Biochemical and molecular characterization of a detergent stable alkaline serine-protease from a newly isolated Bacillus licheniformis NH1

A bacterium producing thermostable alkaline serine-protease was isolated from an activated sludge reactor treating fishery wastewaters and was identified as Bacillus licheniformis NH1. The most appropriate medium for the growth and protease production is composed of (g/l): casein 5; yeast extract 2–4, KCl 1.5, K2HPO4 0.5 and KH2PO4 0.5. The crude extracellular protease produced by the isolate had optimal activity at 65–70 and 70 °C in the absence or presence of 2 mM CaCl2, respectively. The thermostability of the enzyme was considerably enhanced in the presence of Ca2+ at temperature values above 50 °C. The enzyme retained 62 and 100% of its initial activity after heating for 60 min at 60 °C, in the absence or presence of 2 mM CaCl2, respectively. The protease was highly active and stable from pH 7.0 to 12.0, with an optimum at pH 10.0–11.0. The activity was totally lost in the presence of PMSF, suggesting that the preparation contains serine-protease(s). Furthermore, the enzyme showed excellent stability and compatibility with some commercial laundry detergents. The enzyme retained more than 93% of its initial activity after preincubation 60 min at 40 °C in the presence of 7 mg/ml of Dixan, Axion and New Dex.