A chymotrypsin from the Digestive Tract of California Spiny Lobster, Panulirus interruptus: Purification and Biochemical Characterization

Abstract

A chymotrypsin was purified from the gastric juice of California spiny lobster (Panulirus interrutpus), using preparative electrophoresis and affinity chromatography on agarose-p-aminobenzamidine. The molecular mass was estimated by polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions to be 28 kDa. Chymotrypsin activity was totally inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin. Lobster chymotrypsin had optimal pH 7.0-8.0 and temperature of 55 °C. The enzyme is highly stable under a wide range of pH (retaining up to 80 % of activity after 1 h of incubation at pH 3.0, 5.0, and 12.0), showing higher stability at pH 8.0, and was inactivated after 20 min at 55 °C. Lobster chymotrypsin was able to hydrolyze protein substrates at as low as pH 3.0. These results are consistent with the findings of enzyme stability. Activity was assessed after incubation of enzyme with different organic solvents (in the range of 10-50 %); when tested in the presence of acetone, ethanol, propanol, and butanol, lobster chymotrypsin residual activity was >80 %; whereas in the presence of dimethyl sulfoxide (DMSO) and toluene, lobster chymotrypsin residual activity was <80 %. Deduced amino acid sequence, corroborated by mass spectrometry, was determined.

Is digestive cathepsin D the rule in decapod crustaceans?

“…This is partially concordant for invertebrate enzymes as well, lobster cathepsin D, for example, accommodates both hydrophobic and charged amino acids (Asp, Phe, Met, Ala) at P2 (Bibo-Verdugo et al, 2015). Moreover, mammalian cathepsin D shows preference for basic resides at P2' often accepting Arg and Lys.…”

“…Moreover, mammalian cathepsin D shows preference for basic resides at P2' often accepting Arg and Lys. Lobster cathepsin D showed similar specificities when testing the suitability of 7-methoxycoumarin-4-acetyl-Gly-LysPro-Ile-Leu-Phe-Phe-Arg-Leu-Lys-(DNP)-DArg-amide to detect crustacean cathepsin D activity (Bibo-Verdugo et al, 2015). Accordingly, 7-methoxycoumarin-4-acetyl-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys-(DNP)-DArg-amide is an appropriate substrate for detecting cathepsin D activity from lobster.…”

Crustacean Proteases and Their Application in Debridement

“…The tropical lobster trypsins were stable at 55°C for at least 60 min and the same was observed for chymotrypsin ( Perera et al 2012 ). Conversely, a chymotrypsin from the gastric juice of a temperate lobster from the same genus, Panulirus interrutpus , is inactivated after 20 min at the same temperature ( Bibo-Verdugo et al 2015 ). The chymotrypsin from P. interruptus showed collagenase activity because it presents the same residues in the S1 binding pocket, as does the brachyurin from Uca pugilator ( Bibo-Verdugo et al 2015 ).…”

“…Conversely, a chymotrypsin from the gastric juice of a temperate lobster from the same genus, Panulirus interrutpus , is inactivated after 20 min at the same temperature ( Bibo-Verdugo et al 2015 ). The chymotrypsin from P. interruptus showed collagenase activity because it presents the same residues in the S1 binding pocket, as does the brachyurin from Uca pugilator ( Bibo-Verdugo et al 2015 ). The same is likely to be true for chymotrypsin from the tropical lobster P. argus although functional data are not available.…”

Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion

“…In decapod crustaceans, the digestive enzymes are secreted from the midgut gland (or hepatopancreas) and consist mainly of peptidases, glycosidases, lipases and nucleases. Some of these digestive enzymes have been biochemically characterized (Le Chevalier and Wormhoudt 1998;Nilsen et al 2010;Rivera-Perez et al 2011;Bibo-Verdugo et al 2015).…”