Properties of new enzyme glycerol oxidase from Aspergillus japonicus AT 008.

Abstract

Purified glycerol oxidase from Aspergillus faponicus AT 008 was homogeneous by ultracentrifugation and acrylamide gel electrophoresis. The molecular weight was determined to be 400,000 by sedimentation equilibrium, and the isoelectric point was found to be 4.9 by isoelectric focusing. The enzyme showed spectral characteristics of a heme protein. The reduced form possessed absorption maxima at 557 and 430 nm and the oxidized one at 557, 530, 420, 280, and 238 rim. The heme in the enzyme was identified as protoheme IX (one mol per mol of enzyme protein). Glycerol was the best substrate for the enzyme, and the Km value for glycerol was determined to be 10.4 mal. Dihydroxyacetone was oxidized at 59 % of that for glycerol, but glycerol 3-phosphate, dihydroxyacetone phosphate, methanol, and ethanol were not oxidized at all. The enzyme had an optimal pH at 7.0 with glycerol as substrate, and the enzymatic activity increased by treatment in alkaline pH. The enzyme was also activated by addition of several divalent metal ions including Zn2+, Nit+, and Mgt+.

The substrate tolerance of alcohol oxidases

“…Dihydroxyacetone (Table 7 , entry 36) was also oxidised at a fair rate by glycerol oxidase (GlycOx) from Aspergillus japonicus (59 % rel. activity) (Uwajima and Terada 1980 ). Furthermore, galactose oxidase was active on guaran, a galactomannan (Table 7 , entry 38) (47 % rel.…”

“…Glycerol was oxidised to l -glyceraldehyde as a building block for a follow-up aldolase reaction in a multienzyme cascade (Franke et al 2003 ). The latter is also oxidised by the Cu-containing glycerol oxidase which exhibited excellent activity towards glycerol, which was selected as a name-giving substrate (Uwajima and Terada 1980 ; Uwajima et al 1984 ). The building block dihydroxyacetone phosphate (DHAP), which is a popular C donor in asymmetric aldol reactions, can be obtained using flavoprotein glycerol 3-phosphate oxidase (GPO) [EC 1.1.3.21] for the oxidation of l -glycerol 3-phosphate (Table 8 , entry 10) at the sec -OH (Babich et al 2011 ).…”

“…For screening purposes, a spectrophotometric assay based on horse radish peroxidase (HRP) together with a suitable artificial electron acceptor, such as 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) may be employed (Scheme 1 ). The ABTS-radical generated shifts its absorption maximum (Baron et al 1994 ; Uwajima and Terada 1980 ).

Scheme 1 Biocatalytic oxidation of alcohols using an alcohol oxidase

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Purification and Properties of Two Different Dihydroxyacetone Reductases inGluconobacter suboxydansGrown on Glycerol

“…[19][20][21] These enzymes are divided into two groups: GALforming and DHA-forming. With respect to DHAforming glycerol dehydrogenase (GLDH), the NADdependent enzyme has been extensively studied, while only a few preliminary papers are available on the NADP-dependent enzyme, and only from eukaryotic microorganisms such as halophilic alga Dunaliella parva [10][11][12] and Mucor javanicus, 13) not from prokaryotes.…”