No abstract

A southwest Atlantic croaker protease was purified from the pyloric caeca by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The enzyme was classified as a trypsin on the basis of molecular weight, its ability to hydrolyze synthetic substrates N‐α‐benzoyl‐arginine‐p‐nitroanilide (BAPA) and tosylarginine methyl ester (TAME), and its inhibition by known trypsin inhibitors. The isolated enzyme has a single band on SDS‐PAGE with an estimated molecular mass of 24 kDa. Croaker trypsin activity was stable between pH 5 and pH 11 for 30 min at 0C, 10C and 25C and its maximal activity against BAPA was at pH 9.5. Thermostability was observed to about 55C for 30 min at pH 7.8 and its temperature optimum was 60C. Substrate turnover number was 850 BAPA units per μmol trypsin, and the Kmwas 0.081 mM at 25C. For the hydrolysis of TAME, Vmaxwas 9273 units per μmol trypsin and the Km was 0.155 mM at 25C. The catalytic efficiency Vmax/Kmwas higher than that of trypsin from fish living in colder waters.

Two digestive proteases from Parona signata (Palometa) were isolated from gastric and pyloric caeca tissues and characterized. The stomach enzyme was inhibited by pepstatin and was classified as pepsin. Palometa pepsin purified from a Sephadex G‐100 column appeared as two bands on an SDS‐PAGE gel and exhibited optimum activity at pH 3.5, and 37C. Palometa pepsin was inhibited by pepstatin to a similar exient as porcine pepsin. An enzyme isolated from the pyloric caeca was shown to be trypsin based on its molecular weight, its ability to hydrolyze the synthetic substrates, N‐α‐benzoyl‐arginine‐p‐nitroanilide (BAPA), and tosyl‐arginine methyl ester (TAME) and inhibition by the known trypsin inhibitors, SBTI, TLCK, PMSF and benzamidine. The purified trypsin from palometa pyloric caeca yielded a single band with a molecular mass of 24 kDa. Optima trypsin activity was obtained at pH 8.5 and the enzyme was stable over a pH range of 3 to 11. Palometa trypsin activity was stable for 30 min at 50C, but lost 50% of its activity after 30 min at 60C. The optimum temperature for activity was 65C. The biochemical properties of Palometa trypsin and pepsin were discussed in relation to the varying environment of Palometa in the Rio de la Plata estuary.

A protease classified as trypsin was isolated and puriJiedfrom the pyloric caeca of two species offshes: Brevoortia spp (menhaden) and Mugil spp. (mullet). The characterization of both enzymes as trypsin was based on their molecular weight (24 kDa) determined by SDS-PAGE, their inhibition by some known trypsin inhibitors (PMSF, SBTI, TLCK and benzamidine), and their ability to hydrolyze the synthetic tiypsin substrate N-a-Benzoyl-DL-arginine-p-nitroanilide (BAPA). Menhaden trypsin had maximum activity at p H 9.5 and was stable for 30 min between p H 6.0 and 10.0 at 0, 10 and 25C. On the other hand, mullet trypsin showed maximum activity atpH'sfrom 7.8 to 9.0, and was stable over a widerpH range (7.0-10.0). The optimum temperature for menhaden and mullet trypsin was 63C and 60C, respectively. Thermostability for menhaden tiypsin was up to SOC, whereas mullet trypsin was stable up to 60C. The KrniBApA) values for menhaden trypsin were conserved in the temperature range from 10 to 30C, while for mullet trypsin the Krn(BApA) was conserved between 10 and 25C.

Trypsins from the pyloric caeca of menhaden (Brevoortia spp.) and croaker (Micropogonias furnieri) were used for hydrolysis of both myofibrillar and sarcoplasmic fractions from menhaden muscle. Digestion of muscle proteins was carried out at 37C for 20 min with the pH maintained at either 3, 4, 5, 6, 7, 8 or 9; or for 60 min at the pH's of 5, 7, 8 or 9 at 30C and 37C. The hydrolytic action was evaluated based on the concentration of peptides solubilized. Solubility after digestion of myofibrillar, sarcoplasmic fraction and hemoglobin was optimum at basic pH. The sarcoplasmic fraction was solubilized more than the myofibrillar fraction by both fish trypsins. Similar results were obtained with both crude pyloric extracts and purified trypsins from menhaden and white croaker. Thus, the preparation of the crude pyloric caeca may be preferentially used, because of its low cost as well as the simplicity of the preparative procedure.