PURIFICATION AND CHARACTERIZATION OF A MALTOTETRAOSE-FORMING ALKALINEALPHA-AMYLASE FROM AN ALKALOPHILIC BACILLUS STRAIN, GM8901

An alkalophilic bacterium, Bacillus sp. strain GM8901, grown at pH 10. 5 and 50 degrees C, produced five alkaline amylases in culture broth. At an early stage of the bacterial growth, amylase I (Amyl I) was prod uced initially and then, as cultivation progressed, four alkaline amyl ases, Amyl II, Amyl III, Amyl IV, and Amyl V, were produced from prote olytic degradation of Amyl I. A serine protease present in the culture medium was believed to be involved in Amyl I degradation, We purified Amyl I from the culture supernatant by ammonium sulfate precipitation , heparin-Sepharose CL-6B column chromatography, phenyl-Toyopearl colu mn chromatography, and Mono Q HR5/5 high-performance liquid chromatogr aphy. The molecular weight of Amyl I was estimated to be about 97,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amyl I h ad an extremely high optimal pH of 11.0 to 12.0 and was stable in a br oad pH range of 6.0 to 13.0. Amyl I had an optimal temperature of 60 d egrees C and was stable up to 50 degrees C. Thermostability was increa sed in the presence of Ca2+ and soluble starch. The enzyme required me tal ions such as Ca2+, Mg2+, Cu2+, Co2+, Ag+, Zn2+, and Fe2+ for its e nzyme activity and was inhibited by 1 mM EDTA and 1 mM phenylmethylsul fonyl fluoride. According to the mode of action of Amyl I on starch, A myl I was classified as an alpha- and exo-amylase. Amyl I produced mal totetraose predominantly from starch via intermediates such as maltohe xaose and maltopentaose.