Hyaluronan synthase mutant and application thereof

A hyaluronic acid synthase and mutant technology, applied in the field of bioengineering, can solve the problems of HA molecular weight reduction, pore instability, affecting hyaluronic acid synthase HA chain retention, etc., and achieve the effect of increasing production

Active Publication Date: 2016-08-10
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the other two polar amino acids Lys in the cell 48 and Glu 327 Mutations lead to a decrease in the molecular weight of HA, indicating that these two sites can interact to form macromolecular HA. If they are destroyed, the channel formed by the synthase for transporting HA will be unstable, thereby affecting the production of hyaluronic acid synthase. Retention of HA chains

Method used

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  • Hyaluronan synthase mutant and application thereof
  • Hyaluronan synthase mutant and application thereof
  • Hyaluronan synthase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of integrated recombinant plasmid pGZA

[0039] Amplify and obtain the target gene shown in SEQ ID NO.1.

[0040] According to the plasmid map of pP43NMK, the primers P43-F and P43-RBS-R were designed respectively. Using the pP43NMK plasmid as the template, the standard PCR amplification system and procedures were used to amplify the promoter P 43 And RBS.

[0041] According to the pSKIZH plasmid information (Production of specific-molecular-weight hyaluronan by metabolicallyengineered Bacillus subtilis 168, Metabolic Engineering, 2016, Jinpeng), the primers pGZ-F and pGZ-R were designed respectively, and the pSKIZH plasmid was used as the template and standard PCR amplification System and procedures to amplify the vector pGZ, which is a T-carrier derivative with upstream and downstream homology arms (530bp) integrated into the nagA site on Bacillus subtilis 168 and lox71-Zeo-lox66 resistance marker.

[0042] Primer sequence information: 5’-3’ direction

...

Embodiment 2

[0051] Example 2 Selection of mutation sites of hyaluronic acid synthase of Streptococcus zooepidemicus and construction of mutant library

[0052] Attached figure 1 Among them, the topological structure of hyaluronic acid synthase derived from Streptococcus zooepidemicus is predicted based on the structure of hyaluronic acid synthase derived from Streptococcus pyogenes and the homology between the two. F 58 -N 208 And Y 233 -P 318 It is the intracellular domain of Streptococcus zooepidemicus hyaluronic acid synthase, that is, two intracellular macrocyclic structures, and has substrate binding and catalytic activity. At the same time, after BLAST alignment of amino acid sequences, five conserved motifs located in the intracellular domain were found, namely V 158 DSDT 162 , L 198 TRL 201 , S 227 GPL-YRR 235 , G 258 DDR-LTN 265 , L 293 -QQ-RW-KS-FRE 306 . Design the amino acids near these 5 motifs to be mutated at the same time, while the conservative site remains unchanged, that is...

Embodiment 3

[0065] Example 3 High-throughput screening and shake flask rescreening of recombinant Bacillus subtilis mutant library

[0066] The 1500 Bacillus subtilis mutant strains obtained above were picked, and a single colony was inoculated on a 96 shallow-well plate of LB medium, and cultured at 200 rpm and 37°C overnight. Transfer 10% of the inoculum to the fermentation medium of 96 deep-well plates. The fermentation medium is: 20g / L yeast powder, 50g / L sucrose, potassium sulfate 3.9g / L, magnesium sulfate 1.5g / L, 50mM Phosphate buffer, pH 7.0. The amount of liquid in each well should not exceed 1 / 3 of the well volume, and the plate cover with good air permeability should be used and incubated at 200rpm 37℃ for 48h.

[0067] To collect hyaluronic acid in the fermentation broth, first add 1 volume of 0.1% (w / v) SDS solution, let it stand for 10 minutes, and centrifuge at 10000×g for 10 minutes at room temperature to remove cells. Add 2 volumes of absolute ethanol to the supernatant and m...

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Abstract

The invention discloses a hyaluronan synthase mutant and application thereof and belongs to the technical field of bioengineering. The mutant with positive effect in both yield and molecular weight is acquired by subjecting hyaluronan synthase of Streptococcus zooepidemicus origin to intracellular domain mutation in Bacillus subtilis; meanwhile, genes of UDP-GlcUA and UDP-GlcNAc synthetic routes in Bacillus subtilis are subjected to modular assembly expression analysis, and high yield of hyaluronan with wide molecular weight range from Bacillus subtilis is implemented by controlling different UDP-GlcA and UDP-GlcNAc concentrations and ratios. Certain basis is laid for the efficient preparation of hyaluronan from food-grade microorganisms, and this mutant is suitable for industrial production and application.

Description

Technical field [0001] The invention relates to a hyaluronic acid synthase mutant and its application, and belongs to the technical field of bioengineering. Background technique [0002] Hyaluronic acid (HA for short) is a linear polymer acid mucopolysaccharide with β-D-acetylglucosamine (GlcNAc) and β-D-glucuronic acid (GlcUA) as disaccharide repeating units. It is easily soluble in water It has good moisture retention, viscoelasticity, permeability and ductility, can lubricate joints, adjust the permeability of blood vessel walls, regulate the diffusion and operation of electrolytes, water and protein, and effectively promote wound healing. Due to its unique physical and chemical properties, hyaluronic acid is widely used in food, medicine and daily chemical fields. [0003] Currently, the production method of hyaluronic acid is mainly animal extraction, but this method has low yield, poor separation and high cost. In addition, the fermentation of group A and group C microorgan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N1/21C12P19/26C12R1/125
CPCC12N9/1051C12P19/26C12Y204/01212
Inventor 康振陈坚堵国成张琳培黄浩
Owner JIANGNAN UNIV
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