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A kind of cyclodextrin glucosyltransferase production strain and its application

A technology for producing glucose-based strains, which is applied in the field of enzyme engineering and achieves the effects of simple reaction method, low material cost and low energy consumption

Active Publication Date: 2016-05-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The main problem in the current production of AA-2G is the lack of a cyclodextrin glucosyltransferase that can be directed to obtain high conversion rates with cheap substrates

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: strain screening

[0024] From the soil near Changbai Mountain in Jilin Province, 10 soil samples were taken according to the "five-point sampling method", and the strains were isolated from them. Put a small amount of sample into an oven for heat treatment at 80°C for 20 minutes, then weigh 5 g of the sample and add it to a small Erlenmeyer flask filled with 45 mL of sterile water, let it stand for 10 minutes, and take 0.1 mL of the supernatant for gradient dilution (10 -5 , 10 -4 , 10 -3 ) on a separation and screening plate, cultured at a constant temperature in a 37°C incubator for 2-3 days, and the colonies with larger yellow transparent circles on the plate were selected as the strains producing CGTase. Pick a single colony and streak the plate 3-4 times. Afterwards, several strains obtained from the screening were purified and cultured in liquid fermentation at 60°C for 44-48 hours. The culture medium was centrifuged and the supernatant was colle...

Embodiment 2

[0031] Embodiment 2: fermentation produces enzyme

[0032] (1) Fermentation culture

[0033] The cyclodextrin glucosyltransferase-producing strain screened in Example 1 was inoculated in the seed medium, cultured at 60°C for 20-24 hours, then transferred to the fermentation medium with 5% inoculum, and cultured at a constant temperature of 60°C 44-48h to produce enzyme. After the fermentation, the supernatant collected by centrifugation is the crude enzyme liquid.

[0034] Seed medium (g / L):

[0035] Peptone 10, yeast powder 5, sodium chloride 10, pH7.0.

[0036] Fermentation medium (g / L):

[0037] Soluble starch 10, peptone 2, yeast powder 2, ammonium sulfate 2.5, magnesium sulfate heptahydrate 0.3, anhydrous calcium chloride 0.2, 1mL (v / v) trace element solution, pH 7.0.

[0038] (2) Enzyme activity assay

[0039] The activity of the enzyme was determined by methyl orange method with soluble starch as substrate. The enzyme activity assay system is 2.5mL (containing so...

Embodiment 3

[0040] Embodiment 3: the concentration of crude enzyme liquid

[0041] Slowly add ammonium sulfate with a concentration of 26% relative to the mass volume of the enzyme liquid to the enzyme liquid obtained in Example 2 while stirring, stir until the ammonium sulfate is dissolved, and stand at 4°C for 8-10 hours to precipitate protein. The mixture was centrifuged (8000rpm, 10min) to collect the precipitate, and then reconstituted with a minimum volume of 50mM KH2PO4-Na2HPO4 buffer (pH 6.0). After reconstitution, the solid was removed by centrifugation again, and the supernatant was collected and dialyzed to obtain a concentrated enzyme solution.

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PUM

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Abstract

The invention discloses a bacterial strain for cyclodextrin glycosyltransferase production. The cyclodextrin glycosyltransferase obtained by fermenting the bacterial strain is acted on L-ascorbic acid and beta-cyclodextrin with different concentrations to efficiently generate 2-oxo-alpha-D-glucopyranosyl ascorbic acid under a condition that pH is 5.0-6.0 and the temperature is 25-40 DEG C. The production method for generating 2-oxo-alpha-D-glucopyranosyl ascorbic acid by using cyclodextrin glycosyltransferase obtained by fermenting the bacterial strain has the characteristics of simplicity, high conversion rate and high output, and is beneficial for industrialized amplified production.

Description

technical field [0001] The invention relates to a cyclodextrin glucosyltransferase production strain. The cyclodextrin glucosyltransferase fermented by the bacterial strain can be used for efficient production of 2-oxy-α-D-glucopyranosyl ascorbic acid and belongs to the field of enzyme engineering. Background technique [0002] Ascorbic acid (Ascorbic acid) or vitamin C, referred to as VC, is a water-soluble vitamin that cannot be synthesized by the human body itself. It participates in many physiological activities in the body and plays an important role in maintaining and promoting human health. However, the hydroxyl group on the 2-position of the VC molecule is extremely unstable, and is oxidatively degraded by oxygen, water, light, high temperature, and heavy metal ions. Therefore, since the last century, the development of stable VC derivatives has become a research hotspot. Researchers have tried to Looking for a VC derivative, which can not only guarantee the normal p...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/10C12P19/60C12R1/07
Inventor 吴敬熊艳军王蕾
Owner JIANGNAN UNIV
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