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

A technology of substrate specificity and glycosyltransferase, applied in botany equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of maltodextrin substrate specificity (low conversion rate, etc.) , to achieve the effect of improving substrate specificity

Inactive Publication Date: 2013-05-29
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

Examples

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Embodiment 1

[0023] Example 1: Cyclodextrin Glucosyltransferase with Improved Substrate Specificity

[0024] The cyclodextrin glycosyltransferase of the present invention is obtained on the basis of the gene sequence published by GenBank AF047363.1 by performing single or compound mutations on the amino acids at four positions, and specifically obtained 15 mutants, namely K47L, Y89F, N94P, D196Y, K47L / Y89F, K47L / N94P, K47L / D196Y, Y89F / N94P, Y89F / D196Y, N94P / D196Y, K47L / Y89F / N94P, K47L / Y89F / D196Y, K47L / N94P / D196Y, Y N94P / D196Y, K47L / Y89F / N94P / D196Y.

[0025] Amino acid substitutions can be carried out at the four sites in the mature region by chemical total synthesis or site-directed mutagenesis.

Embodiment 2

[0026] Example 2: Preparation method of cyclodextrin glucosyltransferase with improved substrate specificity

[0027] 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. Mutant enzymes K47L, Y89F, N94P, D196Y, K47L / Y89F, K47L / N94P, K47L / D196Y, Y89F / N94P, Y89F / D196Y, N94P / D196Y, K47L / Y89F / N94P, K47L / Y89F / D196Y, K47L / N94 / D196Y, Y89F / N94P / D196Y, K47L / Y89F / N94P / D196Y are prepared as follows:

[0028] 1) Site-directed mutation

[0029] Site-directed mutagenesis of single mutant enzymes K47L, Y89F, N94P and D196Y, using the rapid mutation kit MutanBEST kit to express vector cgt / pET-20b(+) 1 for the template,

[0030] The primers for site-directed mutagenesis introducing the K47L codon are:

[0031] Forward primer: 5′-CCAATTTG CTG CTCTATTTCGGGGG-3′, the underline is the mutant base;

[0032] Reverse primer: 5′-ATCGGTCGCCGCTGAACGCA-3′;

[0033] The...

Embodiment 3

[0087] Example 3: This example illustrates the analysis of enzyme activity and the synthesis and detection of AA-2G.

[0088] 1) Enzyme activity assay method:

[0089] 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 unit of enzyme activity defines the amount of enzyme required to generate 1 μmol α-cyclodextrin per minute under this condition.

[0090] Determination of starch hydrolysis activity: add an appropriate amount of enzyme solution to 50mM phosphate buffer (pH 6.5) containing 2% soluble starch, react at 50°C for 10min, and then use the DNS method to measur...

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Abstract

The invention discloses a cyclodextrin glycosyl transferase with improved maltodextrin substrate specificity and a preparation method thereof and belongs to the fields of genetic engineering and enzyme engineering. According to the invention, for CGTase derived from peanibacillus macerans, lysine at a 47th site, tyrosine at a 89th site, asparaginate at a 94th site and aspartic acid at a 196th site are respectively mutated into leucine K47L, phenylalanine Y89F, praline N94P and tyrosine D196Y, so that AA-2G yields are respectively increased by 30.7%, 10.9%, 10.0% and 31.7%. Complex mutation is carried out on the mutant strains to obtain double mutants namely K47L/Y89F, K47L/N94P, K47L/D196Y, Y89F/N94P, Y89F/D196Y and N94P/D196Y, three-point mutants namely K47L/Y89F/N94P, K47L/Y89F/D196Y, K47L/N94P/D196Y and Y89F/N94P/D196Y and a four-point mutant namely K47L/Y89F/N94P/D196Y. Yields of AA-2G produced by the mutants while maltodextrin is utilized as a glycosyl donor are respectively increased by 42.6%, 11.0%, 37.6%, 43.6%, 43.6%, 41.6%, 44.5%, 50.5%, 20.8%, 35.6% and 64.4%. Compared with the wild type CGTase, the mutants is more beneficial to production of the AA-2G while the maltodextrin is utilized as the glycosyl donor.

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): C12N9/10C12N1/21C12N5/10C12N15/54C12N15/70C12R1/19C12R1/91
Inventor 陈坚堵国成刘龙李江华许乔艳
Owner JIANGNAN UNIV
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