Purification and characterization of two forms of maltotetraose-forming amylase from Pseudomonas stutzeri

Agric Biol Chem. 1990 Mar;54(3):737-43.

Abstract

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.

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / analysis
  • Chromatography, Ion Exchange
  • Electrophoresis, Polyacrylamide Gel
  • Genes, Bacterial
  • Isoenzymes / isolation & purification*
  • Isoenzymes / metabolism
  • Kinetics
  • Molecular Sequence Data
  • Peptide Hydrolases / metabolism
  • Pseudomonas / enzymology*
  • Pseudomonas / genetics
  • Pseudomonas / growth & development
  • Substrate Specificity
  • alpha-Glucosidases / biosynthesis
  • alpha-Glucosidases / isolation & purification*
  • alpha-Glucosidases / metabolism

Substances

  • Amino Acids
  • Isoenzymes
  • alpha-Glucosidases
  • glucan 1,4-maltotetraohydrolase
  • Peptide Hydrolases