Cyclodextrin glycosyl transferase with improved maltodextrin substrate specificity and preparation method thereof

A substrate-specific, maltodextrin technology, applied in glycosyltransferases, botanical equipment and methods, and microbial-based methods, can solve the problems of maltodextrin substrate specificity (low conversion rate, etc., and achieve The effect of improving substrate specificity

Active Publication Date: 2013-03-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Cyclodextrin glycosyl transferase with improved maltodextrin substrate specificity and preparation method thereof
  • Cyclodextrin glycosyl transferase with improved maltodextrin substrate specificity and preparation method thereof
  • Cyclodextrin glycosyl transferase with improved maltodextrin substrate specificity and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0021] Example 1: Cyclodextrin Glucosyltransferase with Improved Substrate Specificity

[0022] The cyclodextrin glycosyltransferase of the present invention is obtained on the basis of the gene sequence published by GenBankAF047363.1 by performing single or combined mutations on the amino acids at three positions, and specifically obtained seven mutants, namely Y260R; Q265K ;Y195S;Y260R / Q265K;Y260R / Y195S;Q265K / Y195S;Y260R / Q265K / Y195S.

[0023] 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

[0024] Example 2: Preparation method of cyclodextrin glucosyltransferase with improved substrate specificity

[0025] 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:

[0026] 1) Site-directed mutation

[0027] 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, and J.Chen.2010.Extracellular expression and biochemical characterization of alpha-cyclodextrin glycosyltransferase from Paenibacillus macerans.Carbohydr Res345:886-892 .) for the template,

[0028] The apex mutation primers that introduce the Y195S codon are:

[0029] Forward primer: 5'-TACAAGAACCTC TCT GACCTGGC-3...

Embodiment 3

[0053] Example 3: This example illustrates the analysis of enzyme activity and the synthesis and detection of AA-2G.

[0054] 1) Enzyme activity assay method:

[0055] Method for measuring α-cyclization activity by methyl orange method: Take 0.1mL of appropriately diluted enzyme solution, add it to 0.9mL of 3% soluble starch solution prepared in advance with 50mM phosphate buffer (pH6.5), 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.

[0056] Determination of starch hydrolysis activity: Add appropriate amount of enzyme solution to 50mM phosphate buffer (pH6.5) containing 1% soluble starch, react at 50°C for 10min, and then use DNS method to measure the concen...

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Abstract

The invention discloses cyclodextrin glycosyl transferase with improved maltodextrin substrate specificity and a preparation method thereof, and belongs to the field of genetic engineering and enzyme engineering. 195-bit tyrosine (Tyr) of CGTase of P.macerans strain JFB05-01 (CCTCC NO:M208063) is replaced by serine (Ser), 260-bit tyrosine (Tyr) is replaced by arginine (Arg), and 265-bit glutamine (Gln) is replaced by lysine (Lys), so that the AA-2G yield is respectively improved by 23 percent, 44 percent and 40 percent. According to combined mutation of the mutant strains, double mutants Y195S/Y260R, Y195S/Q265K and Y260R/Q265K and A three-point mutant Y195S/Y260R/Q265K are obtained. A Glycosyl donor is produced by the maltodextrin, so that the AA-2G yield is respectively improved by 57 percent, 49 percent, 55 percent and 59 percent; and compared with the mild type CGTase, the mutant strains facilitate the production of the AA-2G for glycosyl donors by using maltodextrin.

Description

technical field [0001] 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 [0002] 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 system in the molecule and causing an irreversible cleavage reaction, especially in the presence of air, light, heat and metal ions, the reaction is more rapid , so that the physiological activity of L-AA is weakened or even disappeared, which makes it very limited in application. Therefore, how to enhan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/10C12N1/21C12N5/10C12N15/70C12R1/01
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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