Isolation of a novel uric-acid-degrading microbe Comamonas sp. BT UA and rapid biosensing of uric acid from extracted uricase enzyme

摘要

Uric-acid-utilizing soil bacteria were isolated, and 16s rRNA sequence was studied for strain identification. The most prominent uricase-producing bacterium was identified as Comamonas sp BT UA. Crude enzyme was extracted, freeze-dried and its Km and Vmax were determined as 40 e???M and 0.047 e???M mina?’1mla?’1 using Line-weaver Burke plot. An activity of 80 U/mg of total protein was observed when cultured at 37?°C for 84 h at pH 7. The purified enzyme was used to measure uric acid by spectrophotometric method and electrochemical biosensor. In the biosensing system the enzyme was immobilized on the platinum electrode with a biodegradable glutaraldehyde-crosslinked gelatin film having a swelling percentage of 109?±3.08, and response was observed by amperometry applying fixed potential. The electrochemical process as obtained by the anodic peak current and scan rate relationship was further configured by electrochemical impedance spectroscopy (EIS). The polymer matrix on the working electrode gave capacitive response for the electrodea€“electrolyte interaction. The sensitivity of the biosensor was measured as 6.93 e???Ae???Ma?’1 with a sensor affinity [e???m(app)] of 50 e???M and 95% reproducibility after 50 measurements. The spectrophotometric method could be used in the range of 6a€“1000 e???M, whereas the biosensor generated linear response in the 1.5a€“1000 e???M range with a response time of 24 s and limit of detection of 0.56 e???M. Uric acid was estimated in human blood samples by the biosensor and satisfactory results were obtained.