RESUMO
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. CONCLUSION: 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.
Assuntos
Oxirredutases do Álcool/química , Técnicas Biossensoriais/instrumentação , Eletroquímica/instrumentação , Etanol/análise , Formaldeído/análise , Pichia/enzimologia , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Sequência de Bases , Técnicas Biossensoriais/métodos , Eletroquímica/métodos , Ativação Enzimática , Desenho de Equipamento , Análise de Falha de Equipamento , Dados de Sequência Molecular , Mutação , Pichia/genética , Engenharia de Proteínas/métodos , Sensibilidade e Especificidade , Especificidade por SubstratoRESUMO
An extended definition of the term metabolic engineering is given and its successful use in the construction of biorecognition elements of sensors is demonstrated. It is shown that genetic and chemical modifications of methylotrophic yeast cells provide directed changes in their physiological responses towards methanol, ethanol and formaldehyde resulting in enhanced selectivity and shorter time response of the corresponding potentiometric and amperometric biosensors.
