Search

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]