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This study investigates the effects of gradual or abrupt changes in rearing salinity on food transit time and digestive enzymes activity of Caspian kutum (Rutilus kutum) larvae. The larvae (532 ± 0.05 mg) were supplied and randomly allocated into 12 tanks at a density of 45 fish per tank. Experimental treatments were fresh water (salinity 0) [FW] as control, exposure to salinity 5 [T1], and gradual transfer to salinity 10 in two steps of first to 5 h, then and after 12 h to a salinity of 10 [T2], and abrupt change (direct transfer to a salinity of 10 [T3]). Results showed at 8 h after start of feeding that the larvae intestine was filled with food pellets except in treatment T1. Enzyme activity responded to salinity change as follows: the highest trypsin, amylase, and chymotrypsin activities were observed in T1; however, these were not significantly different to treatment T3 (P > 0.05). Trypsin activity peaks in the FW and T2 groups occurred 8 h after feeding, and in T3 and T1 groups 5 h after feeding. Peak chymotrypsin and alkaline phosphatase activity was observed 5 and 8 h after feeding in all experiments, respectively. The highest a amylase activity in FW and T2 groups occurred 5 h after feeding, while in T3 and T1 these peaks were observed 8 h after feeding. These results indicate that salinity had some noticeable effects on the activities of digestive enzymes after feeding. [ABSTRACT FROM AUTHOR]

Abstract: Chymotrypsin from the hepatopancreas of cuttlefish (Sepia officinalis) was purified to homogeneity, with a 120-fold increase in specific activity and 23% recovery. The molecular weight of the purified chymotrypsin was estimated to be 28kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The optimum pH and temperature for the chymotrypsin activity were pH 8.5 and 55°C, respectively, using succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide (SAAPFpNA) as a substrate. The enzyme was extremely stable in the pH range of 7.0–10.0 and highly stable up to 50°C after 1h incubation. This proteinase was strongly inhibited by chymostatin, soybean trypsin inhibitor, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride, but was not inhibited by tosyl-l-phenylalanine chloromethyl ketone, N-carbobenzoxy-phenylalanine chloromethyl ketone or N α-tosyl-l-lysine chloromethyl ketone. The enzyme hydrolysed long chymotrypsin peptide substrates SAAPFpNA, SAAPLpNA and ZAALpNA and did not hydrolyse short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SAAPFpNA, with k cat 18s−1 and K m 22μM. However, the enzyme had a lower K m for SAAPLpNA, 54μM. The N-terminal amino acid sequence of the first 20 amino acids of the purified chymotrypsin was IVGGQEATIGEYPWQAALQV. [Copyright &y& Elsevier]

Abstract: Two chymotrypsins (chymotrypsin A and B) have been purified to homogeneity from the hepatopancreas of crucian carp (Carassius auratus) by ammonium sulphate fractionation and chromatographies on DEAE-Sepharose, Sephacryl S-200 HR, Phenyl-Sepharose and SP-Sepharose. The molecular masses of chymotrypsin A and B were approximately 28 and 27kDa, respectively, by SDS–PAGE. Purified chymotrypsins also revealed single bands by native-PAGE. Optimum temperatures of chymotrypsin A and B were 40 and 50°C, and optimal pHs were 7.5 and 8.0 using Suc-Leu-Leu-Val-Tyr-AMC as substrate. Both enzymes were effectively inhibited by serine proteinase inhibitors and slightly activated by metal ions such as Ca2+ and Mg2+, while inactivated by Mn2+, Cd2+, Cu2+, Fe2+ to different degrees. Apparent K ms of chymotrypsin A and B were 1.4 and 0.5μM, and K cats were 2.7S−1 and 3.4S−1, respectively. Immunoblotting analysis using anti-chymotrypsin B weakly cross reacted with chymotrypsin A. [Copyright &y& Elsevier]

The effect of change in feed and physical manipulation on the digestive system of white shrimp Penaeus vannamei (Boone, 1931) was examined for proteases secreted by the digestive gland and excreted in feces. Organisms were fed a commercial feed containing 45% protein and separated into two groups. One group served as the control and the other was physically manipulated weekly. Each group was subdivided into two groups, establishing four experimental subgroups, 50% of the organisms in each group used as controls and the others subjected to a change in feed by shifting from a 45% protein feed to a 35% protein feed from different brands to induce alimentary stress. Organisms were individually maintained and feces collected and analyzed on a daily basis for 2 months. Trypsin and chymotrypsin activities decreased in feces and mid-gut gland extracts of organisms fed the 35% protein feed and those physically manipulated during weighting. Two-way ANOVA demonstrated a greater effect of physical manipulation on trypsin and chymotrypsin activities than change in feed. One-way ANOVA demonstrated differences between the two periods analyzed (before and after the beginning of stressors). Composition of proteases in the mid-gut gland and feces were identical, as observed by S-SDS PAGE. Studies of enzyme activities in feces may be helpful, when studying organisms of short digestion periods where very active enzymes and low-efficiency digestion are expected, as in the case of decapods. [Copyright &y& Elsevier]