title: Overexpression and characterization of an extracellular leucine aminopeptidase from Aspergillus oryzae.
abstract: Leucine aminopeptidase (LAP), an enzyme used in the food industry, is an exopeptidase that removes an amino acid residue, primarily leucine (Leu), from the N-terminus of peptides and protein substrates. In this study, we focused on the leucine aminopeptidase A (lapA) gene from Aspergillus oryzae RIB40. To purify and characterize the LapA, lapA was overexpressed in A. oryzae RIB40 using the amyB promoter. LAP activity in the culture supernatant of one transformant harboring the lapA expression plasmid was 33 times that of the host strain. LapA was purified from the culture supernatant of this lapA-overexpressing strain by column chromatography. The purified recombinant LapA had a molecular mass of 33 kDa, and its N-terminal amino acid was the tyrosine at position 80 of the deduced amino acid sequence. Optimal enzyme activity was observed at 60°C and pH 8.5, and the enzyme was stable at temperatures up to 60°C and in the pH range 7.5-11. In transcriptional analysis, lapA was induced under alkaline conditions and expressed at a relatively low level under normal conditions. LapA showed maximum hydrolyzing activity for the substrate leucine para-nitroanilide (Leu-pNA), followed by substrates Phe-pNA (39% activity compared with Leu-pNA), Met-pNA, Lys-pNA, and Arg-pNA. In addition, LapA preferentially hydrolyzed peptides longer than tripeptides.
authors.affiliation: National Food Research Institute, Tsukuba, Ibaraki, 305-8642, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Matsushita-Morita
authors.forename: Sawaki
authors.initials: S
authors.lastname: Tada
authors.forename: Satoshi
authors.initials: S
authors.lastname: Suzuki
authors.forename: Ryota
authors.initials: R
authors.lastname: Hattori
authors.forename: Junichiro
authors.initials: J
authors.lastname: Marui
authors.forename: Ikuyo
authors.initials: I
authors.lastname: Furukawa
authors.forename: Youhei
authors.initials: Y
authors.lastname: Yamagata
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Amano
authors.forename: Hiroki
authors.initials: H
authors.lastname: Ishida
authors.forename: Michio
authors.initials: M
authors.lastname: Takeuchi
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kashiwagi
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Kusumoto
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D003470:Culture Media / Q000737:chemistry; D004795:Enzyme Stability; D015870:Gene Expression; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D007931:Leucyl Aminopeptidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D011401:Promoter Regions, Genetic; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D003470:Culture Media; D007931:Leucyl Aminopeptidase
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