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A genetic engineering strain of Bacillus subtilis and its application in the preparation of small molecule hyaluronic acid

A technology of genetically engineered bacteria and Bacillus subtilis, which is applied in genetic engineering, application, and plant gene improvement, can solve the problems of low expression, inclusion bodies, and low enzyme activity, and achieve simple operation, increased profit margins, and reduced The effect of the production cycle

Active Publication Date: 2021-07-09
南京锐博科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The technical problem to be solved in the present invention is to provide a genetically engineered bacterium producing chondroitin sulfate AC lyase and its construction method to realize the secretion and expression of chondroitin sulfate AC lyase, so as to solve the problem of chondroitin sulfate AC cleavage in the prior art Low enzyme expression, low enzyme activity, inclusion bodies, etc.

Method used

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  • A genetic engineering strain of Bacillus subtilis and its application in the preparation of small molecule hyaluronic acid
  • A genetic engineering strain of Bacillus subtilis and its application in the preparation of small molecule hyaluronic acid
  • A genetic engineering strain of Bacillus subtilis and its application in the preparation of small molecule hyaluronic acid

Examples

Experimental program
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Embodiment 1

[0082] Example 1: Construction of recombinant Bacillus subtilis.

[0083] Utilize the GeneRunner software to design primers, design primers (P1, P2 and P3, P4) according to the plasmid pMA5 gene sequence to synthesize fragments F1 and F2 respectively; design primers (P5, P6) to synthesize fragment F3 according to the pHT43 gene sequence; Sequence design primers (P7, P8) to synthesize fragment F4; design primers (P9, P10) to synthesize fragment F5 according to the gene (csl AC) of chondroitin sulfate AC lyase in the genome of P. heparinus ATCC13125.

[0084] Table 2-3 Primers required for recombinant plasmid construction

[0085] Table 2-3 Primers used for the recombinant plasma construction

[0086]

[0087] (1) Construction of shuttle plasmid pHA03

[0088] Amplification of F1 gene fragments: Add reagents according to Table 2 in a 0.2 mL Eppendorf tube. The pMA5 plasmid used as the template DNA was extracted by a quick plasmid extraction kit. The PCR cycle parameters w...

Embodiment 2

[0102] Example 2: Induced expression of chondroitin sulfate AC lyase

[0103] Inoculate the constructed recombinant Bacillus subtilis B. subtilis WB800-△spoOA-pHA03-csl AC in LB liquid medium containing kanamycin sulfate resistance, culture overnight at 37°C; then transfer with 4% inoculum into 1L of fermentation medium containing kanamycin sulfate, and ferment for 2 to 4 hours to OD 660 When it reaches 0.6, add 8g / L IPTG or lactose to induce expression for 24h, and the activity of chondroitin sulfate AC lyase in the supernatant of the fermentation broth can reach 16U / mL.

Embodiment 3

[0104] Embodiment 3: the chondroitin sulfate AC lyase activity assay of fermented liquid

[0105] 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, pH 7.5) respectively, add them to a 15mL colorimetric tube, place at 37 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 37°C.

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Abstract

The invention discloses a subtilis spore genetically engineered bacterium and its application in the preparation of small molecular hyaluronic acid, belonging to the technical field of bioengineering. The present invention performs heterologous expression of chondroitin sulfate AC lyase, selects the signal peptide amyQ, adopts self-constructed pHA03 carrier, and induces it through lactose or isopropyl-β-D-thiogalactoside (IPTG), and realizes The secretory expression of chondroitin sulfate AC lyase in Bacillus subtilis, and the efficient and continuous production of small molecule hyaluronic acid was realized through the designed chondroitin sulfate AC lyase-enzyme membrane reactor. The invention uses food-grade Bacillus subtilis as a host strain, is safe and reliable, provides an effective reference for industrialized green production of small-molecule hyaluronic acid, saves energy and reduces emissions, and has remarkable economic and social benefits.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a subtilis gene engineering bacterium producing chondroitin sulfate AC lyase and its application in the preparation of small molecule hyaluronic acid. Background technique [0002] Hyaluronic acid (Hyaluronic acid, HA), also known as hyaluronic acid, is a highly viscous substance first isolated from the bovine vitreous by Meyer and Palmer in 1934. Hyaluronic acid is a linear polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine linked by β-(1,3) glycosidic bonds; each disaccharide The unit is connected to another disaccharide unit through a β-(1,4) glycosidic bond; there can be as many as 25,000 disaccharide units, and the molecular weight is between 20,000 and 50,000 kDa. [0003] [0004] Hyaluronic acid has high viscoelasticity and plasticity, super strong water holding capacity and permeability, and good biocompati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/60C12N15/75C12N9/88C12P19/26C12P19/04C08B37/08C12R1/125
CPCC08B37/0003C08B37/0072C12N9/88C12N15/75C12P19/04C12P19/26C12Y402/02004
Inventor 吴凌天徐悦杜悦朱益波丁高杰杨兴旭王成红杨蕾
Owner 南京锐博科技有限公司
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