Isolation and characterization of mutated alcohol oxidases from the yeast Hansenula polymorpha with decreased affinity toward substrates and their use as selective elements of an amperometric biosensor

Hansenula polymorpha (Pichia angusta) mutant strains resistant to allyl alcohol in methanol medium were selected. Such strains possessed decreased affinity of alcohol oxidase (AOX) towards methanol: the KM values for AOX of wild type and mutant strains CA2 and CA4 are shown to be 0.62, 2.48 and 1.10 mM, respectively, whereas Vmax values are increased or remain unaffected. The mutant AOX alleles from H. polymorpha mutants CA2 and CA4 were isolated and sequenced. Several point mutations in the AOX gene, mostly different between the two mutant alleles, have been identified. Mutant AOX forms were isolated and purified, and some of their biochemical properties were studied. An amperometric biosensor based on the mutated form of AOX from the strain CA2 was constructed and revealed an extended linear response to the target analytes, ethanol and formaldehyde, as compared to the sensor based on the native AOX.The described selection methodology opens up the possibility of isolating modified forms of AOX with further decreased affinity toward substrates without reduction of the maximal velocity of reaction. It can help in creation of improved ethanol biosensors with a prolonged linear response towards ethanol in real samples of wines, beers or fermentation liquids.The detection and quantification of alcohols with high selectivity and accuracy is required in many different areas. Accurate and rapid measurement of ethanol is important in clinical and forensic practice to analyse human body fluids, e.g. blood, serum, saliva, urine, breath and sweat, among others. The food, beverage (wine, beer and spirits), and pulp industries also require simple, correct, fast, and economic analytical methods to control fermentation processes and the quality of products.Enzymatic and biosensor approaches are among the most convenient analytical methods for this purpose. Yeast alcohol oxidase (AOX; alcohol:O2 oxidoreductase, EC 1.1.3.13) has been extensively used for the determination of lower p