Purification and characterization of alkaline soda-bleach stable protease from Bacillus sp. APP-07 isolated from Laundromat soil

Abstract

The detergent-compatible alkaline protease was produced from the bacterial strain Bacillus sp. APP-07 isolated from Laundromat soil of Solapur, Maharashtra, India. The culture was grown in 1000 ml capacity baffled flask with a working volume of 100 ml and incubated at 55 °C for 33 h on a rotary shaker. After incubation, alkaline protease was partially purified by the sequential method of acetone precipitation followed by nominal molecular weight limit (NMWL) cut-off ultrafiltration using 50 K and 10 K filters. Finally, Sephadex G-100 gel filtration chromatographic purification was performed to obtain 3.12 fold purified alkaline protease enzyme with a 66.67% final yield. The purified enzyme showed 31907.269 units (U) of enzyme activity containing 8741.718 U/mg of specific enzyme activity. The molecular weight of the enzyme was confirmed about 33.0 kDa (kDa) by the SDS-PAGE analysis. The purified enzyme was stable at higher pH and temperature range, with an optimum pH 10.5 and temperature 55 °C. The enzyme showed excellent stability and compatibility in various detergents, surfactants, bleach, and oxidizing agents. The enzyme activity enhanced in the presence of Ca2+, Cu2+, and surfactants, whereas; the phenylmethylsulphonyl fluoride (PMSF) and Diisopropyl fluorophosphate (DFP) completely inhibit the enzymatic activity, which pointed out that the enzyme affiliated to serine-centered metalloproteases family.In conclusion, the remarkable tolerance and stability of the enzyme explored the promising candidature for the several potential applications in the laundry detergents. The sustainability of the enzyme might serve several possible applications in the laundry detergents, leather industries, and other harsh industrial processes. Keywords: Bacillus sp., Alkaline protease, Purification and characterization, Laundry detergent