A novel fibrinolytic enzyme producer from mangrove soil sediments: Screening, isolation, strain improvement, and fermentation

Abstract

Fibrinolytic enzymes are used for addressing many cardiovascular disease conditions. However, current fibrinolytic enzymes are highly expensive with many side effects which necessitate the development of alternative cost-effective processes for low-cost production of these lifesaving enzymes. Since the availability of a suitable strain is considered the basic requirement for any process development effort, we started our work in this direction with screening and isolation of fibrinolytic enzyme producers. The goal of this study was to screen and isolate fibrinolytic enzyme producers from a relatively unexplored environment, i.e., the mangroves. Mangroves are wetlands and are one of the unique and less studied habitats for the production of therapeutic molecules. Microbiota that produces fibrinolytic enzymes from Coringa mangroves located in Kakinada District, Andhra Pradesh, India, is lacking. Therefore, in this study, fibrinolytic enzyme-producing bacterium was screened from samples obtained from Coringa mangroves. Out of 200, protease enzyme-producing isolates obtained from screening 30 mangrove soil samples, 7 isolates exhibiting fibrinolytic activity were selected. Out of these 7, the highest fibrinolytic enzyme-producing bacterial strain (AIBL_AMSB2) was characterized by biochemical and genomic methods, which was finally identified as Bacillus subtilis subsp. Inaquosorum by 16S rRNA analysis. The strain was also found to be an amylase producer. AIBL_AMSB2 was subjected to strain improvement using random mutagenesis techniques – (i) Ultraviolent radiations (UV) and (ii) Ethyl methyl Sulphonate (EMS), which resulted in an improved mutant strain AIBL_AMSB2_M7E32 exhibiting 54.70% improvement in fibrinolytic activity. Batch fermentation in controlled experimental conditions using the obtained mutant strain resulted in a 133.54% increase in growth OD and a 391.11% increase in enzyme activity. Thus, the study reports an increased fibrinolytic enzyme activity producing mangrove isolate and its production by submerged fermentation. Further studies to prove the potential of the enzyme on blood clots are necessary to utilize for industrial application.