A mutant of hyaluronan synthase and its application

A hyaluronan synthase and mutant technology, which is applied in the field of bioengineering, can solve the problems of reducing the molecular weight of HA, affecting the HA chain retention effect of hyaluronan synthase, and unstable pores, and achieves the effect of increasing yield

Active Publication Date: 2019-03-19
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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  • A mutant of hyaluronan synthase and its application
  • A mutant of hyaluronan synthase and its application
  • A mutant of hyaluronan synthase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of integrated recombinant plasmid pGZA

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

[0040] According to the pP43NMK plasmid map, primers P43-F and P43-RBS-R were designed respectively, and the pP43NMK plasmid was used as a template to amplify and obtain the promoter P 43 and RBS.

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

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

[0043] Has A-F: GGTAAGAGAGGAATGTACACATGAGAACATTAA...

Embodiment 2

[0051] Example 2 Mutation site selection and mutant library construction of Streptococcus zooepidemicus hyaluronan synthase

[0052] attached figure 1 Among them, the topological structure of hyaluronan synthase derived from Streptococcus zooepidemicus is predicted based on the structure of hyaluronan synthase derived from Streptococcus pyogenes and the homology between the two, and it is deduced that F 58 -N 208 and Y 233 -P 318 It is the intracellular domain of hyaluronan synthase of Streptococcus zooepidemicus, that is, two macrocyclic structures in the cell, and has substrate binding and catalytic activity. At the same time, after BLAST comparison 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 . The amino acids near these five motifs are designed to be mutated at the same time, while the conserved sites remain...

Embodiment 3

[0065] Example 3 High Throughput Screening and Shake Flask Rescreening of Recombinant Bacillus subtilis Mutant Library

[0066]The 1500 mutant strains of Bacillus subtilis obtained above were picked, and a single colony was inoculated in the LB medium of a 96-well plate, and cultured overnight at 200 rpm at 37°C. Transplant in the fermentation medium of 96 deep-well plates according to 10% inoculation amount, 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 volume of the well, and a well-permeable plate cover should be used to incubate at 200 rpm at 37°C for 48 hours.

[0067] For the collection of 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 times the volume of ...

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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, belonging to the technical field of bioengineering. Background technique [0002] Hyaluronic acid (abbreviated as HA) is a linear polymeric acidic mucopolysaccharide with β-D-acetylglucosamine (GlcNAc) and β-D-glucuronic acid (GlcUA) as disaccharide repeating units, and is easily soluble in water. , has good moisture retention, viscoelasticity, permeability and extensibility, can lubricate joints, regulate the permeability of blood vessel walls, regulate the diffusion and operation of electrolytes, water and proteins, and effectively promote wound healing. Because of its unique physical and chemical properties, hyaluronic acid is widely used in food, medicine and daily chemical fields. [0003] At present, 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 Str...

Claims

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Application Information

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Patent Type & Authority Patents(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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