Original article Aminopeptidase from jumbo squid (Dosidicus gigas) hepatopancreas: purification, characterisation, and casein hydrolysis Idalia Osuna-Ruı´z, 1,2 Gloria Yepiz-Plascencia, 3 Ofelia Rouzaud-Sa ´ ndez 1 & Josafat Marina Ezquerra-Brauer 1 * 1 Departamento de Investigacio´n y Posgrado en Alimentos, Universidad de Sonora, Blvd. Luis Encinas y Rosales s ⁄ n. Apdo. Postal 1658, 83000 Col Centro, Hermosillo, Sonora 2 Universidad Polite´cnica de Sinaloa, Nin˜os He´roes No. 1413 Esq. Constitucio´n, C.P. 82000. Centro Histo´rico, Mazatla´n, Sinaloa 3 Centro de Investigacio´ n en Alimentacio´ n y Desarrollo, A.C. Carretera a la Victoria Km 0.6, Apdo. Postal 1735. C.P. 83000.Hermosillo, Sonora (Received 20 August 2009; Accepted in revised form 10 December 2009) Summary 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-c-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. Zn 2+ and (or) Ca 2+ seemed to be its metal cofactor(s). Incubation of casein with the partially purified AP resulted in a degree of hydrolysis of 6%. Keywords Aminopeptidase, casein, characterisation, degree of hydrolysis, hepatopancreas, jumbo squid, marine by-products, purification. Introduction About 50–60% of catch total marine animals is used for direct human consumption, therefore about twenty-five to thirty million metric tons of fish and shellfish caught each year are discarded. Much of these discards are potential sources for unique compounds, including marine enzymes (Morrissey & Okada, 2007). Enzymes are used as processing aids and can be produced from by-product material from both new and traditional seafood processing operations (Morrissey & Okada, 2007). The use of proteases from squid hepato- pancreas (HP) can improve the fermentation of brined squid (Lee et al., 1982), accelerate fish sauce preparation (Raksakulthai & Haard, 1992), decrease the hardness of cooked squid, and also improve the flavour of Cheddar cheese (Raksakulthai et al., 2002). Most squid HP proteases studied so far are from European or Asian common squids and there are only very few reports about this enzymes from the digestive gland of the jumbo squid species Dosidicus gigas. Enzyme differences between species are known, and furthermore, there are reports of their variations in animals caught in different regions of the world (Haard, 1992); thus the squid enzyme activity depends on the fishing regions, as well as the species. Jumbo squid is an important fishery in Mexico, but it is normally sold as frozen or cooked frozen gutted mantle (Luna-Raya et al., 2006), consequently the HP is eliminated. This HP could be a good source of enzymes. Jumbo squid HP constitutes 2–8% of the jumbo squid’s body weight and contains 18–20% crude protein (Ezquerra-Brauer et al., 2002) and is a good source for digestive enzymes. A cysteine proteinase from the HP of jumbo squid was purified and characterised (Cardenas- Lopez & Haard, 2009). Also, a trypsin-like, carboxy- peptidase and aminopeptidase activities were detected in crude extracts of the squid D. gigas HP (Ezquerra- Brauer et al., 2002). Aminopepidases (AP) (EC 3.4.11.1-5) hydrolyse amino acid residues from the amino terminus of proteins or peptides and they are classified according to their preference for the amino-terminal amino acid of *Correspondent: Fax: +52 (662) 2 59 22 08; e-mail: ezquerra@guayacan.uson.mx International Journal of Food Science and Technology 2010, 45, 387–394 387 doi:10.1111/j.1365-2621.2009.02164.x Ó 2010 The Authors. Journal compilation Ó 2010 Institute of Food Science and Technology