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.
In vitro assays used porcine or bovine trypsin as models of exogenous enzymes to determine functioning in the presence of enzymatic extracts from the digestive gland of whiteleg shrimp Penaeus vannamei. Using electrophoresis and zymograms, when enzymes from the shrimp were mixed in the absence of protein substrate, they hydrolysed the trypsin from bovine or porcine origin. Porcine or bovine trypsin, when mixed with shrimp enzymes in pHstat assays in the presence of shrimp commercial feed, fish meal, or casein, there was added activity to hydrolyse the protein substrate. Hydrolysis of protein substrate was twofold to threefold stronger if exogenous enzymes were added. Results suggest that porcine or bovine trypsin could be used as feed supplements for whiteleg shrimp P. vannamei to enhance hydrolysis of proteins in feeds, because the commercial enzymes contributed to the hydrolysis of the protein in the three substrates in the presence of shrimp enzymes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.