Aspergillus oryzae acid proteinase. Purification and properties, and formation of π-chymotrypsin

Abstract

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.

Aspergillus oryzaeGB-107 Fermentation Improves Nutritional Quality of Food Soybeans and Feed Soybean Meals

“…It is well documented that A. oryzae secretes proteases, including aminopeptidases, serine endopeptidases, aspartic endopeptidases, etc. 30,31 Sardjono et al 23 demonstrated that protease activity from A. oryzae reached its highest levels after a 48-hour fermentation and had no further increase after a 48-hour fermentation. This suggests that the 48-hour fermentation used in this study provides the most efficient production of small-size peptides, without excessive nutrient use for metabolism.…”

Photoresponsive Peptide and Polypeptide Systems, 14. Biodegradation of Photocrosslinkable Copolypeptide Hydrogels ContainingL-Ornithine andδ-7-Coumaryloxyacetyl-L-ornithine Residues

“…(Figure 6 c). These results suggest that the productive secretions of hydrolytic enzymes, such as proteases [23] from the microorganism during their growth caused the degradation of the gel materials.…”

Cloning and Nucleotide Sequence of the Acid Protease-encoding Gene (pepA) fromAspergillus oryzae

“…Davidson et al 27 ) and Tujita and End0 28 ) have independently purified acid proteases from commercial enzyme preparations of A. oryzae and have analyzed their amino acid compositions, which correlate well. In addition, the amino acid composition of the mature protein deduced from DNA sequence of the cloned pepA gene is almost identical with those reported (Table I).…”