PURIFICATION AND PROPERTIES OF A 140 KDA AMYLOPULLULANASE FROM THERMOPHILIC AND ALKALIPHILIC BACILLUS SP STRAIN TS-23

An amylopullulanase from Bacillus sp. strain TS-23 was purified to hom ogeneity by a combination of ultrafiltration, ammonium sulphate precip itation, anion-exchange chromatography and preparative continuous-elut ion electrophoresis. This purified enzyme had a molecular mass of abou t 140 kDa and had both pullulanase and amylase activities. The enzyme was negative for Schiff staining, suggesting that it was not a glycopr otein, and had a pi value of 3.5. The temperature and pH optima for bo th pullulanase and amylase activities were 70 degrees C and pH 8-9 res pectively. The enzyme remained more than 96% active at temperatures be low 65 degrees C, and both activities were retained at temperatures up to 90 degrees C in the presence of 5% SDS. The enzyme was stimulated by Co2+ ions and inhibited by Mg2+, Mn2+ and Hg2+ ions and EDTA. Under optimum conditions this enzyme catalysed the hydrolysis of alpha-1,6 glucosidic linkages in pullulan with maltotriose as the only product. It also cleaved alpha-1,4 bonds in amylose, starch and related malto-o ligosaccharides larger than maltotriose with maltose and maltotriose a s main products.