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

N-glycosylation algin lyase mutant capable of being efficiently applied and genetic engineering bacterium construction method

A technology of alginate lyase and genetically engineered bacteria, which is applied in the directions of lyase, introduction of foreign genetic material using a vector, recombinant DNA technology, etc., can solve the problems of low expression level and low enzyme activity, and achieves the effect of improving temperature stability

Active Publication Date: 2019-06-07
JIANGNAN UNIV
View PDF3 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still problems such as low expression level and low enzyme activity

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
  • N-glycosylation algin lyase mutant capable of being efficiently applied and genetic engineering bacterium construction method
  • N-glycosylation algin lyase mutant capable of being efficiently applied and genetic engineering bacterium construction method
  • N-glycosylation algin lyase mutant capable of being efficiently applied and genetic engineering bacterium construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Construction of mutant enzyme NACob1 and engineering bacteria introduced with N-glycosylation site

[0025] The present invention introduces an N-glycosylation site 1×DQNAT into the N-terminal of the alginate lyase Aly-Cob, and expresses it by fusion with the carrier protein YebF. After co-expression with the glycosylated pglB plasmid, N-glycosyl is produced Alginate lyase NACob1 (amino acid sequence such as SEQ ID NO.1) modified and transformed.

[0026] The specific method is as follows:

[0027] (1) Digest the plasmid pTrc99A with XbaI and HindIII, introduce the coding glycosylation site DQNAT and 6×His gene sequence, construct the recombinant plasmid, insert the YebF (NCBI: B1847) gene after double digestion with XbaI and SalI Sequence, form pTrc-YebF-gly-His plasmid, after XbaI and EcoR I double digestion, insert Aly-Cob coding gene, form recombinant vector pTrc-YebF-gly-Aly-Cob-His;

[0028] (2) Transform the constructed expression plasmid and the pAC...

Embodiment 2

[0030] Example 2: Construction of mutant enzyme NACob4 and engineering bacteria introduced into N-glycosylation site

[0031] The method is similar to Example 1, except that the glycosylation site is 4×DQNAT, and the obtained mutant enzyme is named NACob4 (amino acid sequence such as SEQ ID NO.2).

Embodiment 3

[0032] Example 3: Comparison of enzyme activity before and after glycosylation modification

[0033] The activity of alginate lyase in the fermentation supernatant was determined by DNS method (Table 1). After glycosylation modification, the activity of alginate lyase was slightly improved, the enzymatic activity NACob4>NACob>Aly-Cob, and the enzymatic activity of NACob4 increased by 12.6%.

[0034] Table 1 Enzyme activity of alginate lyase before and after glycosylation modification

[0035]

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 discloses a N-glycosylation algin lyase mutant capable of being efficiently applied and a genetic engineering bacterium construction method, and belongs to the technical field of biology. Based on algin lyase Aly-Cob, a DQNAT repeated sequence fragment is introduced at an N- terminal to be used as an N-glycosylation site, besides, expression carrier protein YebF is fused to be subjected to exocytosis, and further a glycosylation system pgl from campylobacter jejuni performs glycosylation modification through a sugar chain GalNAc2(Glc)GalNAc3Bac. The enzyme activity and the stability of an obtained mutant enzyme NACob are improved than those of an enzyme before glycosylation. The N-glycosylation modified algin lyase mutant provided by the invention can be widely applied to thefields of foods, medicines, agriculture and the like relevant to seaweed processing and utilizing.

Description

technical field [0001] The invention relates to a glycosylation-modified marine alginic acid lyase and an application thereof, belonging to the field of biotechnology. The alginate lyase provided by the invention can be widely used in the fields of food, medicine, agriculture and the like in seaweed processing and utilization. Background technique [0002] Fucoidan oligosaccharides are a kind of high value-added oligosaccharides produced by the degradation of alginate, which have various biological activities such as anti-tumor, immune regulation, hypoglycemic and blood lipids, etc., and have important applications in food, medicine, feed and other fields. The process of enzymatically hydrolyzing alginate oligosaccharides with alginate lyase as a tool has the advantages of mild reaction conditions, process control, and high yield, and has gradually become the main method for preparing alginate oligosaccharides. [0003] At present, various types of microorganisms producing ...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/70C12P19/04C12P19/00
CPCY02A50/30
Inventor 李恒史劲松许正宏龚劲松郝瑶李星霖
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