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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. [Copyright &y& Elsevier]

Abstract: A bacterial strain LL1 producing κ-carrageenase was isolated from the decayed seaweed collected from Yellow Sea, China and identified as Pseudoalteromonas porphyrae. The extracellular κ-carrageenase in the supernatant of cell culture of the marine bacterium P. porphyrae LL1 was purified to homogeneity with a 202.6-fold increase in specific κ-carrageenase activity as compared to that in the supernatant by ultrafiltration, gel filtration chromatography, and anion-exchange chromatography. According to the data from sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 40.0kDa. The purified enzyme could actively convert κ-carrageenan into tetrasaccharides, but poorly convert λ-carrageenan. The optimal pH and temperature of the purified enzyme were 8.0 and 55°C, respectively. The enzyme was significantly stimulated by Mg2+ and Ba2+. The enzyme was inhibited by phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid, EDTA, EGTA and 1,10-phenanthroline. The K m and V max values of the purified enzyme for κ-carrageenan were 4.4mg/ml and 0.1mg/minml, respectively. The amino acid sequence (NPQPHIAKPGQTWILQEKRS) of N-terminus of the purified enzyme was identical to that of N-terminus of the deduced protein encoded by the gene encoding κ-carrageenase cloned from the marine bacterium. [ABSTRACT FROM AUTHOR]

The purification and characterization of xylanase from Aspergillus ficuum AF-98 were investigated in this work. The extracellular xylanase from this fungal was purified 32.6-fold to homogeneity throughout the precipitation with 50–80% (NH4)2SO4, DEAE-Sephadex A-50 ion exchange chromatography and Sephadex G-100 chromatography. The purified xylanase (specific activity at 288.7U/ mg protein) was a monomeric protein with a molecular mass of 35.0kDa as determined by SDS-PAGE. The optimal temperature and pH for the action of the enzyme were at 45°C and 5.0, respectively. The xylanase was activated by Cu2+ up to 115.8% of activity, and was strongly inhibited by Hg2+, Pb2+ up to 52.8% and 89%, respectively. The xylanase exhibited Km and V max values of 3.267mg/mL, 18.38M/min/mg for beechwood xylan and 3.747mg/mL, 11.1M/min/mg for birchwood xylan, respectively. [Copyright &y& Elsevier]