The preparation of a cell free system from Bacillus subtilis capable of carrying out protein synthesis

Typescript.Thesis (Ph. D.)--University of Hawaii, 1968.Bibliography: leaves 144-149.xiv, 149 l illus., tablesα-Amylases have been obtained in highly purified crystalline forms from Bacillus subtilis (Yamamoto, Bull. Agr. Chem. Soc. Japan 19, 121, 1955; Fillig et al., Helv. Chim. Acta. 40, 529, 1957; Hagihara, Proc. Japan Acad. 27, 346, 1951). A new purification method for obtaining crystalline α-amylase starting with a crude commercial enzyme preparation from the Pacific Laboratories strain B. subtilis has been developed. The purification method employs the use of chromatography on DEAE-cellulose, Duolite A-2, and hydroxylapatite. The crystalline enzyme is shown to be homogeneous by the criteria of ultracentrifugation, electrophoresis, amino terminal amino acid analysis, and rechromatography. Unlike other B. subtilis α-amylase (Stein et al., J. Biol. Chem. 232, 867, 1958) which has been reported to occur as a dimer with molecular weight of 100,000 with a zinc ion holding the subunits together, Pacific Laboratories α-amylase yielded a molecular weight value of 48,700 by the Sephadex gel filtration method. Also, the amylase was shown to contain only trace amounts of zinc (0.11 moles zinc/50,000). Other studies on the chemical and physical properties are also reported. A cell free extract capable of incorporating radioactive amino acids into hot trichloroacetic acid insoluble proteins has been prepared from the Pacific Laboratories strain B. subtilis. The endogenous activity of the subcellular system was dependent on the age of the B. subtilis culture. Thus, subcellular systems that are prepared from the exponentially growing bacteria yielded highly active systems. The amino acid incorporation is dependent upon ATP and ATP generating system, magnesium ions, ammonium ions, an amino acids mixture, and a sulfhydryl reagent. Omission of GTP did not result in a decrease of incorporating activity as reported in other B. subtilis system (Taubman et al., 1964, In Antimicrobial Agents and Chemotherapy, p. 395). The incorporation of amino acids is sensitive to ribonuclease and deoxyribonuclease which inhibited the system 92% and 20% respectively. The antibiotics puromycin (90% inhibition), chloramphenicol (31%), and mitomycin c (18% inhibition), also decreased the incorporation of amino acids. The formation of α-amylase in the subcellular fractions of B. subtilis was demonstrated by following the increase of enzyme activity with time. Increase of α-amylase activity could be demonstrated in subcellular fractions isolated from both exponentially growing cells and stationary phase cells. This is in contrast to a previous report by Oishi et al. (Biochem; Biophys. Res. Comm. 8, 342, 1962) who reported increase of enzyme activity in subcellular fractions from stationary phase cells only. The observed increase of α-amylase in the Pacific Laboratories B. subtilis system is shown to be partly due to an energy dependent system and partly to an energy independent process, presumably, the release of preformed enzyme. That part of the process is not a de novo synthesis of α-amylase is supported by the fact that addition of ribonuclease and puromycin increased the enzyme activity