Isolation and purification of an aminoacylase fromAspergillus oryzae
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An aminoacylase (EC 126.96.36.199) has been isolated from a surface culture of the fungusAspergillus oryzae (amilorizin P10X) with a 764-fold degree of purification, an activity yield of 32.7%, and a specific activity in relation to the hydrolysis of N-acetyl-D,L-methionine of 99.3 a.u./o.u. The scheme of the purification of the aminoacylase fromAspergillus oryzae includes: extraction at pH 6.7, precipitation with ammonium sulfate (30 and 80% saturation), gel filtration on Acrilex P-150 (pH 7.5), ion-exchange chromatography on amino-Silochrom Cx-1,5 (mean pore radius 790 Å); the sorption of the enzyme takes place at pH 6.2 and elution with 0.05 M borate buffer, pH 8.0; ion-exchange chromatography on AH-Sepharose 4B at pH 8.0, with elution by a stepwise increase in the concentration of sodium chloride to 0.25 M; and, finally, gel filtration on Sephadex G-200 (pH 8.0). According to the results of disk electrophoresis in 7.5% polyacrylamide gel in a Tris-glycine systems of buffers with a separation pH of 8.9 in the presence of Co2+ ions (10−5 M) of theAspergillus oryzae aminoacylase, two components possessing enzymatic activity were detected, with Rf 0.53 (major component) and Rf 0.63 (minor component).
KeywordsAmmonium Sulfate Borate Buffer Surface Culture Disk Electrophoresis Silochrom
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