The keratinase production by the thermophilic actinomycete strain Thermoactinomyces candidus was induced by sheep wool as the sole source of carbon and nitrogen in the cultivation medium. For complete digestion of wool by the above strain, both keratinolytic serine proteinase and cellular reduction of disulfide bonds were involved. Evidence was presented that substrate induction was a major regulatory mechanism and the keratinase biosynthesis was not completely repressed by addition of other carbon (glucose) and nitrogen (NH4C1) sources. The enzyme was purified 62-fold by diethylaminoethyl-anion exchange and Sephadex G-75 gel permeation chromatographies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the purified keratinase is a monomeric enzyme with a molecular mass of 30 kDa. The pH and temperature optima were determined to be 8.6 and 70 degrees C, respectively. The purified thermophilic keratinase catalyses the hydrolysis of a broad range of substrates and displays higher proteolytic activity against native keratins than other proteinases. Ca2+ was found to have a stabilizing effect on the enzyme activity at elevated temperatures.

Abstract:The production process for Arthrobacter simplex with high specific hydrocortisone A'-dehydrogenase activity was improved by medium optimization in parallel shake flask experiments. Using a genetic algorithm, the concentrations of 12 medium components (mineral salts and amino acid) were optimized within 144 experiments to give maximum specific hydrocortisone A'-dehydrogenase activity and biomass yield (full experimental plan: 1.5 x loL4 experiments). The specific hydrocortisone A'-dehydrogenase activity was improved by a factor of 4.5 to 1800 U g-'. With the optimized medium composition in a stirred tank reactor for batch production of A. simplex, a specific hydrocortisone A'-dehydrogenase activity of 2000-3000 U g -' dry biomass was achieved depending on harvest time (previously reported data: 18-37 U g-').

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