Isolation and Characterization of a Trypsin from the Slipper Lobster,Thenus orientalis(Lund)

Abstract

Trypsin has been isolated and purified from the digestive glands of the slipper lobster,Thenus orientalis.It is a glycoprotein with a molecular mass of approximately 35 kDa as judged by both SDS–PAGE and gel filtration. The N-terminal amino acid sequence has strong homology to crustacean trypsins. This is confirmed by the cross-reaction of crustacean trypsins with antibodies to theT. orientalisenzyme. Despite a 40% identity with the bovine trypsin N-terminal sequence, there was no cross-reaction with the mammalian serine proteases. The optimumkcatandkcat/Kmvalues forN-α-benzoylarginine-p-nitroanalide were 0.91 s−1and 9.7 × 103M−1s−1, respectively, with this specificity constant being lower than those reported for other crustacean trypsins. Inhibition studies indicated the presence of serine and histidine at the active site and pKaof the catalytic histidine residue was found to be 5.7 in the free enzyme and 4.7 in the Michaelis complex.