title: Characterization of recombinant prolyl aminopeptidase from Aspergillus oryzae.
abstract: OBJECTIVE
  Prolyl aminopeptidase (PAP) degrades only amino-terminal proline from peptides. The food-grade fungus Aspergillus oryzae produces this enzyme only in small amounts. In this paper, we present efficient production of recombinant PAP with an overexpression system of A. oryzae and characterization of its biochemical properties.
  
  
  RESULTS
  The gene encoding PAP was overexpressed as a His-tag fusion protein under a taka-amylase gene (amyB) promoter with a limited expressing condition in A. oryzae. The PAP activity in the mycelia grown in rich medium containing glucose (repressing condition) was twice that in starch (inducing condition). The enzyme prepared as cell-free extract was partially purified through two-step column chromatography. The PAP was estimated to be a hexameric protein and exhibited salt tolerance against NaCl of up to 4 mol l(-1).
  
  
  CONCLUSIONS
  Aspergillus oryzae PAP was produced under the repressing condition of amyB promoter in a PAP-overexpressing strain and purified 1800-folds. Overproduction of PAP under promoter-inducing conditions led to an increase in inactive PAP, possibly because of irregular folding.
  
  
  CONCLUSIONS
  PAP with a high specific activity and salt tolerance may be used effectively in the manufacturing processes of fermented foods.
authors.affiliation: National Food Research Institute, Tsukuba, Ibaraki, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Matsushita-Morita
authors.forename: I
authors.initials: I
authors.lastname: Furukawa
authors.forename: S
authors.initials: S
authors.lastname: Suzuki
authors.forename: Y
authors.initials: Y
authors.lastname: Yamagata
authors.forename: Y
authors.initials: Y
authors.lastname: Koide
authors.forename: H
authors.initials: H
authors.lastname: Ishida
authors.forename: M
authors.initials: M
authors.lastname: Takeuchi
authors.forename: Y
authors.initials: Y
authors.lastname: Kashiwagi
authors.forename: K-I
authors.initials: KI
authors.lastname: Kusumoto
mesh_terms: D000626:Aminopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D003470:Culture Media; D007218:Industrial Microbiology; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D000626:Aminopeptidases; C022450:prolyl aminopeptidase