Novel alcohol oxidase with glycolate oxidase activity from Ochrobactrum sp. AIU 033

• Novel alcohol oxidase, which catalyzes oxidation of glycolate to glyoxylate, was found from Ochrobactrum sp. AIU 033.
• The enzyme showed activity for primary alcohols (C2–C10) and glycolate, but not for glyoxylate.
• The enzyme was composed of an α2β2 structure, in which the α subunit was 52 kDa and the β subunit was 14 kDa.
• The enzyme was a flavoprotein and contained two iron atoms.
• The enzyme would be useful in the enzymatic production of glyoxylate.

We revealed that Ochrobactrum sp. AIU 033, which accumulated a high concentration of glyoxylate from glycolate, produced an enzyme catalyzing oxidation of glycolate to glyoxylate. The enzyme further oxidized lactate and primary alcohols (C2–C10), but did not oxidize glyoxylate, ethylene glycol, glycerol, or methanol. The Km value for glycolate (167 mM) was higher than that for primary alcohols. The glycolate oxidase activity was optimum at pH 5.5, and more than 80% of the enzyme activity remained in the pH range from 5.5 to 6.5 and at below 35 °C. The enzyme had a molecular mass of 130 kDa and was composed of an α2β2 structure, in which the α subunit was 52 kDa and the β subunit was 14 kDa. The enzyme was a flavoprotein and contained two iron atoms. The N-terminal sequences of the 52 kDa subunit and 14 kDa subunit had high similarity to those of putative glucose–methanol–choline oxidoreductases and putative 2-keto-gluconate dehydrogenase. These findings implied that the enzyme was a novel type of alcohol oxidase exhibiting glycolate oxidase activity. The enzyme accumulated glyoxylate with time, but oxalate, which is the oxidation product of glyoxylate, was not detected. This result also indicated that the enzyme catalyzed the formation of glyoxylate in the resting cell-reaction and thus could be useful in the enzymatic production of glyoxylate.

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