title: Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.).
abstract: Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification - by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose - was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0-11.0 and an optimal temperature of approximately 55-60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis-Menten constant (Km) and catalytic constant (Kcat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s(-1), respectively. The catalytic efficiency (Kcat/Km) was 238 s(-1) mM(-1).
authors.affiliation: Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand; Biochemical Research Unit for Feed Utilisation Assessment, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
authors.forename: Sasimanas
authors.initials: S
authors.lastname: Unajak
authors.affiliation: Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
authors.forename: Piyachat
authors.initials: P
authors.lastname: Meesawat
authors.affiliation: National Science and Technology Development Agency, Pathum Thani 12120, Thailand.
authors.forename: Atchara
authors.initials: A
authors.lastname: Paemanee
authors.affiliation: Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand.
authors.forename: Nontawith
authors.initials: N
authors.lastname: Areechon
authors.affiliation: Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand; Biochemical Research Unit for Feed Utilisation Assessment, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
authors.forename: Arunee
authors.initials: A
authors.lastname: Engkagul
authors.affiliation: Biochemical Research Unit for Feed Utilisation Assessment, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand; Department of Zoology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
authors.forename: Uthaiwan
authors.initials: U
authors.lastname: Kovitvadhi
authors.affiliation: Biochemical Research Unit for Feed Utilisation Assessment, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand; Faculty of Science and Technology, Bansomdejchaopraya Rajabhat University, Bangkok 10600, Thailand.
authors.forename: Satit
authors.initials: S
authors.lastname: Kovitvadhi
authors.affiliation: Biochemical Research Unit for Feed Utilisation Assessment, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand; Institute of Marine Research, Ecosystem Processes Research Group, Matre Aquaculture Research Station, N-5984 Matredal, Norway.
authors.forename: Krisna
authors.initials: K
authors.lastname: Rungruangsak-Torrissen
authors.affiliation: Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand; Biochemical Research Unit for Feed Utilisation Assessment, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand; Center for Advanced Studies in Tropical Natural Resources (CASTNAR), National Research University-Kasetsart University (NRU-KU), Kasetsart University, Bangkok 10900, Thailand.
authors.forename: Kiattawee
authors.initials: K
authors.lastname: Choowongkomon
mesh_terms: D000818:Animals; D023681:Cichlids / Q000276:immunology*; D005399:Fishes; D007413:Intestinal Mucosa / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry*
chemical_list: D007527:Isoenzymes; D014357:Trypsin