An acid protease having milk clotting activity has been isolated from Mucor bacilliformis cultures. The enzyme was basically purified by ionic exchange chromatography. An average yield of 29 mg purified product was obtained from 100 mL crude extract. As purity criteria, SDS-PAGE, reverse-phase HPLC, and N-terminal analysis were performed. The protease is a protein composed of a single polypeptide chain with glycine at the N-terminus. The mol wt is approx 32,000, and its amino acid composition is very similar to those of other fungal proteases. As expected, its clotting activity was drastically inhibited by pepstatin A action. On the other hand, its instability against heat treatment and its clotting/proteolytic activity ratio indicate that it may be considered as a potential substitute for bovine chymosin.
Antarctic soil chronically exposed to gas-oil was analysed in order to isolate and study the growth conditions of hydrocarbon degrading bacteria. Soil samples taken near the shoreline in Jubany Station (King George Island, South Shetland Islands) were used as inoculum in liquid culture media with crude oil as sole carbon source. A psychrotrophic Acinetobacter strain was isolated and selected for further investigations. Effects were studied of temperature, initial pH, NaCl concentration and different chemical structure of the hydrocarbon on growth. Degradation rate was determined with n-dodecane and n-hexadecane. Growth of Acinetobacter ADH-1 showed no differences at an initial pH of 7.0, 7.5 and 8.0. Optimum temperature ranged between 25–30°C but the strain was capable of growing on n-dodecane at 4°C. Growth was observed in the presence of 3.5% NaCl. A decrease in the surface tension values was observed in the culture broth during the first 20 h of incubation (from 68 din cm−1 to 31 din cm−1). This proved to be related to the cellular fraction of the culture. The study shows that Acinetobacter ADH-1 is a psychrotrophic bacteria able to grow with hydrocarbons as sole carbon and energy source and could be potentially useful to design bioremediation processes in temperate and cold climate areas.
The fourth-day extract of a solid-state culture of the mesophilic Mucor sp. (M-105) strain showed a high milk-clotting activity and a clotting/proteolytic activity ratio similar to that of commercial preparations from microbial origin used in cheese manufacture. After ultrafiltration of the crude extract, the milk-clotting proteinase was purified in two steps: ion-exchange followed by size-exclusion chromatography. Enzyme homogeneity was assessed by HPLC, SDS-PAGE and N-terminal residue determination. A pI value of 4.21 was obtained and a molecular weight of 33 kDa was calculated from size-exclusion chromatography and SDS-PAGE data. The optimum pH for proteolytic activity towards dimethylcasein was in the 3.0-3.5 range. The proteinase retained 26 and 13% of its proteolytic activity after a 30-min incubation period, at pH 5.0 and 50 and 60 degrees C, respectively. This evidenced a lower heat stability than that of the thermophilic enzymes currently used in the cheese industry and also than that of bovine chymosin. The enzyme was fully inhibited by pepstatin A and no effect was observed with PMSF, p-CMPS or EDTA. The N-terminal amino acid sequence: GTGTVPVTDDGNLNEYYXTVTVGXP was compared with those from other fungal enzymes.
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