Heparinase high-yielding strain and breeding method thereof

Abstract

The invention provides a heparanase high-yielding strain. The strain is Raoultella SP.NX-TZ-3,15. The strain is already preserved in a preserve department specified by the State Intellectual Property Office, the preserve date is March 6 th, 2017, the preserve department is the China Common Microorganism Strain Preserve and Management center, and the preserve serial number is CGMCC No.13723. In breeding of the heparanase high-yielding strain, precipitation generated through the reaction of protamine and heparin is utilized as an indicator, heparanase generated through microorganisms are subjected to a reaction of cracking heparin to make the precipitation disappear, transparent rings are generated around thalli, further an effect of directly indicating heparanase source microorganism strains is achieved, through secondary screening and a 16S r DNA method, the heparanase yielding strain SP.NX-TZ-3,15 is identified. In the heparanase high-yielding strain, the disadvantages that in an original screening method of heparanase source microorganisms, time, labor and materials are consumed is solved, the precipitation generated through the reaction of protamine and heparin is utilized as the indicator, and thus the labor, materials and time are sharply saved.

Description

[technical field] [0001] The invention belongs to the field of biotechnology, and in particular relates to a high-yield heparanase strain Raoultella SP.NX-TZ-3,15 and a breeding method thereof. [Background technique] [0002] Heparinase or heparin lyase is a class of proteins that can specifically cleave the glycosidic bonds of heparin and heparan sulfate. Heparinase was first discovered from Flavobacterium heparinus, a microorganism that can produce oligosaccharides using heparin, and it is mainly used clinically to prevent thrombosis or as an anticoagulant drug in vitro. [0003] Currently, three heparinases have been identified from Flavobacterium heparinus: heparinase I (heparinase, EC 4.2.2.7), heparinase II (heparinase II), and heparinase III (heparinase, EC 4.2. 2.8). Heparanase I mainly cleaves heparin, heparanase III mainly cleaves heparan sulfate, and heparanase II cleaves both heparin and heparan sulfate. Flavobacterium heparinus is mainly used in the productio...

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Problems solved by technology

[0005] At present, the screening method of heparinase-producing microorganisms mainly adopts the domestication method of gradually replacing the original carbon source of the microbial screening medium with heparin, and specifically enriches the microbial strains that have the ability to lyse heparin, and then monitors the enriched culture medium by the Azure A method. Changes in the content of heparin in the medium, the samples containing the strains that can utilize heparin were initially screened, and each colony in the sample was smeared, and a single colony was picked for re-screening. The crude extract is crushed, and the enzyme activity is determined by the Azure A method to determine whether it is a heparanase-producing microorganism. However, this screening method needs to try a variety of formula media, which wastes a lot of manpower and material resources. Therefore, to explore a A simple and rapid method for screening heparanase-producing microorganisms is of great significance

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