The purification and characterization of a chymotrypsin from the hepatopan‐creas of the white shrimp Penaeus vannamei is described. Only one chymotrypsin was detected in contrast to other shrimp that have two major forms. P. vannamei chymotrypsin has a molecular mass of 33.2 kDa and a pI of 3.1. The molecular mass is high relative to other penaeid chymotrypsins. The proteinase is acid labile and exhibits optimum activity at pH 8. The enzyme is thermostable both at 25 and 37C. It is a serine proteinase. Phenylmethylsulphonyl fluoride and soybean trypsin inhibitor blocked the activity of the enzyme, and it was not affected by chymotrypsin inhibitors such as tosyl‐PheCH2Cl or benzyloxycarbonyl‐Phe‐CH2Cl. Protein profiles of the hepatopancreas from two populations varied
Juvenile white shrimp, Penaeus vannamei, were studied using tray feeders in relation to food ingestion and enzymatic activity, with data taken every two hours for five days. The feed ingestion was calculated by subtracting the uningested and the leached material from the food provided. Significant differences (P < 0.05) in the ingestion rate were found during the experiment. The greatest ingestion occurred between 2000 and 2200 hours. The largest feed ingestion coincided with the nocturnal activity of shrimp. Time-series analysis showed a cycle of increased ingestion before midnight and lower ingestion during daylight. Total protease, trypsin, and chymotrypsin activity were measured during the last 24 hours of the study and included both fed and starved organisms. The enzymatic pattern was similar for both groups. There was a decrease of the protease and trypsin activities before and after 1800 hours, just before the ingestion increased. The study of digestive enzymes provided evidence of a close association between behavior and regulation of the digestion system physiology. The results
During shrimp larval development, changes occur in molecular components. Enzyme activity and mRNA expression of proteinases were assayed in Penaeus vannamei during larval development, which consists of 5 nauplius stages, 3 protozoeal stages, 3 mysis stages, and 12 postlarval stages. Trypsin activity reached a maximum at the beginning of postlarval stages 1 and 2, and significantly decreased in subsequent postlarval stages. Chymotrypsin activity increased at the third protozoeal stage, then significantly decreased in subsequent stages. Identification of proteinase by mass spectrometry and inhibitors allowed us to track their appearance in zymograms and to distinguish between isoenzymes. Chymotrypsin BI and BII had a distinguishing pattern of appearance during larval development, which could compensate for the reduction in trypsin activity. The mRNA content of isotrypsin 21, chymotrypsin 1, and zinc proteinase was differentially expressed in larvae. Zinc proteinase and chymotrypsin 1 mRNA were expressed at a basal content at the beginning of the protozoeal stages, increased by the end of the mysis stages and onward, while isotrypsin 21 mRNA had a peak at mysis stage 3. Transcript changes reflect transcriptional regulation of the proteinases tested. Proteinase mRNA in tissues, other than the digestive gland, suggests potentially different roles besides digestion during ontogeny.
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