Mechanism 1

Cell Free System

Several methylotrophic yeasts are able to utilize short-chain alcohols as a sole source of energy and carbon. This involves the function of specific enzymes called Alcohol Oxidases (AOx). AOx is the first enzyme involved in the methanol oxidation pathway of yeast and although its physiological role is the oxidation of methanol, it is also able to oxidise other short-chain alcohols, such as ethanol.


Based on this system we have engineered Escherichia coli to express AOx from Pichia pastoris that will then be used in a cell-free colorimetric system. The nature and specificity of the catalytic activity of AOx makes it an excellent tool for chemical analysis.

This cell-free spectroscopic analysis is achieved without the use of living cells. Instead, all components needed to catalyse the alcohols are provided in solution for use in vitro. Colorimetric methods can be used to detect the production of H₂O₂ by AOx during the oxidation of ethanol by the conversion of a chromogenic substrate into a coloured product.


Our mechanisms consists of two catalytic reaction steps. Firstly, in the presence of oxygen, ethanol is oxidised by AOx to acetaldehyde (ethanal), producing hydrogen peroxide (H₂O₂) as a detectable by-product. In the second step, H₂O₂ is then used by horseradish peroxidase (HRP) as a redox substrate to oxidise different chromagens and generate a colour change, where (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) is the most sensitive one, changing from colourless to green.

Different concentrations of ethanol will produce different concentrations of H₂O₂ in the first step of the total reaction. HRP will oxidise ABTS according to the varying amounts of H₂O₂ produced and therefore oxidising more or less dye. This will result in a gradient of colour intensity correlated to the amount of ethanol.