Purification and Characterization of Ovine Pancreatic Proteases and Comparison with the Equivalent Porcine Enzymes

Abstract

Purification of ovine and porcine pancreatic elastase I was initially achieved using affinity chromatography, ion exchange chromatography and chromatofocusing. The purification of elastase I, elastase II, carboxypeptidase A, carboxypeptidase B, chymotrypsin and trypsin from sheep and pig pancreas is described in conjunction with a proposed industrial process. The characterization and comparison of ovine and porcine enzymes are described. Ovine and porcine proteases were compared using kinetic parameters Km, kcat and kcat/Km. Ovine elastase I was functionally similar to that of porcine elastase I. The specific activities of purified ovine and porcine elastase I were 7.19 µmol/min/mg and 9.61 mmol/min/mg respectively at 25˚C in 100 mM Tris-HCl containing 10 mM Cacl2 at pH 8.0 and 1 mM N-succinyl-Ala-Ala-Ala-para-nitroanilide. The apparent Km for ovine elastase I at 25˚C at pH 8.0 was 2.35 mM± 0.298 and that for porcine elastase I 1.13 mM±0.0598. The comparison of catalytic efficiency kcat/Km shows that those for ovine elastase I and porcine elastase I are generally similar over a range of substrates, temperatures and pH values. Ovine and porcine elastase II were shown to have similar substrate specificity. Porcine elastase II was shown to have a 10-fold higher affinity compared to ovine elastase II toward N-succinyl-Ala-Ala-Pro-Leu-pNA. However, ovine elastase II was approximately 50 to 100-fold more effective at hydrolyzing this substrate than porcine elastase II. The catalytic efficiency for ovine elastase II was eight times greater at pH 8.2 with N-succinyl-Ala-Ala-Pro-Leu-pNA than that for porcine elastase II. The specific activity for ovine and porcine elastase II was 1.6 and 1.34 amol/min/mg at 25˚C at pH 8.2 and 0.1 mM N-succinyl-Ala-Ala-Pro-Leu-pNA. Partially purified carboxypeptidase A and B extracts were prepared for industrial trials. The preliminary kinetic comparison made between the two species indicated that ovine carboxypeptidase B had properties similar to that of porcine carboxypeptidase B. In contrast, porcine carboxypeptidase A was more effective than ovine carboxypeptidase A. The specific activities for ovine and porcine chymotrypsin isolated by affinity chromatography were 32.8 and 31.2 µmol/min/mg respectively at pH 8.5, 25˚C and 0.1 mM N-succinyl-Ala-Ala-Pro-Phe-pNA. Comparison of ovine and porcine chymotrypsin’s kinetic parameters indicated that they were similar. The apparent Km for ovine and porcine chymotrypsin toward N-succinyl-Ala-Ala-Pro-Phe-pNA at pH 8.0 at 20˚C were 0.0464 mM±0.00313 and 0.0494 mM±0.00164 respectively. The comparison of the Michaelis constant for ovine and porcine trypsins toward Nα-benzoyl-DL-arginine-pNA indicated that ovine trypsin had twice the affinity of porcine trypsin toward the substrate. The catalytic efficiency for ovine trypsin was twice that of porcine trypsin. The apparent Km for ovine and porcine trypsin toward Nα-benzoyl-DL-arginine-pNA at pH 8.0 at 25˚C were 0.537 mM±0.00841 and 1.72 mM±0.208 respectively. The specific activities for ovine and porcine trypsins isolated by affinity chromatography were 0.893 and 0.31 µmol/min/mg respectively at pH 8.5, 25˚C and 0.1 mM Nα-benzoyl-DL-arginine-pNA. The effect of temperature on the binding affinity and kcat are described for ovine and porcine elastase I, elastase II, chymotrypsin and trypsin. Physical properties and substrate specificity for all enzymes were compared. Protein hydrolysates of β-lactoglobulin were analyzed and compared between the two species using reverse phase high performance liquid chromatography and matrix-assisted laser desorption / ionization time of flight mass spectrometry. Activation of ovine and porcine proteases was studied to optimize the extraction conditions and to improve yields in the proposed industrial processes. Serine protease yields obtained from ovine and porcine pancreas indicted that porcine pancreas had higher amounts of trypsin and elastase I whereas ovine pancreas had higher levels of elastase II and chymotrypsin.