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.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Dmytruk
Kostyantyn V
KV
Institute of Cell Biology, NAS of Ukraine, Lviv, Ukraine. dmytruk@cellbiol.lviv.ua <dmytruk@cellbiol.lviv.ua>
Smutok
Oleh V
OV
Ryabova
Olena B
OB
Gayda
Galyna Z
GZ
Sibirny
Volodymyr A
VA
Schuhmann
Wolfgang
W
Gonchar
Mykhailo V
MV
Sibirny
Andriy A
AA
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004789:Enzyme Activation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D000431:Ethanol / Q000032:analysis*; D005557:Formaldehyde / Q000032:analysis*; D008969:Molecular Sequence Data; D009154:Mutation; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D005557:Formaldehyde; D000431:Ethanol; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase