Purification and partial characterisation of a trypsin from the processing waste of the silver mojarra (Diapterus rhombeus)

• Published in: Food chemistry 2011-12, Vol.129 (3), p.777-782
• Main Authors: Silva, Janilson F., Espósito, Talita S., Marcuschi, Marina, Ribeiro, Karina, Cavalli, Ronaldo O., Oliveira, Vitor, Bezerra, Ranilson S.
• Format: Article
• Language: English
• Subjects: Alkaline peptidase; Biological and medical sciences; Diapterus rhombeus; Fish; Food industries; Fundamental and applied biological sciences. Psychology; Trypsin; Viscera
• ISSN: 0308-8146; 1873-7072
• DOI: 10.1016/j.foodchem.2011.05.019

► Purification of a trypsin isoform from an economically important fish. ► Application of fish waste as a source of proteases with biotechnological potential. ► Biochemical characterisation of a trypsin from an understudied fish. ► Sequencing of the NH2-terminal region of a trypsin from an understudied fish. ► Study of the effects of metal ion on the trypsin from an understudied fish. An alkaline peptidase was purified from the viscera of the silver mojarra (Diapterus rhombeus) in a three-step process: heat treatment, ammonium sulphate fractionation and molecular exclusion chromatography (Sephadex® G-75), with final specific activity 86-fold higher than the enzyme extract and yield of 22.1%. The purified enzyme had an estimated molecular mass of 26.5kDa and NH2-terminal amino acid sequence IVGGYECTMHSEAHE. Higher enzyme activity was observed at pH 8.5 and between 50 and 55°C. The enzyme was completely inactivated after 30min at 55°C and it was significantly more stable at alkaline pH. Km, Kcat and Kcat·Km-1 values, using BApNA as substrate, were 0.266mM, 0.93s−1 and 3.48mM−1s−1, respectively. Enzyme activity increased in the presence of the ions (1mM) K+, Li+ and Ca2+, but was inhibited by Fe2+, Cd2+, Cu2+, Al3+, Hg2+, Zn2+ and Pb2+ as well as by the trypsin inhibitors TLCK and benzamidine.