title: Heterologous expression and characterisation of the Aspergillus aspartic protease involved in the hydrolysis and decolorisation of red-pigmented proteins.
abstract: BACKGROUND
  Aspergillus repens strain MK82 produces an aspartic protease (PepA_MK82) that efficiently decolorises red-pigmented proteins during dried bonito fermentation. However, further expansion of the industrial applications of PepA_MK82 requires the high-level production and efficient preparation of the recombinant enzyme.
  
  
  RESULTS
  The genomic DNA and cDNA fragments encoding the protease were cloned from strain MK82 and sequenced. Phylogenetic analysis of PepA_MK82 and comparisons with previously reported fungal aspartic proteases showed that PepA_MK 82 clusters with different groups of these enzymes. Heterologous expression of PepA_MK82 in Pichia pastoris yielded preparations of higher purity than obtained with an Escherichia coli expression system. Total protease activity in a 100-mL culture of the P. pastoris transformant was 14 times higher than that from an equivalent culture of A. repense MK82. The recombinant PepA_MK82 was easily obtained via acetone precipitation; the final recovery was 83%. PepA_MK82 and its recombinant had similar characteristics in terms of their optimal pH, thermostability, and decolorisation activity. The recombinant was also able to decolorise flaked, dried bonito and to bleach a blood-stained cloth.
  
  
  CONCLUSIONS
  Given its ability to hydrolyse and decolorise red-pigmented proteins, recombinant PepA_MK8 can be exploited in the food industry and as a stain-removal agent in laundry applications. © 2016 Society of Chemical Industry.
authors.affiliation: Division of Agrobioscience, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, 657-8501, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Takenaka
authors.affiliation: Division of Agrobioscience, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, 657-8501, Japan.
authors.forename: Mayo
authors.initials: M
authors.lastname: Umeda
authors.affiliation: Division of Agrobioscience, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, 657-8501, Japan.
authors.forename: Hisanori
authors.initials: H
authors.lastname: Senba
authors.affiliation: Marutomo Co., Ltd, 1696 Kominato, Iyo, Ehime, 799-3192, Japan.
authors.forename: Dai
authors.initials: D
authors.lastname: Koyama
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, 657-8501, Japan.
authors.forename: Kosei
authors.initials: K
authors.lastname: Tanaka
authors.affiliation: Division of Agrobioscience, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, 657-8501, Japan.
authors.forename: Ken-Ichi
authors.initials: KI
authors.lastname: Yoshida
authors.affiliation: Marutomo Co., Ltd, 1696 Kominato, Iyo, Ehime, 799-3192, Japan.
authors.forename: Mikiharu
authors.initials: M
authors.lastname: Doi
mesh_terms: D057055:Aspartic Acid Proteases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001230:Aspergillus / Q000201:enzymology*; D003001:Cloning, Molecular; D003116:Color; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D006868:Hydrolysis; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D057055:Aspartic Acid Proteases
keywords: Aspergillus repens; aspartic protease; decolorisation; heterologous expression; katsuobushi; pigmented protein