Purification of glucoamylase from Lactobacillus amylovorus ATCC 33621.
An intracellular glucoamylase (E.C. 3.2.1.3) was purified to homogeneity from Lactobacillus amylovorus on a Fast Protein liquid chromatography System (FPLC) with a Mono Q ion-exchanger and two Superose 12 gel filtration columns arranged in series. The enzyme activity was quantified with a specific, chromogenic substrate, p-nitrophenyl-beta-maltoside. Preparative gel electrophoresis was then used to further purify active enzyme fractions. Native polyacrylamide gel electrophoresis (Native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of molecular weight 47 kDa. Glucoamylase activity of the purified protein was confirmed by its ability to degrade starch on a 0.025% starch-polyacrylamide gel stained with I2/KI. Glucoamylase exhibited optimum catalytic activity at pH 6.0 and 45 degrees C, and the enzyme had an isoelectric point near 4.39. The glucoamylase contained high levels of hydrophilic amino acids, comparable to fungal glucoamylases.
James
J A
JA
Department of Food Science and Agricultural Chemistry, Macdonald Campus of McGill University, 21111 Lakeshore, Ste-Anne de Bellevue, Québec, H9X 3V9, Canada.
Berger
J L
JL
Lee
B H
BH
D000595:Amino Acid Sequence; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007778:Lactobacillus / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013696:Temperature
D005087:Glucan 1,4-alpha-Glucosidase