Abstract
Arterial/venous thrombosis is the major cardiovascular disorder accountable for substantial mortality; and the current demand for antithrombotic agents is extensive. Heparinases depolymerize unfractionated heparin (UFH) for the production of low molecular-weight heparins (LMWHs; used as anticoagulants against thrombosis). A microbial strain of Streptomyces sp. showing antithrombotic activity was isolated from the soil sample collected from north India. The strain was characterized by using 16S rRNA homology technique and identified as Streptomyces variabilis MTCC 12266 capable of producing heparinase enzyme. This is the very first communication reporting Streptomyces genus as the producer of heparinase. It was observed that the production of intracellular heparinase was [63.8 U/mg protein (specific activity)] 1.58 folds higher compared to extracellular heparinase [40.28 U/mg protein]. DEAE-Sephadex A-50 column followed by Sepharose-6B column purification of the crude protein resulted 19.18 folds purified heparinase. SDS-PAGE analysis of heparinase resulted an estimated molecular-weight of 42 kDa. It was also found that intracellular heparinase has the ability to depolymerize heparin to generate LMWHs. Further studies related to the mechanistic action, structural details, and genomics involved in heparinase production from Streptomyces variabilis are warranted for large scale production/purification optimization of heparinase for antithrombotic applications.
Highlights
Heparin and its structural analogues such as heparin sulphate (HS) are acidic and linear polysaccharides, belongs to the family glycosaminoglycans[1]
In silico screening results suggest that some specific proteins matching with heparinase or heparinase like proteins or some hypothetical proteins are enlisted in different species of Streptomyces genus such as Streptomyces himastatinicus ATCC 53653 (Streptomyces hygroscopicus), Streptomyces olivochromogen, Streptomyces vindochromogenes, Streptomyces venezuelae, Streptomyces griseoruber, Streptomyces orinoci, Streptomyces acidiscabies 84–104, Streptomyces griseochromogenes etc
This in silico screening of heparinase producing gene step had paved the way to further explore the possibilities of identifying heparinase producing novel isolates from Streptomyces genus
Summary
Heparin and its structural analogues such as heparin sulphate (HS) are acidic and linear polysaccharides, belongs to the family glycosaminoglycans[1] These polysaccharides are composed of 1–4 linked repeating units of β-D-glucuronic acid, N-acetyl-glucosamine and disaccharides with different degrees of sulphation[2]. Known as unfractionated heparins (UFHs), has been used clinically in the prevention and cure of thromboembolism since 19352 The depolymerization of these polymers using various chemicals or enzymatic agents results into low molecular weight heparins (LMWHs), which possess a variety of crucial biological functions and can be used as therapeutic agents[4]. Heparinases are commercially used for the depolymerization of unfractionated heparin (UFH) into disaccharide and oligosaccharide products, known as low molecular weight heparins (LMWHs)[4]. Keeping above facts in view, the present study was started with the aim of screening, isolation, purification, and characterization of heparinase from novel microbial source based on in silico screening of heparinase gene present in the genome of Streptomyces genus
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