Production of Fibrinolytic Protease from a Halobacterium Bacillus licheniformis VITLMS Isolated from Marine Sponges of Rameshwaram Coast, India

Abstract

Background: Marine bacteria serve as excellent sources of therapeutic enzymes, metabolites and natural products, which possess novel therapeutic properties. Increasing death rates due to cardiovascular diseases urge for cost-effective production of the fibrinolytic enzyme. Methods: In this study, marine sponge samples were screened for potent fibrinolytic producing bacteria. The primary screening was done for protease production, and clot lysis activity. The secondary screening was done for casein plasminogen activity and fibrinolytic activity. The strain which had potent fibrinolytic activity among them was further subjected to morphological, biochemical and molecular characterization. Media optimization was carried out to enhance enzyme production. The enzyme produced was subjected to purification using ammonium sulfate precipitation, gel filtration and characterized using HPLC and FTIR analysis. Results: Sponge was identified to be Desmapsamma anchorata. Thirteen bacterial isolates were isolated from the sponge sample. The 16S rRNA sequencing revealed that the potential strain had 99% similarity with Bacillus licheniformis. Amongst the isolates, most were found to be morphologically identical to the Bacillus genus. Gram’s staining and SEM analysis of the potent isolate were performed to identify the spore formation and rod-shaped morphology of the bacteria. The optimal temperature and pH for the production of the enzyme were 37°C and 8, respectively. The carbon source maltose and nitrogen sources were malt extract and yeast extract that were found to be optimal. The optimum incubation time was found to be 4 to 5 days. The crude supernatant was purified with ammonium sulfate precipitation and gel filtration chromatography. The retention time of 11.3 min and the presence of functional groups show the purity of the enzyme. The partially purified enzyme showed 96.4% clot lysis in artificial clot lysis activity. Conclusion: Although the secretion of fibrinolytic enzymes from Bacillus species is not new, based on our investigation, there are no reports regarding Bacillus licheniformis being isolated from marine sponges. However, there are reports of Bacillus licheniformis secreting fibrinolytic enzymes isolated from fermented food samples. This study identifies the marine environment as a potential source of new exploration for drug discovery.