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A kind of chondroitin synthase mutant and its application

A chondroitin and mutant technology, applied in the field of bioengineering, can solve problems such as the inability to meet food medical safety requirements, and achieve the effects of increased yield and large application advantages

Active Publication Date: 2019-08-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the pathogenicity of the above-mentioned strains and the production of endotoxin and pyrogen substances by Escherichia coli, they cannot meet the current food medical safety requirements.

Method used

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  • A kind of chondroitin synthase mutant and its application
  • A kind of chondroitin synthase mutant and its application
  • A kind of chondroitin synthase mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Mutation site selection and mutant library construction of Escherichia coli K4 chondroitin synthase

[0035] By comparing the amino acid sequence of chondroitin synthase derived from Escherichia coli K4 with the amino acid sequence of chondroitin synthase derived from other sources, the following 6 relatively conserved motifs were found, namely L 238 DCDMAP 244 ,D 461 LEVC 465 ,D 231 GS-D 235 , G 516 QLDSDD 522 , P 526 DAVE 530 , N 602 AVDYD 607 . The non-conserved amino acids near these 6 motifs are designed to be mutated at the same time, while the conserved sites remain unchanged, that is, four regions of A are selected 236 -P 246 ,D 461 -D 473 , G 516 -E 530 and I 600 -Y 609 Make combinatorial mutations. Using the "RECODE" mutation strategy, design 4 degenerate primers, in which the stop codon should be avoided when the triplet codon is mutated at the same time, such as using "VNN; V = A, C, G; N = A, T, C, G ", using the constructed pA...

Embodiment 2

[0049] Example 2 High-throughput screening and shake flask re-screening of recombinant Bacillus subtilis mutant library

[0050] Pick 1000 mutant strains of Bacillus subtilis obtained above, inoculate a single colony in LB medium in a 96-well plate, and culture overnight at 200 rpm at 37°C. Transplant in the fermentation medium of 96 deep-well plate by 10% inoculum amount, and add final concentration 2% xylose to induce in the 2nd hour of fermentation, 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.

[0051] For the positive mutants obtained by high-throughput screening, shake flask re-screening was carried out, and the original strain B. subtilis ECH was used as a control, and the single clone was inoculated i...

Embodiment 3

[0056] Embodiment 3 produces the 3L tank fermentation of chondroitin-producing bacillus subtilis mutant strain

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Abstract

The invention discloses a chondroitin synthase mutant and application thereof, and belongs to the technical field of biological engineering technology. Chondroitin synthase from the K4 source of escherichia coli is subjected to combination mutation of non-conservative amino acids in a conservative region in bacillus subtilis, and the mutant with the improved chondroitin yield and reduced molecular weight is obtained. Meanwhile, amplification fermentation is performed through a 3L fermentation tank, chondroitin with high bacillus subtilis yield and low molecular weight is obtained, the yield of products on the 3L fermentation tank is 7.25 g / L, and the average molecular weight of the product is 41.62 kDa. The certain foundation is laid for efficiently producing and preparing chondroitin from food grade microorganisms, and the mutant is suitable for industrial production and application.

Description

technical field [0001] The invention relates to a chondroitin synthase mutant and application thereof, belonging to the technical field of bioengineering. Background technique [0002] Chondroitin is a family of polysaccharides of glycosaminoglycans (GAGs). Glycosaminoglycans are a class of unbranched, negatively charged polysaccharide chains composed of repeating disaccharide units. Due to their unique inflexibility and high negative charge, GAGs exhibit a highly extended conformation, occupying Large spaces that absorb cations and water and form porous gels in the extracellular matrix, GAGs, found in most animals, help hydrate and expand tissues and enable the matrix to withstand compressive forces. Glycosaminoglycans thus constitute a class of compounds with great potential for therapeutic applications. Chondroitin is a kind of 4-GlcA-β-1,3-GalNAc-β-1 (GlcUA: glucuronic acid, GalNAc: acetylgalactosamine) disaccharide monomers connected alternately by β-1,3 bonds. into ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/75C12N1/21C12P19/26C12R1/125
Inventor 康振陈坚堵国成张琳培周正雄
Owner JIANGNAN UNIV