Cyclodextrin glucosyltransferase mutant weakened via product inhibition

A glucose-based and mutant technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low starch conversion rate, decreased CGT enzyme cyclization activity, and limited application of cyclodextrin, and achieves benefits for industrial production and increased yield. Effect

Active Publication Date: 2015-01-21
JIANGNAN UNIV
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Problems solved by technology

During the production of cyclodextrin by the action of wild CGTase on starch, cyclodextrin will combine with CGTase protein molecules, resulting in the decrease of the cyclization activity of CGTase, and the product inhibi

Method used

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  • Cyclodextrin glucosyltransferase mutant weakened via product inhibition
  • Cyclodextrin glucosyltransferase mutant weakened via product inhibition
  • Cyclodextrin glucosyltransferase mutant weakened via product inhibition

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[0024] Example 1 Determination of mutation sites

[0025] There are three maltosyl binding sites in the CGT enzyme, namely maltosyl binding site 1 (abbreviated as MBS1), maltosyl binding site 2 (abbreviated as MBS2) and maltosyl binding site 3 (MBS3). During the reaction of starch and CGTase to produce cyclodextrin, the cyclodextrin product will bind to MBS2 in CGTase, which prevents starch from entering the active center, inhibits the cyclization activity of CGTase, and results in a low yield of cyclodextrin. The MBS2 of CGTase derived from Bacillus circulans STB01 is composed of 39 amino acid residues at positions 598 to 636. Among them, Ala599 and Tyr633 can interact with cyclodextrin through hydrogen bonds, and may interact with It is related to preventing starch from entering the active center. Therefore, if the spatial structure of these two sites is changed, the inhibition of CGTase activity by cyclodextrin may be weakened, so that CGTase can continue to convert starch in...

Example Embodiment

[0026] Example 2 Preparation of mutants A599N, A599N / Y633A

[0027] (1) Site-directed mutation

[0028] Site-directed mutagenesis of single mutant A599N: Using rapid PCR technology, the expression vector pST / cgt (or other common Bacillus subtilis expression vectors, such as pUB110, pC194, etc.) containing wild CGT enzyme gene is used as a template for site-directed mutation.

[0029] Site-directed mutagenesis primers introducing codon Asn599:

[0030] Forward primer: 5’-AACGCGACGACG AAT CTTGGGCA-3’, underlined are the mutated bases,

[0031] Reverse primer: 5’-TGCCCAAG ATT CGTCGTCGCGTT-3', the underlined is the mutated base;

[0032] Site-directed mutation of double mutant A599N / Y633A: Using rapid PCR technology, the expression vector pST / cgt of single mutant A599N gene was used as template for site-directed mutation.

[0033] Site-directed mutagenesis primers introducing codon Ala633:

[0034] Forward primer: 5’-GTCGTTTACCAA GCA CCGAACTG-3’, the underlined is the mutant base,

[0035] Re...

Example Embodiment

[0041] Example 3 Enzyme Activity Assay and Analysis

[0042] (1) Determination of enzyme activity

[0043] Determination of α-cyclization activity: Take 0.1 mL of appropriately diluted enzyme solution and add 0.9 mL of 1% (w / v) maltodextrin (DE=5) solution prepared in 50 mM phosphate buffer (pH 6.0) In the test tube, after reacting at 50℃ for 10min, add 1.0mL 1.0N hydrochloric acid to stop the reaction, then add 1.0mL 0.1mM methyl orange solution prepared with 50mM phosphate buffer solution, incubate at 20℃ for 15min, measure absorbance at 505nm . Using the inactivated enzyme as a blank, the content of α-cyclodextrin was determined corresponding to the α-cyclodextrin standard curve. One unit of enzyme activity is defined as the amount of enzyme required to generate 1 μmol of cyclodextrin per minute under the above conditions.

[0044] Determination of β-cyclization activity: Take 0.1 mL of appropriately diluted enzyme solution and add 0.9 mL of 1% (w / v) maltodextrin (DE=5) solutio...

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Abstract

The invention discloses a cyclodextrin glucosyltransferase mutant weakened via product inhibition, belonging to the fields of gene engineering and enzyme engineering. By using a site-specific mutagenesis method, the cyclodextrin production capacity of a CGTase is improved, and a mutation scheme for strengthening the cyclodextrin production capacity of a beta-CGTase from Bacillus circulans STB01 is provided to obtain a mutant A599N or A599N/Y633A. Compared with the wild CGTase, the cyclodextrin production capacity of the mutant is remarkably strengthened so that the mutant is more suitable for the industrial production of cyclodextrin.

Description

technical field [0001] The invention relates to a cyclodextrin glucosyltransferase mutant weakened by product inhibition, which belongs to the fields of genetic engineering and enzyme engineering. Background technique [0002] Cyclodextrin-based starch and related substrates are cyclic oligomeric compounds obtained by enzymatic cyclization of more than six glucose via α-1,4-glucosidic bonds. In the cyclodextrin family, they are named based on the number of glucose in the ring, and cyclodextrins consisting of 6, 7, and 8 glucose units are called α-, β-, and γ-cyclodextrins, respectively. The three-dimensional structure of cyclodextrin is a hollow cylinder, and there are non-polar C3 and C5 hydrogens and oxygen atoms connected by ether bonds inside the cylinder, so the inner cavity of the cylinder is hydrophobic; while the glucose The secondary hydroxyl groups on C2 and C3 are located at the wide side opening of the cylinder, and the primary hydroxyl group on C6 is located at...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/75C12N1/21C12P19/18C12P19/04
CPCC12N9/1074C12P19/04C12P19/18C12Y204/01019
Inventor 李兆丰顾正彪徐琪李才明洪雁程力
Owner JIANGNAN UNIV
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