ABSTRACT
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 μM and 0.047 μM min−1ml−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 electrode–electrolyte interaction. The sensitivity of the biosensor was measured as 6.93 μAμM−1 with a sensor affinity [Km(app)] of 50 μM and 95% reproducibility after 50 measurements. The spectrophotometric method could be used in the range of 6–1000 μM, whereas the biosensor generated linear response in the 1.5– 1000 μM range with a response time of 24 s and limit of detection of 0.56 μM. Uric acid was estimated in human blood samples by the biosensor and satisfactory results were obtained.
ABSTRACT
Aims: Statistical approach, central composite design (CCD) was used to investigate the complex interaction among temperature (25-37 °C), initial medium pH (5-9), inoculum size (4-10 % (v/v)), concentration of (NH4)2SO4 (0-1 g/L) and concentration of mixed organic acids (5-10 g/L) in the production of polyhydroxyalkanoates by Comamonas sp. EB172. Methodology and Results: Mixed organic acids derived from anaerobically treated palm oil mill effluent (POME) containing acetic:propionic:butyric (ratio of 3:1:1) were used as carbon source in the batch culture of Comamonas sp. EB172 to produce polyhydoxyalkanoates (PHAs). The analysis of variance (ANOVA) showed that all five factors were significantly important in the batch fermentation by shake flask with a P value of less than 0.001. The optimal temperature, initial medium pH, inoculum size, concentration of (NH4)2SO4 and concentration of mixed organic acids were 30 °C, 7.04, 4.0 % (v/v), 0.01 g/L and 5.05 g/L respectively. Conclusion, significance and impact of study: Optimization of the production medium containing mixed organic acids has improved the PHA production for more than 2 folds. Under optimal condition in the shake flask fermentation, the predicted growth is 2.98 g/L of dry cell weight (DCW) with 47.07 wt % of PHA content. The highest yield of PHA was 0.28 g of PHA per g mixed organic acids.