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Fusion heparinase and coding gene and preparation method thereof

A technology of heparinase and gene, applied in the fields of enzyme engineering and biocatalysis, can solve the problems of lack of integrated methods for enzyme production

Inactive Publication Date: 2009-12-23
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In the existing heparanase I production and catalytic process, there is a lack of an integrated method for the entire process of enzyme production-separation-application

Method used

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  • Fusion heparinase and coding gene and preparation method thereof
  • Fusion heparinase and coding gene and preparation method thereof
  • Fusion heparinase and coding gene and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Expression of triple fusion protein of green fluorescent protein, maltose binding protein and heparanase I

[0041] 1. Construction of the expression vector of the triple fusion protein

[0042] 1. Construction of the expression vector pMAL-c2x-HepA containing the gene encoding the double fusion protein of maltose binding protein and heparanase I

[0043] The construction process of the expression vector pMAL-c2x-HepA is as follows: figure 1 As shown, the specific process is as follows: amplify the heparanase I gene from the genomic DNA of Flavabacterium heparinum (Flavabacterium heparinum) (purchased from IAM), and the primers used are respectively:

[0044] Upstream primer: 5′-GCCT GGATCC CAGCAAAAAAAAATCCGGTAAC-3' (the underlined base is the enzyme recognition site of BamHI),

[0045] Downstream primer: 5′-CTTA AAGCTT TTACTATCTGGCAGTTTCGCTGTAC-3' (bases underlined are HindIII enzyme recognition sites), after amplification, BamHI and HindIII enzyme rec...

Embodiment 2 3

[0064] Example 2. Fluorescence quantitative tracking of triple fusion protein heparanase activity

[0065] According to the results of Example 1, we selected Escherichia coli TOP10 / phGMH-L3 to conduct a fluorescence quantitative tracking experiment of the heparanase activity of the triple fusion protein. M9YE medium containing 100 μg / ml ampicillin (17.1 g / L Na 2 HPO 4 12H 2 O, 3.0g / L KH 2 PO 4 , 0.5g / L NaCl; 1.0g / L NH 4 Cl, 12.5g / L yeast extract, 12g / L glucose) to cultivate TOP10 / phGMH-L3 at 37°C for about 3.5 hours (to OD 600 0.735), add IPTG with a final concentration of 0.3mM, and change the culture temperature to 15°C for induction culture. After that, every 2 hours, take 2ml of bacterial solution to measure the cell concentration (OD 600 ), the enzyme activity and fluorescence intensity of heparanase I (fluorescence spectrophotometer HITACHI F-2500), until the induction culture for 30 hours.

[0066] The enzyme activity of heparanase I was determined according to ...

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Abstract

The invention discloses fusion heparinase which is the protein of the following a) or b): a) the protein formed by the amino acid sequences shown by No.8-No.1015 in a sequence 1 in a sequence table; b) the protein which has heparinase activity and is derived from a) by substitution and / or deletion of one or more amino acid in and / or addition of one or more amino acid to the amino acid sequences in the sequence 1 in the sequence table. The coding gene of the protein also belongs to the scope of protection of the invention. The enzyme activity of the triple fusion protein generated by the invention can reach 1214.4IU / L culture medium by shaking culture and the specific activity can reach 2.839IU / mg protein. The invention can employ GFP to realize quick online detection of thalli concentration and enzyme activity.

Description

technical field [0001] The invention relates to the fields of enzyme engineering and biocatalysis, in particular to fusion heparinase, its coding gene and its preparation method. Background technique [0002] Heparinase (heparinase) is a kind of polysaccharide lyase that acts on heparin, found in many kinds of microorganisms, including Corynebacterium sp. .39:64-67), Sphingobacterium sp. (Gao Ningguo et al., Production of Sphingobacterium heparanase, Acta Microbiology 2003 Vol.43:813-816), Bacillus subtilis (Wang Zhongyan etc., Screening of Heparanase-producing Bacteria and Research on the Properties of Crude Enzyme, Journal of Sichuan University (Natural Science Edition) 2002 Vol.39: 777-779), Bacillus circulans (Yasutaka Tahara et al., Purification and characterization of heparinase that degrades both heparin and heparin sulfate from Bacilluscirculans BioSci.Biotechnol.Biochem.2002 Vol.66:1181-1184), Bacteroides heparinase Prevotella heparinolytica (Kazuyuki Sugahara et a...

Claims

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Application Information

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IPC IPC(8): C12N9/96C12N15/62C12N15/63C12N5/10C12N1/00C12N1/21C12R1/19
CPCC12N9/88C08B37/0075
Inventor 邢新会叶逢春张翀蒋培霞
Owner TSINGHUA UNIV
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