Journal of Molecular Catalysis B: Enzymatic, ( ISI ), Volume (115), No (2), Year (2015-2) , Pages (51-58)

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

Authors: Shaghayegh Farhadian , Ahmad Asoodeh , Milad Lagzian ,

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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 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, 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.

Keywords

, HtrA-like protease, Bacillus subtilis, purification, characterization, Structural
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@article{paperid:1046309,
author = {Farhadian, Shaghayegh and Asoodeh, Ahmad and Milad Lagzian},
title = {Purification, biochemical characterization and structural modeling of a potential htrA-like serine protease from Bacillus subtilis DR8806},
journal = {Journal of Molecular Catalysis B: Enzymatic},
year = {2015},
volume = {115},
number = {2},
month = {February},
issn = {1381-1177},
pages = {51--58},
numpages = {7},
keywords = {HtrA-like protease; Bacillus subtilis; purification; characterization; Structural modeling},
}

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%0 Journal Article
%T Purification, biochemical characterization and structural modeling of a potential htrA-like serine protease from Bacillus subtilis DR8806
%A Farhadian, Shaghayegh
%A Asoodeh, Ahmad
%A Milad Lagzian
%J Journal of Molecular Catalysis B: Enzymatic
%@ 1381-1177
%D 2015

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