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Cyclodextrin glycosyltransferase with maltodextrin substrate specificity being improved and preparation method thereof

A substrate-specific, maltodextrin technology, applied in the directions of glycosyltransferase, botanical equipment and methods, microorganism-based methods, etc., can solve the problem of low substrate specificity of maltodextrin and achieve substrate specificity. Sex-enhancing effect

Active Publication Date: 2014-10-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the substrate specificity (conversion rate) of cyclodextrin glucosyltransferase (CGTase) to maltodextrin is low at present, therefore, improving its substrate specificity to maltodextrin through molecular engineering CGTase technology will Promote the rapid development of industries related to L-AA glycosyl derivatives

Method used

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  • Cyclodextrin glycosyltransferase with maltodextrin substrate specificity being improved and preparation method thereof
  • Cyclodextrin glycosyltransferase with maltodextrin substrate specificity being improved and preparation method thereof
  • Cyclodextrin glycosyltransferase with maltodextrin substrate specificity being improved and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1: Cyclodextrin Glucosyltransferase with Improved Substrate Specificity

[0023] The cyclodextrin glycosyltransferase of the present invention is based on the gene sequence published by GenBank AF047363.1, and its three sites are subjected to single or combined mutations of amino acids. Specifically, seven mutants are obtained, namely Y260R; Q265K; Y195S; Y260R / Q265K; Y260R / Y195S; Q265K / Y195S; Y260R / Q265K / Y195S.

[0024] Amino acid substitutions can be carried out at the three sites in the mature region by chemical total synthesis or site-directed mutagenesis.

Embodiment 2

[0025] Example 2: Preparation method of cyclodextrin glucosyltransferase with improved substrate specificity

[0026] 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 Y195S, Y260R, Q265K, Y195S / Y260R, Y195S / Q265K, Y260R / Q265K and Y195S / Y260R / Q265K is as follows:

[0027] 1) Site-directed mutation

[0028] Site-directed mutagenesis of single mutant enzymes Y195S, Y260R and Q265K, using the rapid mutation kit MutanBEST kit to express vector cgt / pET-20b(+) 1 (1.Li,Z.,B.Li,Z.Gu,G.Du,J.Wu,andJ.Chen.2010.Extracellular expression and biochemical characterization of alpha-cyclodextrin glycosyltransferase from Paenibacillus macerans.Carbohydr Res 345:886- 892.) as a template,

[0029] The apex mutation primers that introduce the Y195S codon are:

[0030] Forward primer: 5'-TACAAGAACCTC TCT GACCTGGC-3', the und...

Embodiment 3

[0054] Example 3: This example illustrates the analysis of enzyme activity and the synthesis and detection of AA-2G.

[0055] 1) Enzyme activity assay method:

[0056] 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.

[0057] Method for measuring starch hydrolysis activity: Add appropriate amount of enzyme liquid into 50mM phosphate buffer (pH6.5) containing 1% soluble starch, react at 50°C for 10min, and then measure reducing sugar ...

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Abstract

The invention discloses a cyclodextrin glycosyltransferase with maltodextrin substrate specificity being improved and a preparation method thereof, belonging to the field of genetic engineering and enzyme engineering. By replacing 195-site Tyr of CGTase of a P.macerans strain JFB05-01 (CCTCC NO:M208063) with Ser, 260-site Tyr with Arg and 265-site Gln with Lys, the output of AA-2G can be respectively increased by 23%, 44% and 40%. Combination mutation is conducted to the mutant strains, double mutants Y195S / Y260R, Y195S / Q265K and Y260R / Q265K and three-point mutants Y195S / Y260R / Q265K can be obtained. The maltodextrin is utilized for producing AA-2G for a glycosyl donor, the output of the AA-2G is respectively improved 57%, 49%, 55% and 59%. The mutant strains are more favorable to produce AA-2G for the glycosyl donor by utilizing maltodextrin compared with the wild CGTase.

Description

[0001] This application is a branch of an invention patent application titled "A cyclodextrin glycosyltransferase with improved maltodextrin substrate specificity and its preparation method", the application number is 201210527869.2, and the application date is December 10, 2012. case application. technical field [0002] The invention relates to a cyclodextrin glycosyltransferase and a preparation method thereof, in particular to a cyclodextrin glycosyltransferase with improved maltodextrin substrate specificity and a preparation method thereof. Background technique [0003] L-Ascorbic acid (L-AA, vitamin C) is a water-soluble vitamin that participates in many physiological activities in the body and plays an important role in maintaining and promoting human health. It is an essential nutrient element that the human body cannot synthesize by itself. However, L-AA is extremely unstable and is easily oxidized to dehydroascorbic acid in the air, destroying the conjugated syste...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/10C12N1/21C12N15/70C12R1/19C12R1/01
CPCC12N9/1074C12N15/70C12Y204/01019
Inventor 陈坚堵国成刘龙李江华韩瑞枝
Owner JIANGNAN UNIV
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