Abstract
Carboxypeptidase-B (E.C 3.4.17.2) catalyzes the hydrolysis of peptides and esters at C-terminus of arginine and lysine residues. Our study describes the large scale purification, N-terminal sequence analysis and physiochemical properties of pancreatic enzyme from river buffalo (Bubalus bubalis). The enzyme was purified up to 71 folds by anion-exchange chromatography with 21% final recovery. Purified enzyme displayed two bands on SDS-PAGE with molecular weights of 9 kDa and 26 kDa respectively, the N-terminal sequence of later was EFLDKLDFYV. The enzyme has shown optimum activity at pH 9.0 and 40 degrees C. The K-M, K-cat and K-cat/K-M values of purified carboxypeptidase-B with Hippuryl-L-Arg are 30 mu M, 72sec(-1) and 2.4x10(5) M-1 sec(-1) respectively. A computer based model for the structure of enzyme was proposed by chromatographic studies of component fragments and N-terminal sequence. The enzyme purified in the present study was free of carboxypeptidase A and endoprotease contamination. It was efficiently used in the processing of recombinant buffalo proinsulin, in combination with trypsin. Activation of proinsulin was monitored by MALDI-TOF analysis of peptides before and after the action of enzymes.