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

<p>Abstract</p> <p>Background</p> <p>Accurate, rapid, and economic on-line analysis of ethanol is very desirable. However, available biosensors achieve saturation at very low ethanol concentrations and thus demand the time and labour consuming procedure of sample dilution.</p> <p>Results</p> <p><it>Hansenula polymorpha </it>(<it>Pichia angusta</it>) mutant strains resistant to allyl alcohol in methanol medium were selected. Such strains possessed decreased affinity of alcohol oxidase (AOX) towards methanol: the K_Mvalues 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 V_maxvalues are increased or remain unaffected. The mutant AOX alleles from <it>H. polymorpha </it>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.</p> <p>Conclusion</p> <p>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.</p

Alignment of deduced amino acid sequences for AOX from the mutant strains of CA2, CA4 and the wild type DL1

<p><b>Copyright information:</b></p><p>Taken from "Isolation and characterization of mutated alcohol oxidases from the yeast with decreased affinity toward substrates and their use as selective elements of an amperometric biosensor"</p><p>http://www.biomedcentral.com/1472-6750/7/33</p><p>BMC Biotechnology 2007;7():33-33.</p><p>Published online 13 Jun 2007</p><p>PMCID:PMC1905912.</p><p></p

Comparison of an operational stability of nAOX and mAOX-modified electrodes

<p><b>Copyright information:</b></p><p>Taken from "Isolation and characterization of mutated alcohol oxidases from the yeast with decreased affinity toward substrates and their use as selective elements of an amperometric biosensor"</p><p>http://www.biomedcentral.com/1472-6750/7/33</p><p>BMC Biotechnology 2007;7():33-33.</p><p>Published online 13 Jun 2007</p><p>PMCID:PMC1905912.</p><p></p