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Specific enzyme activity improved directionally modified enzyme of heparinase I as well as molecular modification method and expression engineered bacterium

A molecular modification, high activity heparinase technology, applied in the field of expression engineering bacteria, heparinase I directed modification enzymes and molecular modification, can solve the problems of low activity, low heparinase I yield, etc., to achieve specific enzyme activity. The effect of improving, large economic value, large industrial application potential

Active Publication Date: 2019-02-12
TIANJIN UNIV OF SCI & TECH +1
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  • Claims
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Problems solved by technology

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art, overcome the shortcomings of low yield and low activity of current heparanase I, and provide a directed transformation enzyme and molecular transformation method and expression method of heparanase I with improved specific enzyme activity. Compared with wild-type engineering bacteria, the directional modified enzyme of the present invention has higher specific enzyme activity and greater application potential, and also lays a theoretical foundation for the study of HepI

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  • Specific enzyme activity improved directionally modified enzyme of heparinase I as well as molecular modification method and expression engineered bacterium
  • Specific enzyme activity improved directionally modified enzyme of heparinase I as well as molecular modification method and expression engineered bacterium
  • Specific enzyme activity improved directionally modified enzyme of heparinase I as well as molecular modification method and expression engineered bacterium

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Embodiment Construction

[0050] The present invention will be described in further detail below in conjunction with specific examples. The following examples are only descriptive, not restrictive, and cannot limit the protection scope of the present invention.

[0051] The raw materials used in the present invention, unless otherwise specified, are conventional commercially available products; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

[0052] A Directional Modification Enzyme PhHepI of Heparanase I with Increased Specific Enzyme Activity A259D , the nucleotide sequence of which is SEQ ID NO:1.

[0053] The directional transformation enzyme PhHepI of the heparanase I that specific enzyme activity improves as described above A259D The complete amino acid sequence of the highly active heparanase I encoded is SEQ ID NO:2.

[0054] A Directional Modification Enzyme PhHepI of Heparanase I with Increased Specific Enzyme Activity S169D / A259...

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Abstract

The invention relates to a specific enzyme activity improved directionally modified enzyme PhHepI<A259D> of heparinase I. The nucleotide sequence of the enzyme is SEQ ID NO: 1. The invention providesa design and construction method of two mutant enzymes PhHepI<A259D> and PhHepI<S169D / A259D>, and a method for the construction of a novel mutant-enzyme engineered bacterium and the high-efficiency expression of the mutant enzymes. Compared with a wild type, the mutant enzymes provided by the invention have higher specific enzyme activity and greater application potential, and the theoretical basis is also laid for the research of HepI.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and protein expression, and relates to PhHepI A259D and PhHepI S169D / A259D Construction and expression of the enzyme gene, as well as research on the properties and preparation of the enzyme, especially a directional transformation enzyme and molecular transformation method and expression engineering bacteria of heparanase I with improved specific enzyme activity. Background technique [0002] Heparin exists widely in nature and is a natural anticoagulant substance in animals. It was first found in the liver, hence the name heparin. It is widely distributed in mammalian tissues, such as liver, lung, intestinal mucosa, heart, placenta and muscle, etc., and binds to proteins in the form of covalent bonds. Currently commercialized heparin is mainly extracted from bovine lung and pig intestinal mucosa. Low molecular weight heparin (LMWH) is a small heparin fragment produced by the clea...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N1/21
CPCC12N9/2402C12N15/70C12Y302/01019
Inventor 罗学刚张川杨宝成张同存
Owner TIANJIN UNIV OF SCI & TECH
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