Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for producing extracellular production and recombination beta-cyclodextrin transferase

A cyclodextrin and production method technology, applied in the field of fermentation engineering, can solve the problems of high industrial application cost and low enzyme activity, and achieve the effects of fewer steps, improved production efficiency, and shorter time.

Inactive Publication Date: 2012-11-28
YUNNAN NORMAL UNIV
View PDF3 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a production process for high-efficiency extracellular expression of β-surrounding dextrin transferase to solve the problems of low enzyme activity and high cost of industrial application in the fermentation process

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for producing extracellular production and recombination beta-cyclodextrin transferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Escherichia coli E. Coli Construction of BL21(DE3)(pET28(+) / cgt): source Paenibacillus sp The β-cyclodextrin glucosyltransferase gene of XW-6-66 (Xie Zhenrong, Zhao Sanjun, Tang Xianghua, etc.. Source Paenibacillus sp . Cloning of β-cyclodextrin glucosyltransferase gene and its expression in Escherichia coli[J]. Food Science and Technology, 2010, 35(11): 16~19.) was inserted into plasmid pET28(+) to construct the expression The vector pET28(+) / cgt was transformed into host bacteria E. Coli BL21(DE3);

[0034] 2. Seed culture stage: insert the above-mentioned Escherichia coli strains preserved in -80°C glycerol into the seed medium, the seed medium is: 5g / L yeast powder, 10g / L peptone, 10g / L NaCl, kanamycin 0.05 mg / ml, pH 7.0, cultivated for 12 hours using a constant temperature incubation shaker, the rotation speed was 150 rpm, and the temperature was 37 °C;

[0035] 3. Fermentation stage: inoculate the strain into 200ml liquid fermentation medium according t...

Embodiment 2

[0038] 1 strain: Escherichia coli E. Coli BL21(DE3)(pET28(+) / cgt);

[0039] 2. Seed culture stage: insert the above-mentioned E. coli strains stored in -80°C glycerol into the seed medium, seed medium: 5.5g / L yeast powder, 10.5g / L peptone, 10.5g / L NaCl, kanamycin 0.05mg / ml, pH 7.2, cultivated for 12 hours using a constant temperature incubation shaker, the rotation speed was 150rpm, and the temperature was 37°C;

[0040] 3 Fermentation stage: inoculate the strain into 200ml liquid fermentation medium according to the inoculation amount of 2%, fermentation medium: 5.5g / L yeast powder, 10.5g / L peptone, 10.5g / L NaCl, 1.2mmol / L CaCl 2 , 2.25mmol / L MgSO 4 .7H2 O, kanamycin 0.05mg / ml, pH 7.2, placed in a constant temperature shaker for fermentation and culture, the temperature was controlled at 35°C, the rotation speed was controlled at 150rpm, and cultured for 8 hours;

[0041] 4. Induction stage: when the bacteria OD 600 When it reaches 1.4, add lactose to induce, ensure the ...

Embodiment 3

[0043] 1. Strains: Escherichia coli E. Coli BL21(DE3)(pET28(+) / cgt);

[0044] 2. Seed culture stage: Inoculate the above-mentioned Escherichia coli strains preserved in glycerol at -80°C into the seed medium. The seed culture is: 5g / L yeast powder, 10g / L peptone, 10g / L NaCl, 0.05mg kanamycin / ml, pH7.1, cultivated on a constant temperature culture shaker for 12 hours, with a rotation speed of 150rpm and a temperature of 37°C;

[0045] 3. Fermentation stage: Inoculate the strain into 200ml liquid fermentation medium according to the inoculation amount of 2%. The fermentation medium is: 5g / L yeast powder, 10g / L peptone, 10g / L NaCl, 1.2mmol / L CaCl 2 , 2.5mmol / L MgSO 4 .7H 2 O, kanamycin 0.05mg / ml, pH 7.1, placed in a constant temperature shaker for fermentation and culture, the temperature was controlled at 36°C, the rotation speed was controlled at 150rpm, and cultured for 7 hours;

[0046] 4. Induction stage: when the bacteria OD 600 When it reaches 1.6, add lactose to in...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a method for producing extracellular production and recombination beta-cyclodextrin transferase. Escherichia coli E.ColiBL21 (DE3) (Pet28 (plus) / cgt) is taken as an original strain; Escherichia coli E.ColiBL21 (DE3) (Pet28 (plus) / cgt) is cultured by a constant temperature shaking table for 12 hours during a seed culture period; during a fermentation stage, the strain is inoculated to 200 milliliters of liquid fermentation medium according to the inoculum size of 1-2%, and is put in the constant temperature shaking table to be fermented and cultured for 6-8 hours; and when the strain optical density (OD600) reaches 1.4-1.6, lactose is added to induce for 8-9 hours and then filtering is carried out, and the obtained liquid is crude liquid beta-cyclodextrin glucosyl transferase. The total fermentation period takes about 18 hours, and the cyclase activity of the extracellular beta-cyclodextrin transferase reaches above 7 U / ml. The method adopts two stages of control technologies, so the beta-cyclodextrin glucosyl transferase is effectively expressed outside the recombination escherichia coli. Compared with the existing technology, the method has few steps and short time, is suitable for mass production and has effective extracellular expression, and the production efficiency is greatly improved.

Description

technical field [0001] [0002] The invention belongs to the technical field of fermentation engineering, in particular to a production method for producing β-cyclodextrin glucosyltransferase (β-CGTase) by culturing recombinant Escherichia coli. Background technique [0003] Cyclodextrin glucosyltransferase (CGTase for short) is an extracellular enzyme that can convert starch and related substrates into cyclodextrins (CDs) through intramolecular transfer of glycosyl reaction. According to the main products generated, it can be roughly divided into α-, β-, γ- three types. Cyclodextrins are generally classified as α-, β-, γ-, δ-, ε-, ζ- and η-CD, which are composed of 6, 7, 8, 9, 10, 11 and 12 glucose units, respectively. The most studied and widely used are α-, β-, γ-CD. β-CD is currently the most widely used, which is due to the moderate pore size and stable properties of β-CD. β-Cyclodextrin is a cylindrical structure with internal hydrophobicity and external hydrophili...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N9/10C12R1/19
Inventor 黄遵锡宋拓李俊俊唐湘华许波杨云娟周峻沛
Owner YUNNAN NORMAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com