Abstract
An attempt was made to isolate thermophilic bacteria growing at temperatures above 60°C and secreting β-amylase using immunoreaction between anti-jS-amylase serum or IgG prepared from Bacillus cereus β-amylase and unknown amylases. Among 170 strains isolated from various sources like soils, hot springs, etc., only one strain was selected by dot-ELISA and Western blotting methods. Morphological and physiological properties of the strain indicated that the strain belongs to Bacillus stearothermophilus. The amylase fraction was obtained from the culture filtrate by salting out with ammoniumsulf ate (0.7 saturation) and purified by ion-exchange chromatography on DEAE-cellulofine A-500 and gel filtration on Sephadex G-100. Further, by first protein liquid chromatography (FPLC) on Mono-Q HR5/5 column, the three amylase fractions (Fracts. I, II, and III) were obtained. Analysis of their hydrolytic products from starch showed that the amylases are classified to aamylase. Their optimum pH and temperature were around 60 and 70°C, respectively, and the enzymes were stable around 70°C. Among the three a-amylases only Fract. III had immunoreactivity with anti-β-amylase serum or IgG, and gave different products in starch hydrolysis; mainly glucose from soluble starch, and maltose and maltotriose from raw starch.
