Purification and characterization of two chymotrypsin-like proteases from the pyloric caeca of rainbow trout (Oncorhynchus mykiss)

Magnus M. Kristjánsson*, Henrik H. Nielsen

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

57 Citations (Scopus)

Abstract

1. 1. Two chymotrypsins, called chymotrypsin I and II, were purified from the pyloric caeca of rainbow trout, by (NH4)2SO4 fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). 2. 2. The approximate molecular weights of chymotrypsin I and II were 28,200 (± 1200) and 28,800 (±900), respectively, as determined by SDS-PAGE and their isoelectric points were about 5. 3. 3. The pH optima of the enzymes were centered around nine, when assayed for succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroailide (Suc-AAPF-NA) as substrate and both enzymes were unstable at pH values below 5. 4. 4. The amidase activity of both enzymes increased with temperature up to about 55°C. Chymotrypsin I was found to be more heat stable than chymotrypsin II, an effect most likely explained by stronger calcium binding of the former. 5. 5. The trout chymotrypsins were significantly more active than bovine α-chymotrypsin when assayed against Suc-AAPF-NA at 25°C and casein at low temperatures (10-20°C), indicating an adaptation of the activities of the trout chymotrypsins to the habitation temperatures of the fish.

Original languageEnglish
Pages (from-to)247-253
Number of pages7
JournalComparative Biochemistry and Physiology -- Part B: Biochemistry and
Volume101
Issue number1-2
DOIs
Publication statusPublished - 1992

Bibliographical note

Funding Information:
Acknowledgements--The authors would like to thank Mrs Hanne Jacobsen and Mrs Karin H. Reimers for excellent technical assistance in parts of this study. This work was supported by the Danish Biotechnological Research and Development Program.

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