Purification and Characterization of a Carboxypeptidase from Squid Hepatopancreas (Illex illecebrosus)

Abstract

The hepatopancreas of squid (Illex illecebrosus) extract contains a wide range of carboxypeptidase (CP) activities based on hydrolysis of N-CBZ-dipeptide substrates. SDS-PAGE zymograms with N-CBZ-Phe-Leu substrate revealed three activity zones (CP-I, 23 kDa; CP-II, 29 kDa; CP-III, 42 kDa). CP-I was purified 225-fold with 86.20% recovery based on N-CBZ-Ala-Phe activity by chromatography on DEAE-cellulose, gel filtration, and chromatofocusing. The purified enzyme had broad specificity toward N-CBZ-dipeptides; however, it preferred substrates with a hydrophobic amino acid at the C terminus. CP-I had greatest activity with N-CBZ-Ala-Phe (specific activity = 7104 units/mg of protein, K(m) = 0.40 mM, and physiological efficiency = 22863). CP-I had a pI of 3.4 and is a metalloprotease that is activated by Co(2+) and partially inhibited by Pefabloc, a serine protease inhibitor. With N-CBZ-Ala-Phe and Gly-Phe, it had optimum activity at pH 8 and 70 degrees C. The amino acid composition of squid CP-I is similar to that of CP A from other species.

Purification of serine carboxypeptidase from the hepatopancreas of Japanese common squid Todarodes pacificus and its application for elimination of bitterness from bitter peptides

“…When the penultimate amino acid from the C‐terminus was a hydrophobic or bulky amino acid such as Phe, Tyr, Leu or Glu, the hydrolysis rates of the substrates were relatively high. Similarly, CPase produced from Aspergillus saitoi 4–6 exhibited the same specificity in terms of the changing relative activity for the substrate.…”

“…From hepatopancreas of T. pacificus , CPase Tpa, having a specificity for hydrophobic or bulky amino acid residue in the penultimate position of the C‐terminus, was found monitoring the hydrolytic activity of Z‐Glu‐Tyr at pH 4.0. However, from hepatopancreas of the Atlantic short‐finned squid 4 Illex illecebrosus , carboxypeptidase (CP‐I) was found by monitoring the hydrolytic activity of Z‐Phe‐Leu at pH 7.5. Comparing the two carboxypeptidases, CPase Tpa differs from the CP‐I, the former being a serine carboxypeptidase, with the latter being a metalloprotease.…”

“…Recently, the annual haul of Japanese common squid Todarodes pacificus has averaged approximately 300 000 ton, comprising one of the most important marine resources for consumption in Japan. To the authors knowledge, few biochemical studies on the proteases in the internal organs of the squid, which are largely discarded but account for large part of their weight, have been published 1–4 …”

“…To the authors knowledge, few biochemical studies on the proteases in the internal organs of the squid, which are largely discarded but account for large part of their weight, have been published. [1][2][3][4] Of the proteases, the carboxypeptidases are distributed widely among microorganisms, 5-13 animals [14][15][16][17][18] and plants. 19 More types are being reported as their various characteristics come to light.…”

Proteome-derived Peptide Libraries to Study the Substrate Specificity Profiles of Carboxypeptidases

“…The most popular CPs being the SCPs C, P and Y (10). In addition, the food industry uses different SCPs to process protein products to reduce their bitter taste (11)(12)(13).…”