Overproduction and characterization of prolyl aminopeptidase from Aspergillus oryzae

Ken-Ichi Kusumoto, Satoshi Suzuki

Author address: 

National Food Research Institute


Overproduction and characterization of prolyl aminopeptidase from Aspergillus oryzae Ken-Ichi Kusumoto1, Mayumi Matsushita-Morita1, Ikuyo Furukawa1, Youhei Yamagata2, Yoshinao Koide3, Hiroki Ishida4, Michio Takeuchi2, Yutaka Kashiwagi1, Satoshi Suzuki1 1National Food Research Institute, Tsukuba, Ibaraki, Japan; 2Department of Agriscience and Bioscience, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan; 3Gifu R & D Center, Amano Enzyme Inc., Kagamihara, Gifu, Japan; 4Research Institute, Gekkeikan Sake Company Ltd., Fushimi-ku, Kyoto, Japan Introduction: Prolyl aminopeptidase (PAP) degrades only amino-terminal proline from peptides. The food-grade fungus Aspergillus oryzae produces this enzyme only in small amounts. Therefore, we present here an efficient production of recombinant PAP with an overexpression system of A. oryzae and characterization of its biochemical properties. Methods and Results: The gene encoding PAP was overexpressed as a His-tag fusion protein under a amyB promoter with a limited expressing condition in A. oryzae. The PAP activity in the mycelia grown in rich media 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. A. oryzae PAP was purified 1800-folds. The PAP was estimated to be a homo hexameric protein and exhibited salt tolerance against NaCl of up to 4 mol l-1. Discussion: Overproduction of PAP under promoter inducing conditions led to an increase of inactive PAP, possibly because of irregular folding. PAP with a high specific activity and salt tolerance may be used effectively in the manufacturing processes of fermented foods.

abstract No: 


Full conference title: 

    • ECFG 10th (2010)