Purification and biochemical properties of SDS-stable low molecular weight alkaline serine protease from Citrullus colocynthis

• Published in: Natural product research 2016-04, Vol.30 (8), p.935-940
• Main Authors: Khan, Muhammad Bashir, Khan, Hidayatullah, Shah, Muhammad Usman, Khan, Sanaullah
• Format: Article
• Language: English
• Subjects: biochemical characterisation; catalytic activity; Citrullus colocynthis; Citrullus colocynthis - enzymology; detergent stability; detergents; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Hydrogen-Ion Concentration; industrial applications; Molecular Weight; polyacrylamide gel electrophoresis; purification; seeds; Seeds - chemistry; serine protease; Serine Proteases - chemistry; Serine Proteases - isolation & purification; serine proteinases; soybeans; Substrate Specificity; Temperature; thermostability; trypsin inhibitors; urea
• ISSN: 1478-6427; 1478-6419; 1478-6427
• DOI: 10.1080/14786419.2015.1079909

A low molecular weight serine protease from seeds of Citrullus colocynthis was purified to electrophoretic homogeneity with high level of catalytic efficiency (22,945 M ⁻¹ S ⁻¹). The enzyme was a monomer with molecular mass of 25 kDa estimated by SDS–PAGE. The enzyme was highly active over a pH range of 6.5–9.0 and temperature range of 20–80 °C, with maximum activity at pH 7.5 and at 50 °C. The K ₘ and K cₐₜ were 73 μg/mL and 67/s, respectively. The enzyme was strongly inhibited by PMSF, moderately by soybean trypsin inhibitor, indicating that the enzyme was a serine protease. The enzyme retained 86 and 73% of its activity in the presence of urea and DTT, respectively, and its activity was slightly enhanced in the presence of anionic detergent (SDS). Thus, the enzyme is a novel SDS-stable protease with high catalytic efficiency over wide ranges of pH and temperature which is commercially promising for various industrial applications.