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Purification and characterization of a digestive alkaline protease from the larvae of Spilosoma obliqua
Author(s) -
Anwar Adil,
Saleemuddin M.
Publication year - 2002
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/arch.10046
Subject(s) - protease , biology , trypsin , biochemistry , chromatography , casein , affinity chromatography , size exclusion chromatography , molecular mass , hydrolysis , enzyme , chemistry
A digestive protease from Spilosoma obliqua (Lepidoptera: Arctiidae) fifth instar larval guts was purified and characterized. The protease was purified using ammonium sulfate fractionation, ion‐exchange chromatography, and hemoglobin‐sepharose affinity chromatography. The purification procedure resulted in a 37‐fold increase in the specific activity of the protease. Protease thus obtained was found to be electrophoretically pure under native and denaturing conditions. The purified protease had a molecular mass of 90 kDa as determined by gel filtration, and a pH optimum of 11.0. The purified protease optimally hydrolyzed casein at 50°C. A Km of 2 ×10 –6 M was obtained using BApNA as a substrate for the purified alkaline protease. The ability of S. obliqua protease and bovine trypsin to hydrolyze various synthetic substrates (BApNA, BAEE, and BAME), and the inhibition patterns of S. obliqua and bovine trypsin with “classical” trypsin inhibitors are also reported. Arch. Insect Biochem. Physiol. 51:1–12, 2002. © 2002 Wiley‐Liss, Inc.