Method for improving beta-cyclodextrin production capability of cyclodextrin glycosyltransferase by calcium ion binding site amino acid residue mutation

A technology of glucosyl and binding sites, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of separation and purification of unfavorable products, increase the production cost of cyclodextrin, etc., and achieve the effects of being beneficial to industrial production and improving specificity

Inactive Publication Date: 2014-04-23
JIANGNAN UNIV
View PDF2 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is known that the products of wild CGTase cyclization reactions are mixtures of α-, β- and γ-cyclodextrins, which is not conducive to the separation...

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 improving beta-cyclodextrin production capability of cyclodextrin glycosyltransferase by calcium ion binding site amino acid residue mutation
  • Method for improving beta-cyclodextrin production capability of cyclodextrin glycosyltransferase by calcium ion binding site amino acid residue mutation
  • Method for improving beta-cyclodextrin production capability of cyclodextrin glycosyltransferase by calcium ion binding site amino acid residue mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: This example specifically illustrates the preparation of mutants A315R and A315D

[0026] (1) Site-directed mutation

[0027] Using rapid PCR technology, site-directed mutagenesis was carried out with the expression vector pST / cgt containing the wild CGTase gene as a template.

[0028] The mutation primers introducing the Arg315 codon are:

[0029] Forward primer: 5'-GCAGCCGATTAC CGC CAGGTGGATG-3', the underline is the mutant base,

[0030] Reverse primer: 5'-GTCATCCACCTG GCG GTAATCGGCTG-3', the underline is the mutant base;

[0031] The mutation primers that introduce the Asp315 codon are:

[0032] Forward primer: 5'-GCAGCCGATTAC GAC CAGGTGGATG-3', the underline is the mutated base.

[0033] Reverse primer: 5'-GTCATCCACCTG GTC GTAATCGGCTG-3', the underline is the mutated base.

[0034] The PCR reaction system is: 5×PrimeSTAR Buffer (Mg 2+ Plus) 10 μL, dNTPs (2.5 mM each) 4 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, template DN...

Embodiment 2

[0040] Embodiment 2: This example specifies enzyme activity analysis

[0041] (1) Determination of enzyme activity

[0042] Method for measuring α-cyclization activity by methyl orange method: Take 0.1 mL of appropriately diluted enzyme solution, add 0.9 mL of 1% (w / v) soluble starch solution prepared in advance with 50 mM phosphate buffer (pH 6.5) After reacting at 40°C for 10min, add 1.0mL of 1.0N hydrochloric acid to stop the reaction, then add 1.0mL of 0.1mM methyl orange solution prepared with 50mM phosphate buffer, keep warm at 16°C for 20min, and measure the absorbance at 505nm . One enzyme activity unit is defined as the amount of enzyme required to generate 1 μmol α-cyclodextrin per minute under the above conditions.

[0043] The method for measuring β-cyclization activity by phenolphthalein method: take 0.1 mL of appropriately diluted enzyme solution and add it to a test tube containing 0.9 mL of 1% (w / v) soluble starch solution prepared in advance with 50 mM phospha...

Embodiment 3

[0049] Example 3: This example specifically illustrates the use of HPLC to measure the amount of cyclodextrin produced

[0050] To prepare 5% (wet basis, water content 8%, w / v) maltodextrin (DE3) solution as substrate, 5g maltodextrin (DE3) was dissolved in 90mL sodium phosphate buffer (pH6.0), fixed Make up to 100mL, boil in boiling water for 30min. Add a certain amount of wild CGTase, mutants A315R and A315D to make the enzyme activity in the reaction system 1U / mL, place it at 50°C for 9 hours, sample 600 μL at intervals, boil the enzyme for 10 minutes, centrifuge at 12000rpm for 10 minutes, and take the supernatant 500 μL, add 5 μL glucoamylase (70 U / mL), saccharify at 30°C for 1 hour, boil for 10 minutes to inactivate, centrifuge at 12000 rpm for 30 minutes, take 20 μL of the supernatant and filter it through a 0.45 μm ultrafiltration membrane for HPLC analysis.

[0051] HPLC determination conditions are: Waters600 high performance liquid chromatograph (with differential ...

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 provides a mutant of cyclodextrin glycosyltransferase (CGT enzyme for short) with capability for high-yield of beta-cyclodextrin and mutating method thereof, and belongs to the fields of gene engineering and enzyme engineering. The invention employs a site-directed mutagenesis method for improving beta-cyclodextrin production capability of CGT enzyme, and provides a mutating scheme for improving beta-cyclodextrin production capability of Bacillus circulans STB01CGT, that is alanine at the 315th position of the calcium ion binding site in CGT enzyme is changed into arginine (Arg) or aspartic acid (Asp), and the mutants A315R and A315D are obtained. Compared with the wild CGT enzyme, the two mutants have higher beta-cyclodextrin production capability, and are suitable for industrial production of beta-cyclodextrin.

Description

technical field [0001] The mutant of cyclodextrin glucosyltransferase with high ability of producing β-cyclodextrin and its method belong to the fields of genetic engineering and enzyme engineering. Specifically, the present invention uses site-directed mutation technology to improve the product specificity of cyclodextrin glucosyltransferase. Background technique [0002] Because the ring-shaped hollow conical structure of cyclodextrin has the characteristics of internal hydrophobicity and external hydrophilicity, it can form inclusion complexes with various hydrophobic substances. Therefore, it has a wide range of applications in the fields of medicine, chemical industry, food, materials and analytical chemistry. applications, of which α-, β- and γ-cyclodextrins are the most common. [0003] At present, cyclodextrin is usually synthesized by the action of cyclodextrin glucosyltransferase (CGTase for short) on starch. CGTase is a multifunctional enzyme, which can catalyze...

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): C12N9/10C12N15/75
CPCC12N9/1074C12N15/1031C12N15/75C12Y204/01019
Inventor 李兆丰顾正彪班宵逢程力洪雁
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap