Abstract
An extracellular alkaline protease (Prot-2) selectively secreted by Botrytis cinerea growing in medium containing Spirulina algae as inducer was purified to homogeneity by a combination of ammonium sulfate precipitation, gel filtration and ion-exchange chromatography, followed by size-exclusion chromatography. Prot-2 presented a single 30-kDa band on SDS-PAGE, which showed proteolytic activity following renaturation. Prot-2 has a monomeric structure, is active in the pH range 5.0–9.0 and shows an optimal temperature of activity at 50°C. Prot-2 is thermostable, and activated by Ca2+. The inhibitory action of reducing agents (dithiothreitol or β-mercaptoethanol) was suppressed by dithiobis-nitrobenzoic acid (DTNB) addition, indicating the role played by disulfide in enzyme activity and/or stability. Prot-2 showed extreme stability towards non-ionic surfactants (5% Tween 20, 5% Triton X-100 and Nonidet P-40). It was relatively stable in 25% aqueous/organic solvent mixtures and was activated by oxidizing agents (H2O2 and sodium perborate). A broad specificity of Prot-2 was tested using a range of natural and synthetic oligopeptide substrates. It was further supported by the hydrolysis profile of the insulin B chain by Prot-2.
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