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Construction of uricase-overproducing strains of Hansenula polymorpha and its application as biological recognition element in microbial urate biosensor

BACKGROUND: The detection and quantification of uric acid in human physiological fluids is of great importance in the diagnosis and therapy of patients suffering from a range of disorders associated with altered purine metabolism, most notably gout and hyperuricaemia. The fabrication of cheap and re...

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Detalles Bibliográficos
Autores principales: Dmytruk, Kostyantyn V, Smutok, Oleh V, Dmytruk, Olena V, Schuhmann, Wolfgang, Sibirny, Andriy A
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3128571/
https://www.ncbi.nlm.nih.gov/pubmed/21612631
http://dx.doi.org/10.1186/1472-6750-11-58
Descripción
Sumario:BACKGROUND: The detection and quantification of uric acid in human physiological fluids is of great importance in the diagnosis and therapy of patients suffering from a range of disorders associated with altered purine metabolism, most notably gout and hyperuricaemia. The fabrication of cheap and reliable urate-selective amperometric biosensors is a challenging task. RESULTS: A urate-selective microbial biosensor was developed using cells of the recombinant thermotolerant methylotrophic yeast Hansenula polymorpha as biorecognition element. The construction of uricase (UOX) producing yeast by over-expression of the uricase gene of H. polymorpha is described. Following a preliminary screening of the transformants with increased UOX activity in permeabilized yeast cells the optimal cultivation conditions for maximal UOX yield namely a 40-fold increase in UOX activity were determined. The UOX producing cells were coupled to horseradish peroxidase and immobilized on graphite electrodes by physical entrapment behind a dialysis membrane. A high urate selectivity with a detection limit of about 8 μM was found. CONCLUSION: A strain of H. polymorpha overproducing UOX was constructed. A cheap urate selective microbial biosensor was developed.