Exploration for Thermostable β-Amylase of a Bacillus sp. Isolated from Compost Soil to Degrade Bacterial Biofilm.

Abstract

ABSTRACTIn an attempt to explore biofilm degradation using extracellular amylase, a potent amylase-producing bacterium of compost origin, B. subtilis B1U/1, was found to grow suitably in a simple medium of pH 7.5 for 48 h at 37°C under agitation of 140 rpm. This bacillary amylase was recovered by ammonium sulfate precipitation and purified to near homogeneity by membrane filtration and DEAE cellulose column chromatography. The amylase was purified to 4.5-fold with almost 50% yield and 26 kDa of molecular weight. Stable enzyme activity was found in a pH range of 5.2 to 9.0, while 90% residual activity was recorded at 90°C, indicating its thermostable nature. In the presence of 1 mM Fe++ and Ca++, the activity of amylase improved; however, it is inhibited by 1 mM Cu++. In the presence of 5% NaCl concentration, amylase showed 50% residual activity. The end product analysis identified the enzyme as β-amylase, and a crystal violet assay ensured that it can degrade Pseudomonas aeruginosa (78%) and Staphylococcus aureus biofilm efficiently (75%). The experiments carried out with the compost soil isolate were promising not only for biotechnological exploitation due to its pH flexibility during growth but also for high efficiency in the degradation of biofilms, which makes the organism a potent candidate in the fields of food industries and biomedical engineering, where it can be used as a prosthetic and hip joint cleaner. The β-amylase is highly thermostable since it withstands an elevated temperature for a prolonged period with a minimum loss of activity and is also moderately salt and metal tolerant.IMPORTANCE More than 85% of nosocomial infections are due to the development of bacterial biofilms. Recent research proposed that biofilm-like structures are not only visible in autopsies, biopsies, patients with chronic wounds, and exudates in animal models but are also present in biomedical devices, implants, prosthetic valves, urinary catheters, etc. Because complete eradication of biofilm is highly challenging, alternative methods, such as enzymatic damage of extracellular matrix and mechanical removal, are being implemented due to their easy availability, low cost, and high yield. Organisms from compost piles are rich sources of diverse extracellular enzymes with a high level of stability, which makes them able to withstand the different conditions of their environments. Under diverse environmental conditions, the enzymes are active to continue degradation processes, making them potential candidates in waste management, medicine, and the food and agriculture industries.