Purification, biochemical characterization and structural modeling of a potential htrA-like serine protease from Bacillus subtilis DR8806

The production of an extracellular htrA-like serine protease by Bacillus subtilis DR8806 was studied in this study. The enzyme was purified using ammonium sulphate precipitation and Sephacryl S-200 size exclusion chromatography. The analysis by SOS-PAGE and zymogram of enzyme showed a molecular weight of 37 kDa. Isoelectric focusing revealed a pI value of 6.6 for the enzyme. By the use of casein as substrate, the enzyme was active and stable at the wide range of temperatures with maximum activity at 45 degrees C and pH 8. The enzyme activity was increased by Ca2+,K+, Mg2+, Fe2+, dimethylsulfoxide (DMSO), whereas its activity was decreased by Hg2+, Ba2+, Cu2+, Zn2+, H2O2, CTAB (cetyltrimethylammonium bromide) and SDS (sodium dodecyl sulphate). In addition, Mn2+, Na+, Triton X-100, beta-mercaptoethanol, EDTA (ethylenediaminetetraacetic acid) had no significant effect on the enzyme activity. Among organic solvents, ethanol and methanol enhanced the activity. The gene of the protease showed a 1200 bp open reading frame with 97% similarity to other htrA-like proteases. The computational modeling of the protease showed two distinct domains: a PDZ domain and protease core domain. The catalytic triad also demonstrated a degree of discrepancy in comparison to other serine proteases. It is composed of a serine residue as a nucleophile and a praline as a base center, while the acidic center was not fully identified. The obtained results suggested a new type of htrA-like protease with no previous records in bacillaceae family. (C) 2015 Elsevier B.V. All rights reserved.