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Maltose substrate specificity improved cyclodextrin glycosyltransferase and preparation method thereof

A substrate specificity, glycosyltransferase technology, applied in the fields of botanical equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of maltose substrate specificity (low conversion rate, etc.) The effect of improved substrate specificity

Active Publication Date: 2013-03-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low substrate specificity (conversion rate) of cyclodextrin glucosyltransferase (CGTase) to maltose, improving its substrate specificity to maltose through molecular modification of CGTase technology will promote the combination with L- The rapid development of related industries of AA glycosyl derivatives

Method used

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  • Maltose substrate specificity improved cyclodextrin glycosyltransferase and preparation method thereof
  • Maltose substrate specificity improved cyclodextrin glycosyltransferase and preparation method thereof
  • Maltose substrate specificity improved cyclodextrin glycosyltransferase and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Example 1: Cyclodextrin Glucosyltransferase with Improved Substrate Specificity

[0020] The cyclodextrin glycosyltransferase of the present invention is based on the gene sequence published by GenBank AF047363.1, and the lysine at position 47 is mutated into phenylalanine, tyrosine or proline, respectively K47F , K47Y and K47P.

[0021] Amino acid substitution can be carried out at position 47 of the mature region by chemical total synthesis or site-directed mutagenesis.

Embodiment 2

[0022] Example 2: Preparation method of cyclodextrin glucosyltransferase with improved substrate specificity

[0023] 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 enzymes K47F, K47Y and K47P is as follows:

[0024] 1) Site-directed mutation

[0025] Site-directed mutagenesis of single mutant enzymes K47F, K47Y and K47P, using the rapid mutation kit MutanBEST kit to express vector cgt / pET-20b(+) 1 for the template,

[0026] The apex mutation primers introducing the K47F codon are:

[0027] Forward primer: 5'-CCAATTTG TTC CTCTATTTCGGGGG-3', the underline is the mutant base;

[0028] Reverse primer: 5'-ATCGGTCGCCGCTGAACGCA-3';

[0029] The apex mutation primers introducing the K47Y codon are:

[0030] Forward primer: 5'-CCAATTTG TAC CTCTATTTCGGGGG-3', the underline is the mutant base;

[0031] Reverse ...

Embodiment 3

[0041] Example 3: This example illustrates the analysis of enzyme activity and the synthesis and detection of AA-2G.

[0042] 1) Enzyme activity assay method:

[0043] 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.

[0044] 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 concentr...

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Abstract

The invention discloses maltose substrate specificity improved cyclodextrin glycosyltransferase and a preparation method thereof, and belongs to the field of genetic engineering and enzyme engineering. The 47th lysine (Lys) of CGTase of P.macerans strain HFB05-01 (CTCC NO:M208063) is respectively replaced by phenylalanine (Phe), tyrosine (Tyr) and proline (Pro), so that the output of AA-2G is respectively improved by 17.1%, 22.1% and 32.9%. Compared with the wild CGTase, the mutant strains are more conductive to producing AA-2G for glycosyl donors by utilizing maltose, so that important industrial application prospect is provided.

Description

technical field [0001] The invention relates to a cyclodextrin glycosyltransferase and a preparation method thereof, in particular to a cyclodextrin glycosyltransferase with improved maltose 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 enhance th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N1/21C12N5/10C12N15/54C12N15/70C12R1/19
Inventor 陈坚堵国成刘龙李江华韩瑞枝
Owner JIANGNAN UNIV
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