An amylopullulanase enzyme with the ability to hydrolyze different polymeric substrates (starches) was purified from culture supernatants of Lactobacillus amylophilus GV6 during direct fermentation of starch to L(+) lactic acid. The total cell free and cell bound amylase and pullulanase enzyme activities were 0.59 and 0.34 U/(ml min), respectively. Amylopullulanase was active towards soluble starch, raw starch, antylopectin, amylose, glycogen and pullulan. Highest activity was displayed towards the polysaccharide amylopectin. The crude enzyme was purified by ammonium sulphate fractionation, dialysis and Sephacryl S-200 column chromatography. The molecular mass of purified enzyme as estimated by SDS-PAGE and gel filtration chromatography is 90 kDa. Purified enzyme gave an apparent single protein band in SDS-PAGE. This band was found to have both amylase and pullulanase activities. In addition to glucose, maltose and maltotriose were detected as the products of starch hydrolysis by this enzyme. Maximum activity of antylopullulanase was observed at pH 6.5 and temperature 37 degrees C in acetate buffer. The enzyme activity was observed to be higher at 2.5 mM Ca2+ in comparison with other metals and surfactants tested. Presence of both amylase and pullulanase activities in amylopullulanase enzyme is a characteristic feature of the fermenting organism, L. amylophilus GV6. This is advantageous for direct fermentation of starch to lactic acid and there are no reports on Lactobacillus spp. producing amylopullulanase enzyme. (c) 2005 Elsevier Inc. All rights reserved.