title: Purification and characterization of an aminopeptidase from Lactobacillus casei subsp. rhamnosus S93.
abstract: An aminopeptidase of broad specificity was extracted by cell lysis of a selected strain of Lactobacillus casei subsp. rhamnosus during the late exponential phase. The enzyme was purified 195-fold from crude extract by using an f.p.l.c. system. Native and SDS/PAGE of the purified enzyme showed a single protein band of 89 kDa. The maximum aminopeptidase activity was observed at pH 7.0 and 39 degrees C. The enzyme hydrolysed a range of nitroanilide-substituted amino acids, as well as dipeptides, and accounted for most of the aminopeptidase activity found in cell-free extracts. The enzyme activity was inhibited by metal chelators such as EDTA and 1,10-phenanthroline. Cobalt ions only stimulated aminopeptidase activity and were also able to re-activate the enzyme previously inhibited by metal chelators. The Km and Vmax. values of the aminopeptidase for leucine p-nitroanilide were 0.06 mM and 12.6 mmol/min per mg of protein respectively. This enzyme was stable over the pH range of 5-9 and below 45 degrees C.
authors.affiliation: Agriculture Canada Food Research and Development Center, St. Hyacinthe, Québec.
authors.forename: G
authors.initials: G
authors.lastname: Arora
authors.forename: B H
authors.initials: BH
authors.lastname: Lee
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003035:Cobalt / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008970:Molecular Weight; D010618:Phenanthrolines / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000596:Amino Acids; D010618:Phenanthrolines; D003035:Cobalt; D004492:Edetic Acid; D000626:Aminopeptidases; C025205:1,10-phenanthroline