PURIFICATION OF GLUCOAMYLASE FROM LACTOBACILLUS-AMYLOVORUS ATCC-33621

An intracellular glucoamylase (E.C. 3.2.1.3) was purified to homogenei ty from Lactobacillus amylovorus on a Fast Protein Liquid Chromatograp hy System (FPLC) with a Mono Q ion-exchanger and two Superose 12 gel f iltration columns arranged in series. The enzyme activity was quantifi ed with a specific, chromogenic substrate, p-nitrophenyl-beta-maltosid e. Preparative gel electrophoresis was then used to further purify act ive enzyme fractions. Native polyacrylamide gel electrophoresis (Nativ e-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of mole cular weight 47 kDa. Glucoamylase activity of the purified protein was confirmed by its ability to degrade starch on a 0.025% starch-polyacr ylamide gel stained with I-2/KI. Glucoamylase exhibited optimum cataly tic activity at pH 6.0 and 45 degrees C, and the enzyme had an isoelec tric point near 4.39. The glucoamylase contained high levels of hydrop hilic amino acids, comparable to fungal glucoamylases.