Purification and characterization of leucyl aminopeptidase from the Todarodes pacificus hepatopancreas

Abstract

Here, leucyl aminopeptidase (LAP) was purified from the hepatopancreas extract (HPE) of Todarodes pacificus, with 63.7-fold purification and a yield of 6.5%. Ammonium sulfate fractionation and sequential chromatographic steps, involving Sephacryl S-300, Toyopearl DEAE-650 M, DEAE-Sepharose CL-6B, and Sephadex G-50, were used for purification. The bitter-taste improvement of trypsin casein hydrolysate (TCH) by the purified LAP was also investigated. The LAP was characterized as a monomer; its molecular mass was 29.0 kDa under denatured conditions and 27.1 kDa under native conditions. With an optimum pH of 7.5 and optimum temperature of 45 °C, it remained stable up to ∼50 °C, within a pH range of 6–8. Its activity was inhibited by Cu2+ and Hg2+, bestatin, and the metal-chelating agents EDTA and 1,10-phenanthroline, and slightly enhanced by Fe2+, Zn2+, Mn2+, and Mg2+. Moreover, the purified enzyme was classified as a leucyl aminopeptidase, as it preferentially hydrolyzed Leu, followed by Met, Phe, Ala, and Lys p-nitroanilide. The apparent Km and Vmax values of the enzyme were 0.385 mM and 796.2 U/mg, respectively, for LeuPNA. Additionally, LAP-treated TCH showed lower bitterness than untreated TCH. The bitterness improvement effect of LAP could be attributed to the hydrolysis of hydrophobic amino acids at the N-terminus of the peptide; this confirms the substrate specificity of LAP. It has been confirmed that enzymes such as LAP that effectively reduce the bitter taste of bitter hydrolysates are distributed in the hepatopancreas of Todarodes pacificus, and this protein hydrolysate will be used as a bitter taste improver and food industry.