Genetically engineered bacterium for producing heparinase and application of genetically engineered bacterium in preparation of small-molecular heparin

A technology of genetically engineered bacteria and molecular heparin, which is applied in the field of preparation of small molecule heparin, can solve the problems of product biological activity impact, complex reaction conditions, product purification difficulties, etc., to ensure biological activity and physical and chemical properties, simple operation, avoid physical The effect of changing traits

Pending Publication Date: 2021-12-24
CHANGSHU INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the chemical degradation method introduces chemical reagents, the reaction conditions are complicated, it is easy to affect the biological activity of the product and the purification of the product is difficult, and a large amount of industrial wastewater is generated

Method used

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  • Genetically engineered bacterium for producing heparinase and application of genetically engineered bacterium in preparation of small-molecular heparin
  • Genetically engineered bacterium for producing heparinase and application of genetically engineered bacterium in preparation of small-molecular heparin
  • Genetically engineered bacterium for producing heparinase and application of genetically engineered bacterium in preparation of small-molecular heparin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1: Construction of recombinant Bacillus subtilis

[0070] Amplification of the signal peptide amyQ: the gene fragment of the signal peptide amyQ was amplified using the plasmid pHT43 as a template. The PCR amplification system is 1 μL of genome, 4 μL 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 62°C for 30 s, extension at 72°C for 0.2 min, cycle 35 times; extension at 72°C for 1 min. After gel electrophoresis, the PCR product was gel-cut and recovered with a column-type gel-slicing recovery kit to obtain a purified amyQ gene fragment.

[0071] Amplification of the heparinase gene: using the genome of P.heparinus ATCC13125 as a template, the gene fragment of the heparinase gene was amplified. The PCR amplification system is 1 μL of genomic DNA, 4 μL of primer 3 and primer 4, 50 μL of KOD polymerase, ddH 2 O 41 μL, PCR reaction program: ...

Embodiment 2

[0079] Example 2: Induced expression of heparanase

[0080]Inoculate the constructed recombinant Bacillus subtilis B. subtilis WB800-△spoOA-pHT01-amyQ-Hap in LB liquid medium containing chloramphenicol resistance, culture overnight at 37°C; then transfer to In 1L fermentation medium containing chloramphenicol, ferment for 2-4 hours to OD 660 When it reaches 0.8, add 20g / L lactose or 80mg / L IPTG to induce expression for 24h, and the activity of heparanase in the supernatant of the fermentation broth can reach 3200U / mL.

Embodiment 3

[0081] Embodiment 3: the heparanase activity assay of fermented liquid

[0082] Take 1 mL of fermentation broth and centrifuge, take 0.5 mL of fermentation supernatant and 1.5 mL of 25 g / L heparin (prepared with 0.02 mol / LTris-HCL, pH 7.5) respectively, add them to a quartz cuvette, and incubate the reaction at 30 ° C. Scan at 232nm for 1min, and calculate the slope of the reaction k(min -1 ). The formula for calculating the enzyme activity (U / L) of heparanase is as follows:

[0083]

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Abstract

The invention discloses a genetically engineered bacterium for producing heparinase and an application of the genetically engineered bacterium in preparation of small-molecular heparin, and belongs to the technical field of bioengineering. According to the invention, heparinase derived from Pedobacter vulgaris ATCC13125 is subjected to heterologous expression, a signal peptide amyQ and a pHT01 carrier are selected, induction is carried out through lactose or isopropyl-beta-D-thiogalactoside (IPTG), secretory expression of the heparinase in bacillus subtilis is achieved, through a designed heparinase-enzyme membrane reactor, efficient and continuous production of the mall-molecular heparin is realized, heparinase can be recycled, and production cost is greatly reduced. According to the invention, the food-grade bacillus subtilis is used as a host strain, is safe and reliable, provides effective reference for industrial green production of micromolecular heparin, and has advantages of energy conservation, emission reduction, and remarkable economic and social benefits.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a heparanase-producing genetically engineered bacterium and a method for preparing small molecule heparin. Background technique [0002] Heparin is a natural anticoagulant. It is widely used as medical intervention equipment in medicine. In addition, heparin and extracorporeal circulation are used as an adjuvant for chemotherapy drugs and anti-inflammatory drugs, and a regulator of growth factors. Heparin is used to treat hemodynamic disorders, enteritis diseases, cancer, venous thrombosis and other diseases. [0003] [0004] Heparin has a polymeric structure, and thus heparin compositions typically contain heparin with a molecular weight of typically 5 kDa to 40 kDa. The molecular weight of small molecule heparin is generally 3kDa ~ 8kDa, and the average molecular weight is about 5kDa. LMWH is better absorbed and can be administered subcutaneously; remains in the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12N9/24C12P19/26C12R1/125
CPCC12N9/2402C12N15/75C12Y302/01019C12P19/26
Inventor 吴凌天闫如玉国兆宇周茹芮蝶包天铮刘思瑜
Owner CHANGSHU INSTITUTE OF TECHNOLOGY
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