Purification and Characterization of a Collagenolytic Protease from the Filefish, Novoden modestrus

Abstract

A serine collagenolytic protease was purified from the internal organs of filefish, Novoden modestrus, by ammonium sulfate, ion-exchange chromatography on a DEAE-Sephadex A-50, ion-exchange rechromatography on a DEAE-Sephadex A-50, and gel filtration on a Sephadex G-150 column. The molecular mass of the filefish serine collagenase was estimated to be 27.0 kDa by gel filtration and SDS-PAGE. The purified collagenase was optimally active at pH 7.0-8.0 and 55 o C. The purified enzyme was rich in Ala, Ser, Leu, and Ile, but poor in Trp, Pro, Tyr, and Met. In addition, the purified collagenolytic enzyme was strongly inhibited by N-P-toluenesulfonyl-L-lysine chloromethyl ketone (TLCK), diisopropylfluorophosphate (DFP), and soybean trypsin inhibitor.

Colagenase de pescada branca: extração, purificação parcial, caracterização e teste de especificidade ao colágeno para aplicação industrial

“…In relation to the susceptibility to metal ions, the data obtained from our study corroborate those reported by KIM et al (2002) andWU et al (2010a) which observed increased activity of collagenolytic proteases of filefish N. modestrus and sea bream P. major when exposed to the ions Ca 2+ and Mg 2+ , respectively. HAYET et al (2011) .…”

“…The abundance of type V is low, but it is found to be associated with types I and II, bone and cartilage, and other tissues, with important participation in the function of tensile strength (DABOOR et al, 2010). LIMA et al (2013) observed that the cleavage of type I collagen by collagenase produced by Penicillium aurantiogriseum, was similar to those already described with aquatic organisms KIM et al, 2002;DABOOR et al, 2012). According to HERREIRO-HERNANDEZ et al (2002), once one collagenolytic enzyme works degrading particular collagen types, it is likely that other proteases may follow the process.…”

“…HAYET et al (2011) . ROY et al (1996), BYUN et al (2002, KIM et al (2002) andWU et al (2010a) also observed increased activity of collagenolytic proteases from greenshore crab (C. maenas), tuna (T. thymus), mackerel (S. japonicus), filefish (N. modestrus) and sea bream P. major in the presence of the ions Ca 2+ and Mg…”

“…The studies of KIM et al (2002) and HAYET et al (2011) describe the employment of a specific trypsin inhibitor, TLCK. KRISTJÁMSSON et al (1995) reported strong inhibition of collagenolytic protease from Atlantic cod Gadus morhua exposed to the specific inhibitor of chymotrypsin, TPCK, in contrast to the reports by BYUN et al (2002) which studied enzymes from tuna T. thymus.…”

“…Hydrolysis of type I collagen has been detected by using collagenolytic proteases produced by various aquatic organisms, such as for the species of fish described by: KRISTJÁMSSON et al (1995) for Atlantic cod (G. morhua); TERUEL and SIMPSON, (1995) for winter flounder (P. americanus); BYUN et al (2002) for tuna (T. thymus); PARK et al (2002) for mackerel (S. japonicus); KIM et al (2002) for filefish (N. modestrus); HERREIRO-HERNANDEZ et al (2002) for iced cod (G. morhua); HAYET et al (2011) for sardinelle (S. aurita);and ROY et al (1996) for greenshore crab (C. maenas). In our studies, the intestinal protease of smooth weakfish was able to cleave the collagen type I tested, as shown in Figure 3.…”