Purification and characterization of two forms of maltotetraose-forming amylase from Pseudomonas stutzeri.
Pseudomonas stutzeri MO-19 produced two active forms of extracellular maltotetraose-forming amylase. Both forms, G4-1 and G4-2, were purified to electrophoretic homogeneity. The molecular masses of G4(-1) and G4(-2) were 57 kd and 46 kd by SDS-polyacrylamide gel electrophoresis, respectively. An identical N-terminal sequence up to 20 amino acid residues and similar amino acid compositions were obtained from both forms, but different C-terminal amino acids, leucine from G4(-1) and alanine from G4(-2), were released by carboxypeptidase Y. By in vitro incubation with a culture supernatant containing protease activity, G4(-1) was converted into G4(-2) without any loss of the amylase activity. It was concluded that G4(-2) was a product derived by the limited proteolysis of G4(-1), and that the proteolysis occurred in the C-terminal region of G4-1. G4-2 was more thermostable than G4(-1), and had a 20-fold higher Michaelis constant value for glycogen, which was 50 mg/ml against 2.3 mg/ml of G4(-1). G4(-1) adsorbed onto raw starch granules while G4(-2) did not.
Hayashibara Biochemical Laboratories, Inc., Okayama, Japan.
T
T
Nakada
M
M
Kubota
S
S
Sakai
Y
Y
Tsujisaka
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D007527:Isoenzymes; D000520:alpha-Glucosidases; C020135:glucan 1,4-maltotetraohydrolase; D010447:Peptide Hydrolases