevent tumors from becoming cancer cells. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/60C12P19/18
CPCC12N9/1074C12N15/70C12P19/18C12P19/60C12Y204/01019
Inventor 韩瑞枝倪晔葛彬彬姚栋董晋军许国超
Owner JIANGNAN UNIV
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