Method for preparing cycloamylose 4-alpha-glycosyl transferase production

A technology of glycosyltransferase and macrocyclodextrin, which is applied in the field of enzyme engineering, can solve the problems of few research results on enzymes producing macrocyclodextrin, achieve huge potential commercial value, high heat resistance, and easy separation and purification steps line effect

Inactive Publication Date: 2010-01-27
JIANGNAN UNIV
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Problems solved by technology

Research on glycosyltransferases producing macrocyclodextrins abroad has been carried out for decades, while domestic research has only been carried out in recent years,

Method used

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  • Method for preparing cycloamylose 4-alpha-glycosyl transferase production
  • Method for preparing cycloamylose 4-alpha-glycosyl transferase production
  • Method for preparing cycloamylose 4-alpha-glycosyl transferase production

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Embodiment 1、4

[0023] Preparation and purification of embodiment 1, 4-alpha-glycosyltransferase

[0024] 1. Enzyme preparation

[0025] 1) Single colony selection: Dip a small amount of bacterial solution from the genetically engineered strains with an inoculation loop to draw an LB plate containing 100 μg / mL ampicillin sodium, and place the plate in a biochemical incubator at 37°C for 12 hours at a constant temperature;

[0026] 2) Seed culture: Pick a single colony and inoculate it in a 50 mL conical flask containing 100 μg / mL ampicillin sodium LB medium, fix the conical flask on a constant temperature shaker at 37°C, 200 rpm, and vibrate for 8 hours to make seeds Culture medium;

[0027] 3) Fermentation culture: Inoculate the seed medium into a fermenter of 3L medium containing ampicillin sodium 100 μg / mL, adjust the temperature to 37°C, stir at 200 rpm, pH to 7.5, and ventilation to 10L / h , the inoculum size was 10%, adding 10mL / L soybean oil as an antifoaming agent, and fermented and ...

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Abstract

The invention provides a method for preparing cycloamylose 4-alpha-glycosyl transferase production, belonging to the technical field of enzyme engineering. The method uses Escherichia coli CGMCC No. 3093 as a parent strain to prepare pure enzyme 4-alpha-transferase through preparing a seed culture medium by jar fermentation, fermenting and culturing the culture medium in a fermentation cylinder, collecting strains, extracting crude enzyme by ultrasonication and purifying. The transferase prefers a temperature at 75 DEG C and pH of 7.5, and has the characteristics of high temperature resistance, high enzyme activity and the like, wherein, vitality of the 4-alpha-transferase which is primarily purified, frozen and dried can reach 7,000-10,000U/g. The 4-alpha-transferase can act on amylase to generate cycloamylose, and can modify amylopectin to generate slowly digestible starch. Therefore, the transferase can be widely applied in foods, pharmacy and other industries, and has enormous potential commercial value.

Description

technical field [0001] The invention discloses a method for preparing macrocyclodextrin 4-alpha-glycosyltransferase, which belongs to the technical field of enzyme engineering. Background technique [0002] Macrocyclodextrin is a cyclic glucan with a degree of polymerization ranging from 9 to several hundred, which is different from common cyclodextrins. Because macrocyclodextrin has the characteristics of high water solubility, low viscosity and non-retrogradation, it can be widely used in food industry, chemical industry and pharmaceutical industry. Macrocyclodextrins are mainly produced by glycosyltransferases. Research on glycosyltransferases producing macrocyclodextrins abroad has been carried out for decades, while domestic research has only been carried out in recent years, and great achievements have been made in the production of common cyclodextrins, but for the production of macrocyclodextrins There are few research results on dextrinase. The macrocyclodextrin-...

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Application Information

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IPC IPC(8): C12N1/21C12N9/10C12R1/19
Inventor 王金鹏徐学明金征宇谢正军陈寒青晋雪霞曹旭
Owner JIANGNAN UNIV
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