caa a22 4500 605498369 CHVBK 20210128100545.0 cr unu---uuuuu 210128e20150801xx s 000 0 eng 10.1007/s00253-015-6428-1 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6428-1 Recombinant production and evaluation of an antibacterial l -amino acid oxidase derived from flounder Platichthys stellatus [Elektronische Daten] [Kosuke Kasai, Kenro Hashiguchi, Hiroki Takahashi, Ayano Kasai, Sadanori Takeda, Manabu Nakano, Takashi Ishikawa, Toshiya Nakamura, Tomisato Miura] Fish produce mucus substances as a defensive outer barrier against several bacterial infections. We have recently identified an antibacterial l-amino acid oxidase (psLAAO1) in the mucus layer of the flounder Platichthys stellate. In this study, the antibacterial protein psLAAO1 was expressed as a secretory bioactive recombinant protein in the methylotrophic yeast Pichia pastoris. The recombinant psLAAO1 inhibited the growth of bacteria to the same levels as native psLAAO1 present in mucus. In particular, Staphylococci and Yersinia were strongly suppressed, showing the highest growth retardation of the 21 species and strains tested. Moreover, Staphylococcus epidermidis was most sensitive to psLAAO1 with a minimum inhibitory concentration (MIC) of 0.078μg/mL, whereas Escherichia coli was essentially resistant to psLAAO1 with a MIC of >10μg/mL. Interestingly, psLAAO1-treated E. coli were found to upregulate the expression of the btuE gene, which encodes glutathione peroxidase (GPx). The biochemical function of GPx is to reduce freehydrogen peroxide and is induced under response to reactive oxygen species (ROS). Thus, E. coli confers resistance to the reduced free hydrogen peroxide produced by psLAAO1 by increasing GPx levels. Furthermore, the growth of Staphylococcus aureus was completely inhibited in the presence of recombinant psLAAO1. The morphology of psLAAO1-treated S. aureus showed cell surface damage, the formation of large aggregates and the cells showed severe deformations. Western blot analysis showed that psLAAO1 binds to the surface of S. aureus. Therefore, psLAAO1 binds to the surface of LAAO-sensitive S. aureus and directs peroxidative activity at the surface of the bacterial membrane. Springer-Verlag Berlin Heidelberg, 2015 l -Amino acid oxidase nationallicence Antibacterial protein nationallicence Staphylococcus aureus nationallicence Platichthys stellatus nationallicence Glutathione peroxidase nationallicence MRSA nationallicence Kasai Kosuke Department of Pathologic Analysis, Division of Medical Life Sciences, Graduate School of Health Sciences, Hirosaki University, 66-1 Hon-cho, Hirosaki, Aomori, Japan aut Hashiguchi Kenro Department of Clinical Laboratory, Yokohama City University Medical Center, Kanagawa, Japan aut Takahashi Hiroki I'ROM Co., Ltd, Tokyo, Japan aut Kasai Ayano Department of Clinical Laboratory, Hirosaki Municipal Hospital, Aomori, Japan aut Takeda Sadanori Department of Medical Technology, School of Health Sciences, Hirosaki University, Aomori, Japan aut Nakano Manabu Department of Biomedical Sciences, Division of Medical Life Sciences, Graduate School of Health Sciences, Hirosaki University, Aomori, Japan aut Ishikawa Takashi Department of Biomedical Sciences, Division of Medical Life Sciences, Graduate School of Health Sciences, Hirosaki University, Aomori, Japan aut Nakamura Toshiya Department of Biomedical Sciences, Division of Medical Life Sciences, Graduate School of Health Sciences, Hirosaki University, Aomori, Japan aut Miura Tomisato Department of Pathologic Analysis, Division of Medical Life Sciences, Graduate School of Health Sciences, Hirosaki University, 66-1 Hon-cho, Hirosaki, Aomori, Japan aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/16(2015-08-01), 6693-6703 0175-7598 99:16<6693 2015 99 253 https://doi.org/10.1007/s00253-015-6428-1 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6428-1 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kasai Kosuke Department of Pathologic Analysis, Division of Medical Life Sciences, Graduate School of Health Sciences, Hirosaki University, 66-1 Hon-cho, Hirosaki, Aomori, Japan aut NATIONALLICENCE 700 1- Hashiguchi Kenro Department of Clinical Laboratory, Yokohama City University Medical Center, Kanagawa, Japan aut NATIONALLICENCE 700 1- Takahashi Hiroki I'ROM Co., Ltd, Tokyo, Japan aut NATIONALLICENCE 700 1- Kasai Ayano Department of Clinical Laboratory, Hirosaki Municipal Hospital, Aomori, Japan aut NATIONALLICENCE 700 1- Takeda Sadanori Department of Medical Technology, School of Health Sciences, Hirosaki University, Aomori, Japan aut NATIONALLICENCE 700 1- Nakano Manabu Department of Biomedical Sciences, Division of Medical Life Sciences, Graduate School of Health Sciences, Hirosaki University, Aomori, Japan aut NATIONALLICENCE 700 1- Ishikawa Takashi Department of Biomedical Sciences, Division of Medical Life Sciences, Graduate School of Health Sciences, Hirosaki University, Aomori, Japan aut NATIONALLICENCE 700 1- Nakamura Toshiya Department of Biomedical Sciences, Division of Medical Life Sciences, Graduate School of Health Sciences, Hirosaki University, Aomori, Japan aut NATIONALLICENCE 700 1- Miura Tomisato Department of Pathologic Analysis, Division of Medical Life Sciences, Graduate School of Health Sciences, Hirosaki University, 66-1 Hon-cho, Hirosaki, Aomori, Japan aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/16(2015-08-01), 6693-6703 0175-7598 99:16<6693 2015 99 253