Two isoenzymes of a new thermostable Amylase from the obligate thermophile Bacillus stearothermophilus, LN 90

ABSTRACT

Amylase of an obligate thermophilic Bacillus stearo-thermophilus L.N. 90 was purified from the cell free filtrate of the culture previously grown at 55C by ethanol precipitation and fractionation on Sephadex G-200 column chromatography. The enzyme was purified 28 fold with 48% yield. The purified enzyme exhibited a maximal activity at pH 5.8 using phosphate buffer and optimum temperature of 55C. The enzyme was also sensitive to high concentration of the substrate. Both C2+ and Mg2+ relatively activated the enzyme, whereas Pb2+, Zn2+ and Li+ exhibited no effect. Ag2+, Sn2+, Fe2+, Cu2+, Mn2+, and KCN exerted 100% inhibition, whereas Co2+, Hg2+, Ni2+ and arsenate exerted only 10, 17, 9 and 40% inhibition, respectively. The molecular weight of purified thermostable amylase was found to be 11,220 as determined by the polyacrylamide gels electrophoresis technique. Data of the HPLC spectrum of the purified amylase[s] resulted in having 2 distint peaks with two different retention times/min. Viz. 10.40 and 11.30 indicating the presence of two isoenzymes [I and II]. Amylase I resembles amylase II in all amino acid composition except in its leucine content which gives another criterion to the phenomenon of multienzymes [isoenzymes] biosynthesized by the same strain acting amultaneously on the same substrate. Data indicate also that the polyacrylamide gel electrophoresis technique is less effective than that of the HPLC