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 degrees 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 degrees C. The enzyme retained 62 and 100% of its initial activity after heating for 60 min at 60 degrees 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 degrees C in the presence of 7 mg/ml of Dixan, Axion and New Dex. The aprN gene encoding the alkaline serine-protease was isolated and its DNA sequence was determined. The aprN gene consisted of 1137 bp encoding a protein of 379 amino acids organized into a signal peptide (29 amino acids), a pro-protein (76 amino acids), and mature enzyme (274 amino acids). The deduced amino acid sequence indicates only three amino acid differences between NHI enzyme and subtilisin Carlsberg from Bacillus licheniformis NCIMB 6816. (c) 2006 Elsevier Inc. All rights reserved.