Stability and effects of organic solvents on endopeptidases from the gastric fluid of the marine crab Cancer pagurus

Abstract

Activities of proteolytic enzymes, represented by azocasein digestion (total protease), trypsin, and chymotrypsin from the gastric fluid of the marine crab, Cancer pagurus, were evaluated for operational parameters like pH-optimum, thermal stability, and effectors such as metal ions and organic solvents. Total protease activity was 98.5 ± 4.3 E 366 min −1 ml −1 . Trypsin and chymotrypsin activities amounted to 14.7 ± 1.5 U ml −1 and 197 ± 59 U ml −1 , respectively. The maximum activity of total protease appeared at pH 5-7, that of trypsin at pH 7-9, and that of chymotrypsin at pH 6.5-7. Trypsin and chymotrypsin activities were slightly activated by Ca 2+ , but were drastically reduced by Zn 2+ . In the presence of 2-propanol, trypsin activity was enhanced 8-fold at 30• C. Highest amplification of activity of up to 30-fold, however, appeared below 12• C. The effects of ethanol and acetone were less distinct. These caused a 2.5-fold increase of activity. Methanol was the least activating solvent.Activities of all enzymes remained stable for up to 127 days at 5• C, keeping up to 70% of the initial activity. When stored at 25• C for 120 days, total protease decreased to about 40% and trypsin and chymotrypsin activities kept more than 60% of initial activity. Zymograms showed five major and several minor proteolytic bands with apparent molecular weight of 20-45 kDa. After long-term storage at room temperature, the major bands remained active, while some bands with minor activity disappeared. This proteolytic enzyme preparation from the gastric fluid of crab seems suitable for biotechnological application at neutral pH and in the presence of organic solvents.

Digestive enzymes of two brachyuran and two anomuran land crabs from Christmas Island, Indian Ocean

“…Using the methods of Teschke and Saborowski (2005) and Saborowski et al (2004) total protease, trypsin, chymotrypsin and alanine amino peptidase activities were measured in the digestive fluid (Table 2). Inhibition assays were also conducted on the digestive fluid using the methods of Teschke and Saborowski (2005) to determine the percentage of serine, cysteine and other proteases, possibly metalloproteases within it (Table 2).…”

Crystallization and preliminary X-ray diffraction studies of trypsin-like proteases from the gastric fluid of the marine crabCancer pagurus

“…Based on similarity searches including the available structures of trypsins from different species, a typical trypsin-like fold containing two -barrel domains is expected for the structures of both C. pagurus trypsins. Thus, it is specific sequence modifications and smaller structure adjustments that must explain the extraordinary properties of crab trypsins (Saborowski et al, 2004). In terms of C.p.TryIII, the expected 12 molecules per asymmetric unit will complicate the determination of the molecular structure.…”

“…In addition to high activity, the trypsin-like enzymes from the gastric fluid of the edible marine crab Cancer pagurus display some unique properties such as long-term stability and enhanced activity in organic solvents (Saborowski et al, 2004). C. pagurus inhabits wide areas of the northeast Atlantic.…”

Food utilisation and digestive ability of aquatic and semi-terrestrial crayfishes, Cherax destructor and Engaeus sericatus (Astacidae, Parastacidae)

“…The assays for total proteinase, trypsin-like and chymotrypsin-like activities were as outlined by Saborowski et al (2004). Total proteolytic activity was determined by the release of azo dye from the hydrolysis of azocasein (Fluka, 11615).…”