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

• An htrA-like protease was characterized from thermophilic Bacillus subtilis DR8806.
• This 37 kD protease was active in a wide range of pH and temperature.
• The potential gene of the protease was also identified.
• 3D model predicted for the enzyme showed a PDZ-like domain and protease domain.

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 SDS-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 °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, β-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 proline 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.

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