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 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 ىM was found. Conclusion: A strain of H. polymorpha overproducing UOX was constructed. A cheap urate selective microbial biosensor was developed. Keywords: urate; oxidoreductase; methylotrophic yeast; amperometric biosensor; cell-based biosensor. Background Urate is the final purine intermediate of the purine nucleotide catabolism in human [1]. Normally, urate is not accumulated in human body fluids and hence its concentration is a valuable indicator in clinical diagnosis [2] indicating gout, hyperuricemia, or Lesch-Nyhan syndrome [3]. Elevated urate levels are also related to e.g. increased alcohol and cholesterol consumption, obesity, diabetes, kidney and heart diseases, and they are considered to be a risk factor for cardiovascular diseases [4]. Uricase (urate oxidase, UOX, EC 1.7.3.3) is a key enzyme in the purine degradation pathway. It catalyzes the oxidation of urate in the presence of oxygen, producing allantoin and CO2. Uricase does not require an additional cofactor other than oxygen for the enzymatic reaction and is therefore widely used for the amperometric determination of urate to diagnose gout and the above mentioned diseases and conditions. For urate determination in serum and urine, several biosensors have been reported [5-8]. Numerous amperometric urate biosensors based on uricase or uricase coupled with peroxidase varying in the type of electrode, immobilization technique, ability and type of redox mediator(s), conductive polymers etc. have been described [9-15]. Several new amperometric uric acid biosensors were fabricated by immobilizing uricase onto gold nanoparticles or carbon nanotubes [16-18]. These urate-selective biosensors are using expensive commercially available uricases purified from Bacillus fastidiosus, Arthrobacter globiformis, or Candida utilis. Therefore, the fabrication of cheap urate-selective amperometric biosensors is still a challenging task. In this work, we introduce a novel microbial urate-selective amperometric biosensor based on the cells of the uricase-overproducing recombinant yeast H. polymorpha.