Abstract
BackgroundThe 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.ResultsA 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.ConclusionA strain of H. polymorpha overproducing UOX was constructed. A cheap urate selective microbial biosensor was developed.
Highlights
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
Urate is not accumulated in human body fluids and its concentration is a valuable indicator in clinical diagnosis [2] indicating gout, hyperuricemia, or Lesch-Nyhan syndrome [3]
We introduce a novel microbial urateselective amperometric biosensor based on the cells of the uricase-overproducing recombinant yeast H. polymorpha
Summary
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. Uricase (urate oxidase, UOX, EC 1.7.3.3) is a key enzyme in the purine degradation pathway. Several new amperometric uric acid biosensors were fabricated by immobilizing uricase onto gold nanoparticles or carbon nanotubes [16,17,18]. These urate-selective biosensors are using expensive commercially available uricases purified from Bacillus fastidiosus, Arthrobacter globiformis, or Candida utilis. The fabrication of cheap urate-selective amperometric biosensors is still a challenging task
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