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CGTase high in temperature stability and gamma-cyclodextrin prepared from CGTase

A cyclodextrin and maltodextrin technology, applied in the directions of transferase, fermentation, etc., can solve the problems of residue, low conversion rate, poor temperature stability, etc., and achieve the effect of reducing use and improving starch conversion rate

Active Publication Date: 2019-06-28
JIANGNAN UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0006] Therefore, at present, there are few types of cyclodextrin glucosyltransferases for the production of γ-CD, and the temperature stability is poor, or the conversion rate of the enzyme action product with good temperature stability is not high, and it is usually broken by adding organic solvents or other substances. Reaction balance to increase the generation of products, which leads to the problem of residual organic solvents in the final γ-CD, mining more γ-CGTase with known sequences and better temperature stability, and synthesizing γ without adding organic solvents -CD is of great significance to promote the industrial production of γ-CD

Method used

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  • CGTase high in temperature stability and gamma-cyclodextrin prepared from CGTase
  • CGTase high in temperature stability and gamma-cyclodextrin prepared from CGTase
  • CGTase high in temperature stability and gamma-cyclodextrin prepared from CGTase

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Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1: the preparation of enzyme

[0032] The inventors conducted a lot of research and experiments, and unexpectedly found a gene annotated as α-amylase on NCBI (its amino acid sequence is shown in SEQ ID NO.1), and its expression product has the function of synthesizing γ-CD. Under normal circumstances, the general α-amylase can only hydrolyze the α-1,4-glucosidic bond in the starch molecular chain, and does not have the function of catalyzing the synthesis of γ-CD from starch.

[0033] The gene fragment whose nucleotide sequence is shown in SEQ ID NO.2 (amino acid sequence is shown in SEQ ID NO.1) was coded and synthesized at Beijing Huada Gene Co., Ltd., using pET-15b as a plasmid, NcoI and XhoI as enzymes site, connect the gene sequence to the plasmid, and transfer it to E.coli BL21(DE3) for expression. Place E.coli BL21(DE3) in LB broth medium containing 100 μg / mL ampicillin, and culture it on a shaker at 37°C and 200r / min until OD 600 Reach 0.4 to 0.6. ...

Embodiment 2

[0034] Example 2: Temperature Stability of Enzymes

[0035] Add Ca at a final concentration of 5mmol / L 2+ To the enzyme purified in Example 1, incubate at different temperatures of 50, 55, and 60° C. for 0 min, 20 min, 40 min, 60 min, 90 min, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, and 9 h. The residual cyclodextrin glucosyltransferase activity of γ-CGTase was measured at different temperatures and different incubation times.

[0036] The results show that (see figure 2 ): The enzyme has a residual enzyme activity of 80% after incubation at 50°C for 9 hours, a half-life of 8 hours at 55°C, and a half-life of 90 minutes at 60°C, indicating that it has good temperature stability.

Embodiment 3

[0037] Embodiment 3: Preparation of γ-CD

[0038] (1) Mix 15 mg of soluble starch solution with 1 mL of distilled water (1.5% substrate concentration), and stir for 15 min at 90°C;

[0039] (2) Lower the temperature to 50°C, adjust the pH to 9.0, add 0.05U of the enzyme purified in Example 1, react for 0.5h, then boil in a water bath at a high temperature of 100°C for 10min to kill the enzyme, filter at normal pressure, evaporate and concentrate, and Dry to obtain γ-cyclodextrin. The conversion of γ-cyclodextrin was measured to be 10%.

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Abstract

The invention discloses CGTase high in temperature stability and gamma-cyclodextrin prepared from the CGTase, and belongs to the technical field of cyclodextrin production. The novel CGTase acts on starch and a derivative thereof to produce gamma-CD; due to the fact that the gamma-CGTase is good in temperature stability, the residual enzyme activity is 80% after heat preservation is carried out for 9 h at the temperature of 50 DEG C, the half-life period is 8 h at the temperature of 55 DEG C, the half-life period is 90 min at the temperature of 60 DEG C, the enzyme quantity does not need to bereplenished in real time in the reaction process, the starch is not likely to retrograded due to the good temperature stability, and the starch conversation rate is increased. In addition, under thecondition of not adding an external organic solvent, the starch conversion rate can reach 24% to the maximum, and use of an organic solvent can be reduced. More choices are provided for production ofgamma-cyclodextrin.

Description

technical field [0001] The invention relates to a CGTase with high temperature stability and its preparation of gamma-cyclodextrin, which belongs to the technical field of cyclodextrin production. Background technique [0002] Cyclodextrins (cyclodextrins, CD) is a general term for a class of cyclic maltoligosaccharides produced by cyclodextrin glucosyltransferase (CGTase) produced by Bacillus. It is composed of several α-D glucopyranose units linked by α-1,4 glycosidic bonds. The most common cyclodextrins contain 6, 7, and 8 glucose molecules, which are α-, β-, and γ-cyclodextrin, respectively. Refined. The analysis results of infrared spectrum and nuclear magnetic resonance spectrum show that the glucose unit constituting the cyclodextrin is in a chair structure, and because each sugar group cannot rotate around the glycosidic bond, the cyclodextrin molecule is in the shape of a cone and a cylinder. The small port is composed of the primary hydroxyl group at the C-6 posi...

Claims

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Application Information

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IPC IPC(8): C12P19/18C12P19/04C12N9/10
Inventor 金征宇柏玉香郑丹妮纪杭燕
Owner JIANGNAN UNIV
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