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

Mutation method for enhancing beta-cyclodextrin production capacity of beta-cyclodextrin glycosyltransferase

A glucosyl and cyclodextrin technology, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of inconvenient separation and purification of a single type of cyclodextrin product, and achieve the effect of improving specificity and facilitating industrial production.

Inactive Publication Date: 2014-02-05
JIANGNAN UNIV
View PDF7 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the wild CGTase acts on starch, the products obtained are all mixtures of α-, β- and γ-cyclodextrins, which brings a lot of inconvenience to the separation and purification of a single type of cyclodextrin products

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
  • Mutation method for enhancing beta-cyclodextrin production capacity of beta-cyclodextrin glycosyltransferase
  • Mutation method for enhancing beta-cyclodextrin production capacity of beta-cyclodextrin glycosyltransferase
  • Mutation method for enhancing beta-cyclodextrin production capacity of beta-cyclodextrin glycosyltransferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: This example illustrates the preparation of mutants A31R, A31P and A31T.

[0029] (1) Site-directed mutation

[0030] Using rapid PCR technology, site-directed mutagenesis was carried out with the expression vector pST / cgt containing the wild CGTase gene as a template.

[0031] Primers for site-directed mutagenesis introducing the Arg31 codon:

[0032] Forward primer: 5'-GACGGCAATCCC CGC AACAATCC-3', the underline is the mutated base,

[0033] Reverse primer: 5'-GGATTGTT GCG GGGATTGCCGTC-3', the underline is the mutant base;

[0034] Primers for site-directed mutagenesis introducing the Pro31 codon:

[0035] Forward primer: 5'-GACGGCAATCCC CCC AACAATCC-3', the underline is the mutated base,

[0036] Reverse primer: 5'-GGATTGTT GGG GGGATTGCCGTC-3', the underline is the mutant base;

[0037] Primers for site-directed mutagenesis introducing the Thr31 codon:

[0038] Forward primer: 5'-GACGGCAATCCC ACC AACAATCC-3', the underline is the mutated bas...

Embodiment 2

[0046] Example 2: This example illustrates an enzyme activity assay.

[0047] (1) Determination of enzyme activity

[0048] Determination of α-cyclization activity: 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 phosphate buffer (pH 6.0). After reacting at 50°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 solution and incubate at 20°C for 15min, and measure the absorbance at 505nm. Using the inactivated enzyme as a blank, the content of α-cyclodextrin was determined corresponding to the α-cyclodextrin standard curve. One enzyme activity unit is defined as the amount of enzyme required to generate 1 μmol of cyclodextrin per minute under the above conditions.

[0049] Determination of β-cyclization activity: Take 0.1 mL of appropriately diluted enzyme solut...

Embodiment 3

[0055] Example 3: This example illustrates the use of HPLC to analyze the amount of cyclodextrin produced

[0056] 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 A31R, A31P, and A31T 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 Add 5 μL of glucoamylase (70 U / mL) to 500 μL of supernatant, saccharify at 30°C for 1 hour, boil for 10 minutes to inactivate, centrifuge at 12,000 rpm for 30 minutes, and take 20 μL of the supernatant for HPLC analysis after filtering through a 0.45 μm ultrafiltration membrane.

[0057] HPLC determination conditions are: Waters600 high performance liquid chromatograph (with differenti...

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 mutation method for enhancing beta-cyclodextrin production capacity of beta-cyclodextrin glycosyltransferase (short for beta-CGT enzyme), and belongs to the field of gene engineering and enzyme engineering. According to the present invention, a site-specific mutagenesis method is adopted to increase beta-cyclodextrin production capacity of the CGT enzyme, the mutation scheme for enhancing beta-cyclodextrin production capacity of the beta-CGT enzyme derived from Bacillus circulans STB01 is provided, mutation of alanine on the site 31 in the CGT enzyme into arginine (Arg), proline (Pro) or threonine (Thr) is performed to obtain mutants A31R, A31P and A31T, beta-cyclodextrin production capacity of the mutant is significantly enhanced compared with beta-cyclodextrin production capacity of the wild CGT enzyme, and the mutation method is more suitable for beta-cyclodextrin industrial production.

Description

technical field [0001] The invention relates to a mutation method for enhancing the ability of beta-cyclodextrin glucosyltransferase to produce beta-cyclodextrin, which belongs to the fields of genetic engineering and enzyme engineering. The invention is a technique for enhancing product specificity of cyclodextrin glucosyltransferase by site-directed mutation method. Background technique [0002] Cyclodextrin is composed of D-glucopyranose connected by α-1,4-glycosidic bonds, and has a ring-shaped hollow conical structure, in which α-, β- and Gamma-cyclodextrin is the most common. Due to its external hydrophilic and internal hydrophobic properties, cyclodextrin can form inclusion complexes with many hydrophobic guest molecules, thereby changing the physical and chemical properties of the guest molecules. Therefore, it is widely used in food, medicine and other industrial fields. [0003] The industrial production of cyclodextrin adopts enzymatic process, that is, it is sy...

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): C12N9/10C12N15/54C12N15/10C12N15/75C12R1/09C12R1/125
CPCC12N9/1074C12N15/70C12Y204/01019
Inventor 顾正彪李兆丰班宵逢程力洪雁李才明
Owner JIANGNAN 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