Aminopeptidase from jumbo squid (Dosidicus gigas) hepatopancreas: Purification, characterisation, and casein hydrolysis

An aminopeptidase (AP) was partially purified from jumbo squid (Dosidicus gigas) hepatopancreas with 154.24-fold and yield of 6.15%. The purification procedure consisted of ammonium sulphate fractionation and DEAE-Sephacel chromatography. The enzyme was approximately 48-53 kDa as estimated by SDS- PAGE. With l-leu-p-NA, it had optimum activity at pH 8.0 and 30 °C. The K_m and V_max/K_m values of the enzymes for l-leu-p-NA were 0.326 mm and 2787 at 37 °C, respectively. Activation energy (E_a) of the enzyme was 53.50 kJ M^-1. The AP showed activity against seven synthetic substrates: L-proline > L-methionine > Ac. l-γ-glutamic > L-glycine > L-leucine > L-alanine > L-lysine-p-NA. The enzyme was strongly inhibited by Bestatin, partially inhibited by a metal-chelating agent and by PCMB, a cystein protease inhibitor. Zn²⁺ and (or) Ca²⁺ seemed to be its metal cofactor(s). Incubation of casein with the partially purified AP resulted in a degree of hydrolysis of 6%.