Purification and Characterization of Collagenolytic Proteases from the Hepatopancreas of Northern Shrimp (Pandalus eous)

Abstract

Three gelatinolytic proteases (A1, A2, and B) were purified using a synthetic substrate, DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, from the hepatopancreas of Northern shrimp (Pandalus eous) by several chromatographic steps involving hydroxyapatite column chromatography, gel filtration on Superdex75, and ion-exchange chromatography on a MonoQ column. Collagenolytic proteases A2 and B, but not protease A1, were demonstrated to digest native porcine type I collagen at 25 degrees C and pH 7.5. Further characterizations of these two collagenolytic proteases showed that the pH optimum of enzyme A2 against DNP-peptide was found to be 11, whereas that of enzyme B was 8.5. The optimum temperature ranged between 40 and 45 degrees C for both enzymes, although enzyme B appeared to be thermally more stable than enzyme A2 at pH 7.5. Both enzymes were strongly inhibited by PMSF and antipain, which suggests that they belong to collagenolytic serine proteases.

Characterization of gelatinolytic enzymes in the skeletal muscle of red sea bream Pagrus major

“…These results are similar to those of white croaker serine proteinase (WSP), the sarcoplasmic serine proteinase of white croaker reported by Yanagihara et al [15]. Collagenolytic serine proteinases were also found in fiddler crab [16] and Northern shrimp [17]. Hence, collagenolytic activity of G1 should also be investigated.…”

Recovery and Characterization of a Serine Collagenolytic Extract from Snow Crab (Chionoecetes opilio) By-products

“…The yields of tryptic activity in excess of 100% could be explained by the elimination of inhibiting or interfering substances. Previous reports on marine collagenases typically indicate purification factors lower than 30 and yields lower than 10% [8,14,28,29,40]. In these studies, acetone and (or) ammonium sulfate precipitations were used as a first fractionation step, followed by one or two chromatographic techniques.…”

“…Tryptic, Chymotryptic, and Elastolytic Activities Synthetic peptides ZAANA, BTNA, and STANA were used to measure tryptic, chymotryptic, and elastolytic activities, respectively [14,18,31]. Stock solutions of ZAANA (10 mM) and BTNA (12.3 mM) in N,N-dimethylformamide, and STANA (11 mM) in dimethyl sulfoxide were prepared.…”

“…They have been categorized as a special subgroup of chymotrypsin-like serine endopeptidases (EC 3.4.21.32) [4,5]. Those isolated from the fiddler crab Uca pugilator [6][7][8], king crab Paralithodes camtschatica [9][10][11], greenshore crab Carcinus maenas [12], snow crab C. opilio [13] and many other crustaceans and fish [14,15] resemble mammalian collagenases in splitting telopeptide fragments of native collagen and then slowly digesting alpha chains. This activity differs from that of bacterial collagenases, which attack over 200 positions of the collagen triple helix [10].…”

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

“…After these initial and specific cleavages, 3 = 4 -and 1 = 4 -cleavage products are formed, and collagen is spontaneously denatured (helix-to-coil transition) to gelatin (Miller et al, 1976). Collagenases are classified into two major groups, metallocollagenases and serine collagenases (Aoki, Ahsan, Matsuo, Hagiwara, & Watabe, 2003). Furthermore, non-collagenase proteinases can cleave the collagen molecule in the telopeptide region and contribute to hydrolysis of the collagen molecule by disrupting the regions, in which intermolecular cross-links are formed (Bornstein & Traus, 1979).…”