Purification and Characterization of Gelatinase-like Proteinases from the Dark Muscle of Common Carp (Cyprinus carpio)

Abstract

Gelatinolytic proteinases from common carp dark muscle were purified by 30-60% ammonium sulfate fractionation and a combination of chromatographic steps including ion exchange on DEAE-Sephacel, gel filtration on Sephacryl S-200, ion exchange on High-Q, and affinity on gelatin-Sepharose. The molecular masses of these proteinases as estimated by SDS-PAGE were 75, 67, and 64 kDa under nonreducing conditions. The enzymes revealed high activity at a slightly alkaline pH range, and their activities were investigated using gelatin as substrate. Metalloproteinase inhibitors, EDTA, EGTA, and 1,10-phenanthroline, almost completely suppressed the gelatinolytic activity, whereas other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca (2+) is essential for the gelatinolytic activity. Furthermore, these gelatinolytic proteinases hydrolyze native type I collagen effectively even at 4 degrees C, strongly suggesting their involvement in the texture softening of fish muscle during the post-mortem stage.

Purification and Characterization of a Gelatinolytic Metalloproteinase from the Skeletal Muscle of Red Sea Bream (Pagrus major)

“…The optimal pH value for the present proteinase was around pH 8.0 ( Figure 5B), which is almost the same as those of the above-mentioned metalloproteinases (18,(31)(32)(33).…”

Indigenous proteases in the skin of unicorn leatherjacket (Alutherus monoceros) and their influence on characteristic and functional properties of gelatin

“…Proteolysis induced by heat-activated and heat-stable indigenous proteases associated with skin matrix can contribute to the destabilisation as well as disintegration of collagen structure by disrupting the intra-and intermolecular cross-links (Wu et al, 2008). Collagenases have the unique ability to degrade the major structural extracellular matrix proteins, especially the triple helical strand of type I and type II collagens at Gly 775 -Leu (Ile) 776 .…”

Effect of legume seed extracts on the inhibition of proteolytic activity and muscle degradation of fresh water prawn (Macrobrachium rosenbergii)

“…According to the hydrolysis mechanism, proteases are classified as serine, cysteine, aspartic and metallo proteases (Garcia-Carreño, 1992). Among these proteases, serine-like proteases are mostly associated with muscle softening in fish and shellfish (Ezquerra-Brauer, Salazar-Leyva, Bringas-Alvarado, & Rouzaud-Sandez, 2003) including common carp (Cyprinus carpio) (Wu et al, 2008), red sea bream (Pagrus major) (Wu et al, 2010) and Mexican flounder (Cyclopsetta chittendeni) (Ramirez, Garcia-Carreño, Morales, & Sanchez, 2002). Recently, serine protease has been reported to contribute to the softening of fresh water prawn (Macrobrachium rosenbergii) during iced storage (Sriket, Benjakul, Visessanguan, & Kishimura, 2011).…”