A rapid method to detect and estimate the activity of the enzyme, alcohol oxidase by the use of two chemical complexes - acetylacetone (3,5-Diacetyl-1,4-dihydroludin) and acetylacetanilide (3,5-Di-N-phenylacetyl-1,4-dihydroludine).

A rapid and sensitive method has been devised in order to detect and estimate the synthesis of the alcohol oxidase (AOX) enzyme by the fungi, by way of the use of two chemical complexes namely, acetylacetone (3,5-Diacetyl-1,4-dihydroludine) and acetylacetanilide (3,5-Di-N-phenylacetyl-1,4-dihydroludine). This method involves the use of the AOX enzyme that could specifically oxidize methanol, giving rise to a μmol equivalent each of formaldehyde (HCHO) and hydrogen peroxide (H2 O2 ) as the end products. Further, the formaldehyde, thus produced was allowed to interact with the neutral solutions of acetylacetone and the ammonium salt, gradually developing a yellow color, owing to the synthesis and release of 3,5-Diacetyl-1,4-dihydroludine (Yellow product; λ = 420 nm; λex/em  = 390/470 nm) and the product, so generated was quantified spectrophotometrically by measureing its absorbance at 412 nm. In an other set up, the amount of formaldehyde produced as a sequel to the oxidation of methanol by the AOX enzyme was determined by allowing it to react with the acetylacetanilide reagent, after which the volume of the fluorescent product - 3,5-Di-N-phenylacetyl-1,4-dihydroludine (colorless product; λex/em  = 390/470 nm) that was generated was estimated by measuring its emission at 460 nm (excitation wavelength at 360 nm) in a spectrophotometer. Of the various substrates tested, a commercial source of the AOX enzyme appreciably oxidizes methanol, thereby generating formaldehyde, and further reacts with acetylacetone, to give rise to a bright yellow complex, displaying a maximum activity of 1402 U/mL. Determination of the AOX activity by the use of acetylacetone and acetylacetanilide could serve as a viable alternative to the conventional alcohol oxidase-peroxidase- 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (AOX-POD-ABTS) based method. In view of this, this method appears to be invaluable for application at the various food, pharmaceutical, fuel, biosensor, biorefinery, biopolymer, biomaterial, platform chemical, and biodiesel industries.