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Construction of uricase-overproducing strains of Hansenula polymorpha and its application as biological recognition
Kostyantyn V Dmytruk1, Oleh V Smutok, Olena V Dmytruk
1Institute of Cell Biology, NAS of Ukraine, Drahomanov Street 14/16, Lviv 79005, Ukraine. dmytruk@cellbiol.lviv.ua
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.
