Chondroitinase ABC producing recombinant yeast strain and structuring and applying methods thereof

A chondroitin sulfate, construction method technology, applied in the field of recombinant yeast producing chondroitin sulfate ABC lyase and its construction, can solve the problems of increased production cost, low ChSaseABC enzyme activity, etc., and achieves resource saving, production cost reduction, Avoid the effects of changing the physical properties of the product

Active Publication Date: 2019-07-09
CHANGSHU INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] To sum up, the main problems in the fermentation production of ChSase ABC in China are: the enzyme activity of ChSase ABC produced by fermentation is relatively low, and most of them are expressed intracellularly, and the crushing process will definitely increase the production cost

Method used

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  • Chondroitinase ABC producing recombinant yeast strain and structuring and applying methods thereof
  • Chondroitinase ABC producing recombinant yeast strain and structuring and applying methods thereof
  • Chondroitinase ABC producing recombinant yeast strain and structuring and applying methods thereof

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Experimental program
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Effect test

Embodiment 1

[0065] Embodiment 1: Construction of recombinant Pichia pastoris GS115

[0066] Amplification of the chondroitin sulfate ABC lyase gene: the genome of Proteus vulgaris ATCC33420 was used as a template to amplify the chondroitin sulfate ABC lyase gene fragment. The PCR amplification system was 1 μL of genomic DNA, 4 μL each of primer 1 and primer 2, 50 μL of KOD polymerase, ddH 2 O 41 μL, PCR reaction program: 94°C pre-denaturation for 4 min, 94°C denaturation for 2 min; then annealing at 60°C for 30 s, extension at 72°C for 3 min, cycle 35 times; extension at 72°C for 1 min. The PCR product was subjected to gel electrophoresis and then gel-cut, and recovered with a column-type gel-tapping recovery kit to obtain a purified chondroitin sulfate ABC lyase gene fragment.

[0067] Table 1 Primers required for the construction of recombinant Pichia pastoris GS115

[0068]

[0069] Double digestion of chondroitin sulfate ABC lyase gene: the double digestion system is 10×QuickCut ...

Embodiment 2

[0073] Embodiment 2: the induced expression of chondroitin sulfate ABC lyase (see attached figure 2 )

[0074] The constructed recombinant Pichia pastoris GS115-pPIC9K-csl ABC was inoculated in YPD liquid medium and cultured overnight at 28°C; then transferred to 1L fermentation medium with 4% inoculum size, and fermented to OD 660 At 5 o'clock, centrifuge the fermentation broth under sterile conditions, discard the fermentation supernatant, resuspend the recombinant Pichia pastoris GS115-pPIC9K-csl ABC cell enzyme production medium, add 10g / L methanol (supplemented every day) to induce sulfate cartilage Secreted expression of ABC lyase, after 5 days of culture, the enzyme activity in the fermentation broth can reach 8U / mL.

Embodiment 3

[0075] Embodiment 3: the chondroitin sulfate ABC lyase enzyme activity assay of fermented liquid

[0076] Take 1mL of fermentation broth and centrifuge, take 0.1mL supernatant and 7.9mL 1g / L chondroitin sulfate A (prepared with 0.02mol / LTris-HCL, pH7.5) respectively, add them to a 15mL colorimetric tube, place at 28 React in a water bath at ℃ for 20 minutes, immediately place it in a boiling water bath and boil for 5 minutes, add inactivated fermentation broth supernatant to the control tube under the same conditions, and measure the light absorption value at 232 nm. The enzyme activity unit U is defined as the amount of enzyme required to catalyze the formation of 1 μmol of unsaturated disaccharide per minute at 28°C.

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Abstract

The invention discloses a chondroitinase ABC producing recombinant yeast strain and application thereof and belongs to the technical field of bioengineering. According to the chondroitinase ABC producing recombinant yeast strain, chondroitinase ABC from Proteus vulgaris ATCC33420 is subjected to heterogeneous expression, and with pPIC9K carriers and through induction of methanol, secretory expression of the chondroitinase ABC in pichia pastoris GS115 can be achieve; by means of a designed chondroitinase ABC-enzymatic membrane reactor, efficient and continuous production of small-molecular chondroitin sulfate can be achieved, the chondroitinase ABC can be recycled and reutilized, and the production cost can be greatly reduced. By taking food-grade yeast as host strains, the chondroitinase ABC producing recombinant yeast strain can be safe and reliable, provide effective references for industrialized and green production of small-molecular chondroitin sulfate A, B and C, and meanwhile, save energy, reduce emission and achieve significant economic and social benefits.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant yeast producing chondroitin sulfate ABC lyase, a construction method and application thereof. Background technique [0002] Chondroitin Sulfate Sulfate salt of a copolymer of D-glucuronic acid and N-acetylgalactosamine, the six-carbon sugar in the copolymer passes through β-(1,4) and β-(1,3) glycosides The bonds are alternately connected, generally containing about 50-70 disaccharide units, and the molecular weight is between 10kDa and 50kDa. As a medical drug, chondroitin sulfate has the effects of lowering blood fat, antithrombotic, antitumor, treating arthritis, arteriosclerosis, cardiovascular and cerebrovascular diseases, hearing impairment, nephritis, hepatitis and neuralgia, and can also be used as eye drops; As a food additive, chondroitin sulfate can be used for emulsification, moisturizing and odor removal of food. [0003] [0004] Table 1 Ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/60C12N15/81C12P19/26C12M1/40C12M1/34C12M1/02C12M1/00C12R1/84
CPCC12M21/18C12M27/02C12M29/04C12M41/12C12N9/88C12N15/815C12P19/26C12Y402/02004
Inventor 吴凌天郗栋南赖淑涵朱益波刘欣雨霍薪宇刘学润
Owner CHANGSHU INSTITUTE OF TECHNOLOGY
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