Isolation of a trypsin-like enzyme from Streptomyces paromomycinus (paromotrypsin) by affinity adsorption through Kunitz inhibitor-sepharose.
A trypsin-like enzyme has been isolated from the filtrate of a Streptomyces rimosus forma paromomycinus culture. Purification involves acetone fractionated precipitation, ultrafiltration on a Diaflo UM 10 membrane and affinity adsorption on to Kunitz pancreatic trypsin inhibitor linked to Sepharose. The trypsin-like enzyme (paromotrypsin) appears homogeneous by zone electrophoresis on gelatinized cellulose acetate. Specific activity toward Tos-Arg-OMe, calculated from amino acid analysis, is about 220 mu mg-1. The overall yield in activity is about 30%. The molecular weight of the trypsin-like enzyme, determined by gel filtration, is around 22,000-25,000 daltons. Electrophoretic migration on cellulose acetate strips indicates an isoelectric point around 8. Amino acid composition has been determined; the protein comprises about 210 residues on the basis of a single histidine residue per molecule. Paromotrypsin is unstable in acidic medium and is not stabilized by calcium ions. Enzymic activity towards Bz-Argo-OEt is not increased by the addition of calcium ion in contrast to the activating effect observed on bovine trypsin. Paromotrypsin is inhbited by TLCK and NPGB; it interacts with naturally occurring bovine trypsin inhibitors such as soya bean and Kunitz pancreatic inhibitors, but not with chicken ovomucoid. Proteolytic specificity, examined by hydrolysis of oxidized Kunitz pancreatic inhibitor and characterization of resulting peptides, seems similar to that of bovine trypsin.
D000327:Adsorption; D000596:Amino Acids / Q000032:analysis; D002118:Calcium / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D008970:Molecular Weight; D010446:Peptide Fragments; D010447:Peptide Hydrolases / Q000032:analysis / Q000302:isolation & purification*; D013302:Streptomyces / Q000201:enzymology*; D014360:Trypsin Inhibitor, Kunitz Soybean
D000596:Amino Acids; D010446:Peptide Fragments; D014360:Trypsin Inhibitor, Kunitz Soybean; D010447:Peptide Hydrolases; D002118:Calcium