Gene cloning,purification and characterization of thermostable alanine dehydrogenase of Bacillus stearothermophilus |
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| Institution: | 1. State Key Laboratory of Elemental Organic Chemistry, Nankai University, Tianjin, 310074, China;2. College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China;3. Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin, China |
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| Abstract: | The alanine dehydrogenase (l-alanine: NAD+ oxidoreductase, deaminating, EC 1.4.1.1) gene of Bacillus stearothermophilus IFO12550 was cloned and expressed in Escherichia coli C600 with a recombinant plasmid, pICD301, which was constructed from pBR322 and the alanine dehydrogenase gene derived from B. stearothermophilus. The enzyme overproduced in the clone was purified about 30 fold to homogeneity by heat treatment and two subsequent steps with a yield of 46%. The enzyme of E. coli-pICD301 was immunochemically identical with that of B. stearothermophilus. The enzyme has a molecular weight of about 240,000 and consists of six subunits identical in molecular weight (40,000). The enzyme is not inactivated by heat treatment: at pH 7.2 and 75°C for 30 min; at 55°C and various pHs between 6.0 and 11.5 for 10 min. The enzymological properties are very similar to those of the mesophilic B. sphaericus enzyme (Ohshima, T. and Soda, K., Eur. J. Biochem., 100, 29–39, 1979) except for thermostability. |
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