Aspergillus oryzae acid proteinase. Purification and properties, and formation of pi-chymotrypsin.
An acid proteinase from Aspergillus oryzae was isolated from a commercial powder by successive (NH4)2SO4 fractionation, acetone precipitation, and ion-exchange chromatography on phosphate- and DEAE-cellulose columns. The purified enzyme was found to be homogeneous by ultracentrifuge-sedimentation analysis (S20, W equal 3.63S), but electrofocusing in polyacrylamide gels and electrophoresis at pH 3.2 revealed that it consists of two very closely migrating bands. No difference in the amino acid composition and enzymic activities of the two partially separated bands could be detected, and it was concluded that the acid proteinase exists in two molecular forms. The enzyme activates bovine trypsinogen and chymotrypsinogen at pH 3.5 (the kappacat. and Km values at 35degrees C are 11.3S- minus 1, 0.10mM and 1.14S- minus 1, 0.18mM respectively). It hydrolyses the Phe-Phe bond of the synthetic pepsin substrates Z-His-Phe-Phe-OEt (kappacat. equal 1.65S- minus 1, Km equal 0.640mM at pH 3.5, 30degrees C) and Z-Ala-Ala-Phe-Phe-OPy4Pr (kappacat. equal 0.37S- minus 1, Km equal 0.037 mM at pH2.9, 39degrees C), where Z represents benzyloxycarbonyl and OPy4Pr represents 3-(4-pyridyl)-propyl 1-ester. Activation of bovine chymotrypsinogen results from the cleavage of the Arg(15)-Ile(16) bond in the zymogen. No other cleavages were observed. The use of A. oryzae proteinase provides a simple tool for the production of pi-chymotrypsin in good yield and purity.
D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate; D001230:Aspergillus / Q000201:enzymology*; D001236:Aspergillus oryzae / Q000201:enzymology*; D005591:Chemical Fractionation; D002918:Chymotrypsin / Q000302:isolation & purification*; D002919:Chymotrypsinogen; D010450:Endopeptidases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D010434:Pepsin A; D010446:Peptide Fragments; D014362:Trypsinogen
D000596:Amino Acids; D010446:Peptide Fragments; D014362:Trypsinogen; D002919:Chymotrypsinogen; D010450:Endopeptidases; D002918:Chymotrypsin; D010434:Pepsin A; D000645:Ammonium Sulfate