Isolation of Protease Producing Bacteria

Abstract

One of the most significant industrial enzymes, proteases find extensive use in biotechnology, pharmacology, and therapeutic procedures, especially in thrombolytic treatments. The isolation, generation, purification, and characterisation of protease-producing bacteria from soil samples taken from heavy metal-contaminated industrial areas in Coimbatore, Tamil Nadu, India, are the main objectives of this work. For the initial screening of proteolytic activity, soil samples were serially diluted and plated on skim milk agar. Protease synthesis was demonstrated by sixteen bacterial isolates that showed distinct zones of casein hydrolysis. Isolates BP12, BP15, and BP16 were chosen for additional research because they showed noticeably greater protease activity. The chosen isolates were cultivated in protease production media, and the effects of several environmental and nutritional factors, such as temperature, pH, incubation duration, and various sources of carbon and nitrogen, were assessed in order to maximize enzyme synthesis. After 72 hours of incubation, the highest level of protease synthesis was seen. The ideal pH was between neutral and slightly alkaline, and higher enzyme activity was encouraged by temperatures between 47 and 57 degrees Celsius. The most efficient suppliers of carbon and nitrogen among the nutrients examined were sucrose and ammonium nitrate, respectively. Protease test and protein estimation were performed on the crude enzyme extracts, and then ammonium sulfate precipitation, dialysis, and gel filtration chromatography were used for partial purification. There were different levels of thrombolytic and proteolytic activity in the isolated enzyme fractions. The chosen isolates showed considerable clot lysis potential in in vitro thrombolytic experiments, with some fractions exhibiting activity above 90%, indicating the presence of strong fibrinolytic enzymes. Nevertheless, it was found that increased thrombolytic effectiveness was not always correlated with higher general protease activity, indicating the existence of functionally different enzyme components. Subsequent analysis showed that the enzymes' molecular weight varied from 15 to 31 kDa, which is in line with previously documented bacterial fibrinolytic proteases. In the presence of EDTA, the action of metal inhibitors demonstrated a decrease in enzyme activity, suggesting that the enzymes were likely metalloproteases. An increase in the purification fold suggested better enzyme specificity and activity, even if the purification technique produced lower yields. The study concludes by showing that contaminated soil bacteria are an important source of proteases with substantial industrial and medicinal potential. The chosen isolates' potent proteolytic and thrombolytic capabilities demonstrate their potential use in enzyme-based businesses and the creation of therapeutic medicines for the management of thrombotic diseases.

Keywords: Protease, Protease-producing bacteria, Enzyme isolation, Microbial enzymes, Proteolytic activity, Enzyme production, Biotechnology, Bacterial screening