Novel alkaline serine proteases from alkalophilic Bacillus spp.: purification and some properties |
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| Affiliation: | 1. Centre for Molecular Biology, Federal Research Centre for Nutrition, Engesserstrasse 20, D-76131 Karlsruhe, Germany;2. Dairy Microbiology Division, National Dairy Research Institute, Karnal 132 001, India;1. Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, 9500 Gilman Drive, MC0755, La Jolla, CA 92093-0755, USA;2. The Scripps Research Institute, Scripps Florida, 130 Scripps Way #3A2, Jupiter, FL 33458, USA;1. School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, PR China;2. Guangdong Key Laboratory of Animal Conservation and Resource Utilization, Guangdong Public Laboratory of Wild Animal Conservation and Utilization, Guangdong Institute of Applied Biological Resources, Guangzhou 510260, PR China;1. College of Food and Biological Engineering, Jimei University, Xiamen, Fujian 361021, China;2. Hefei National Laboratory for Physical Sciences at Microscale, CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine, University of Science & Technology of China, Hefei 230027, China;3. Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen, Fujian 361100, China;1. Synergetic Innovation Center of Food Safety and Nutrition, Wuxi 214122, China;2. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China;3. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China;4. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China;5. School of Biotechnology, Jiangnan University, Wuxi 214122, China |
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| Abstract: | Two extracellular alkaline proteases produced by an alkalophilic Bacillus isolate were purified and characterized using acetone precipitation, DEAE- and CM-Sepharose CL-6B ion exchange and Sephacryl S-200 gel filtration chromatographic techniques. Analysis of the purified proteases by SDS–PAGE revealed that both proteases, AP-1 and AP-2 were homogenous with molecular weight estimates of 28 and 29 kDa, respectively. The optimum activity of AP-1 and AP-2 were at temperatures of 50 and 55°C and pHs of 11 and 12, respectively. The enzymes were also stable in the pH range of 6.0–12.0 for a period of 4 h with and without Ca2+ (5 mM) and temperatures of up to 50°C. The half-lives of the enzymes recorded at 50°C were 50 and 40 min for proteases AP-1 and AP-2, respectively. The inhibition profile of the enzymes by phenylmethanesulphonyl fluoride, confirmed these enzymes to be alkaline serine proteases. The purified proteases hydrolysed native protein substrates such as casein, elastin, keratin, albumin and the synthetic chromogenic peptide substrates Glu-Gly-Ala-Phe-pNA and Glu-Ala-Ala-Ala-pNA. The Km values for the purified proteases were calculated as 1.05 mM and 1.29 mM, respectively, for Glu-Gly-Ala-Phe-pNA, and 3.81 mM and 4.79 mM, respectively, for Glu-Ala-Ala-Ala-pNA as substrates. The kinetic data also indicated that small aliphatic and aromatic amino acids were the preferred residues at the P1 position. |
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