Preparation and application of cyclodextrin glycosyltransferase mutant

A glucosyl and cyclodextrin technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low AA-2G conversion rate and low enzyme yield, and achieve the effects of high yield and simple purification

Active Publication Date: 2018-05-15
JIANGNAN UNIV
View PDF1 Cites 12 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, there are many studies on the genetic modification and high-efficiency expression of cyclodextrin glucosyltransferase, but there are still big

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
  • Preparation and application of cyclodextrin glycosyltransferase mutant
  • Preparation and application of cyclodextrin glycosyltransferase mutant
  • Preparation and application of cyclodextrin glycosyltransferase mutant

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0037] Example 1: Preparation of mutants of cyclodextrin glucosyltransferase

[0038] (1) Preparation of mutants

[0039] According to the gene sequence of the cyclodextrin glucosyltransferase shown in the amino acid sequence position SEQ ID NO.1, primers were designed and synthesized to introduce K124E, G450S, I465F, I641T, K647E, and I631T mutations to transfer cyclodextrin glucosyl The enzyme gene undergoes site-directed mutation, and the DNA coding sequence is determined. The 124th Lys codon is changed to Glu codon, the 450th Gly codon is mutated to Ser codon, and the 465th Ile codon is mutated to Phe. The 641st Ile codon becomes a Thr codon, the 647th Lys codon becomes a Glu codon, and the 631th Ile codon becomes a Thr codon. The mutant gene is placed in an appropriate expression vector and introduced into Escherichia coli for expression to obtain cyclodextrin glucosyltransferase.

[0040] Site-directed mutagenesis of mutants K124E, G450S, I465F, I641T, K647E, I631T: Using rap...

Example Embodiment

[0062] Example 2 Analysis of single mutant cyclodextrin glucosyltransferase activity and soluble expression

[0063] (1) Expression of mutant enzyme

[0064] Pick the positive clones transferred into the expression host Escherichia coli BL21(DE3) and grow them in LB liquid medium (containing 100μg / mL ampicillin) for 8-10h, and then connect the seed fermentation broth to TB medium (containing 100μg / mL ampicillin), after culturing in a shaker at 25°C for 60h, the fermentation broth was centrifuged at 4°C and 8000rpm for 10min to remove bacteria, and the centrifuge supernatant was collected. The wild cgt / pET20b(+) / E.coli BL21(DE3) fermented supernatant was used as the wild enzyme control.

[0065] The enzyme activities of wild-type cyclodextrin glucosyltransferase and mutant enzyme cultured for 60 hours are listed in the table. I641T and K647E have the most significant effects, and the enzyme activities are 2.5 times and 2.4 times that of the wild enzyme, respectively.

[0066] Table 1...

Example Embodiment

[0070] Example 3: Preparation and expression of double mutants of cyclodextrin glucosyltransferase

[0071] (1) Preparation of mutants

[0072] Combine the above three sites with mutations and design double mutations to obtain mutants I641T / K647E, I641T / I631T, I631T / K647E. The preparation method of double mutant enzymes is based on the single mutant enzymes I641T, I631T, K647E The gene sequence is used as a template, and primers are designed and synthesized to introduce double mutations, the sequence is determined, the mutant gene is placed in an appropriate expression vector and introduced into Escherichia coli for expression to obtain cyclodextrin glucosyltransferase. Site-directed mutagenesis of mutants I641T / K647E, I641T / I631T, I631T / K647E: Using rapid PCR technology, using the expression vector cgt / pET20b(+) as a template.

[0073] The site-directed mutagenesis primers for introducing the K647E mutation are:

[0074] Forward primer: 5’-CGAGTTTAAATTCATC GAA AAAGACTCTCAGGGC-3' (m...

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 preparation and application of a cyclodextrin glycosyltransferase mutant, and belongs to the field of gene engineering and enzyme engineering. The amino acid of cyclodextrin glycosyltransferase is subjected to mutation; the enzyme activity of the obtained mutant can reach 2.5 times of that of wild enzyme; the purification of the obtained cyclodextrin glycosyltransferase mutant is simple; the industrial production can be realized.

Description

technical field [0001] The invention relates to the preparation and application of a cyclodextrin glucosyltransferase mutant, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Vitamin C (VC) is a water-soluble vitamin that cannot be synthesized by the human body itself. It participates in many physiological activities in the body, such as promoting the conversion of cholesterol into bile acids, promoting the synthesis of adrenal cortex hormones, participating in the metabolism of aromatic amino acids, promoting iron absorption and Participate in various redox reactions in the body and play an important role in maintaining and promoting human health. In addition, vitamin C can also promote the synthesis of collagen, restore the formed melanin, and play a certain role in maintaining skin elasticity, whitening, and wrinkle removal. Widely used in food, medicine, cosmetics and other fields. However, the hydroxyl group of the 2...

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): C12N9/10C12N15/70C12P19/60
CPCC12N9/1074C12N15/70C12P19/60C12Y204/01019
Inventor 吴敬宿玲恰陶秀梅王昱升董俊辰
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