Amine Oxidases of Microorganisms

Part VII. An Improved Purification Procedure and Further Properties of Amine Oxidase of Aspergillus niger

Abstract

An improved purification procedure is described for preparation of crystalline amine oxidase in higher yields from the mycelial extract of Aspergillus niger. The amine oxidase was accumulated in mycelia of the fungus when it was grown in a medium containing n-butylamine as a sole nitrogen source. The n-butylamine was fed from the alkali tank of a pH stat connected with the jar fermenter, to maintain pH of the culture medium at 5.0_??_5.5 during the incubation.
The amine oxidase was shown to be inhibited by guanidine hydrochloride and dissociation of the enzyme occurred when it was dialyzed against 6M guanidine hydrochloride containing 0.1M mercaptoethanol. The native enzyme of its molecular weight of 252, 000 dissociated into three subunits of each molecular weight of 85, 000 during the dialysis.
Pyridoxal derivatives were detected in digest of the native enzyme by proteolysis followed by acid hydrolysis. The derivatives showed a biological activity to support the growth of Saccharomyces carlsbergensis. Fluorescence property of these derivatives rather resembled to that of the known pyridoxal derivatives. A ¹⁴C-labelled protein was prepared from a solution containing the enzyme and ethylamine-1-¹⁴C, through the reductive action of sodium borohydride. On acid hydrolysis of the protein, a radioactive compound was obtained that has chromatographic and fluorescence properties of pyridoxylethylamine.