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

Chitosan deacetylase combined mutant with optimal pH reduction and application

A technology of deacetylase and chitobiose, which is applied in the field of bioengineering, can solve the problems of poor stability of the product GlcN, reduce the optimal pH of enzyme catalysis, etc., and achieve the effect of improving conversion rate and enzyme activity

Pending Publication Date: 2021-09-07
JIANGNAN UNIV +1
View PDF0 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the above-mentioned technical problems, the present invention reduces the optimum pH of enzyme catalysis by modifying the surface charge of chitobiose deacetylase, so as to solve the problem that the optimum pH of chitobiose deacetylase is too high and the product GlcN is in production. The problem of poor stability

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
  • Chitosan deacetylase combined mutant with optimal pH reduction and application
  • Chitosan deacetylase combined mutant with optimal pH reduction and application
  • Chitosan deacetylase combined mutant with optimal pH reduction and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Site-directed mutant library construction for predicted sites

[0044] The amino acid sequence of chitobiose deacetylase (as shown in SEQ ID NO.1) derived from extreme thermophilic archaea was subjected to site-directed mutation, the mutations were specifically G74D, H152E, F168A, W232A, and the gene encoding the mutant (sequence shown in SEQ ID NO.2) was connected with the p43NMK vector to obtain the recombinant expression vector p43NMK-M14.

[0045] The surface charge of Dac wild type and the prediction of related sites are as follows figure 1 and 2 , the initial surface charge is -35, and the predicted 23 sites are Q29, N30, H86, K94, K106, N112, S121, K134, N176, T177, I181, N184, N187, S188, K215, R221, I228, K231, K246, H264, L271 and I272. Using p43NMK-M14 as a template, design site-directed mutagenesis primers, as shown in Table 1, for PCR amplification and Escherichia coli BL21 transformation.

[0046] Table 1 Primer Sequence

[0047]

[0048] ...

Embodiment 2

[0050] Embodiment 2 high-throughput screening

[0051] Pick a single colony and inoculate it in a sterilized 96-deep well plate containing liquid LB medium (600 μL per well) containing Kan antibiotics. Cultivate at 37° C., 900 rpm for 12 hours to obtain seed liquid. The next day, transfer 200 μL of the resulting seed solution to a new sterilized 96 deep-well plate equipped with a medium (600 μL per well). The medium composition of the new 96 deep-well plate is: Kan antibiotics, the final concentration of 0.05mmol / L IPTG LB liquid medium. At 37°C, culture was induced for 5-8 hours on a shaking shaker at 900 rpm. Use a 96-well plate centrifuge to centrifuge the 96-well plate of cultured bacteria at 3,500×g for 5 min, discard the supernatant, and add 100 μL of 50 g / L GlcNAc solution (solvent is PB buffer at pH 6.0-10.0) to each well. The bacteria were suspended and reacted at 40° C. on a shaking table at 900 rpm for 20 minutes. Then 50 μL of terminator (0.5 mol / L HCl) was add...

Embodiment 3

[0053] Embodiment 3 combined mutant specific enzyme activity detection

[0054] Combined mutations were performed on the 5 sites screened in Example 2. Using p43NMK-M14 as a template, the sequence is shown in SEQ ID NO.2. Design site-directed mutagenesis primers (as shown in Table 1) for PCR amplification and transformation of Escherichia coli JM109. After extracting the plasmid, it was transformed into B. subtilis WB600. The combined mutants and mutation sites are shown in Table 3.

[0055] Table 3 Combined mutants and mutation sites

[0056]

[0057]

[0058] The monoclonal transformants of the five combined mutant strains constructed were picked into 50 mL centrifuge tubes containing 5 mL liquid LB medium for seed culture, and 10 mg / mL kanamycin was added to each tube. The seed solution was cultured on a spring shaker at 37°C for 12 hours, and then transferred to a 500mL Erlenmeyer flask containing 96mL liquid TB medium with a 4% inoculum size for fermentation, an...

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

PropertyMeasurementUnit
Specific enzyme activityaaaaaaaaaa
Login to View More

Abstract

The invention relates to a chitosan deacetylase combined mutant with optimal pH reduction. The chitosan deacetylase combined mutant is prepared by the following steps: analyzing and predicting chitosan deacetylase surface charge and enzyme activity related sites through a molecular model and a computer, constructing mutants of the related sites through a site-directed mutagenesis technology, screening out single mutants with optimal pH, enzyme activity and product GlcN stability during mutation of different sites, and constructing a combined mutant, thereby obtaining the combined mutant with the optimal pH reduced to 6.0 and the obviously improved enzyme activity.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a combined mutant of chitobiose deacetylase with reduced optimum pH and its application. Background technique [0002] At present, using N-acetylglucosamine (GlcNAc) as a raw material, the reaction conditions are mild, and the research on the biocatalytic method to produce glucosamine (GlcN) has received extensive attention. Chitobiose deacetylase Dac is derived from the extreme thermophilic archaea Pyrococcus horikoshii and has good thermostability. Chitobiose deacetylase (Dac) has a high catalytic activity on acetylglucosamine monomer, and can use GlcNAc as a substrate to carry out a single-enzyme catalytic reaction to deacetylate to generate GlcN, realizing the one-step production of GlcN. [0003] At present, through molecular model construction and molecular docking prediction, a series of rational and irrational modifications have been used to modify Dac, and the ca...

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): C12N9/80C12N15/55C12N15/75C12N1/21C12P19/26C12R1/125
CPCC12N9/80C12N15/75C12P19/26
Inventor 刘龙陈坚吕雪芹卢健行堵国成李江华刘延峰张弘治黄子洋
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