Preparation, Purification And Characterization Of An Acid Urease

Since urea is a precursor of ethylcarbamate, a potential carcinogenic compound, feasible measures must be taken to control the level of urea. Acid urease has special properties of showing its catalytic activity in low pH range and a tolerance to ethanol. It has been demonstrated that acid urease is capable of decomposing urea in fermented beverage and foods. The objects of this study were to purify and characterize the enzyme from Enterobacter sp. R-SYB082.In cells lysate phase, harvested cell was lysed by a high-pressure homogenizes. Under experimental conditions: 80MPa, 3 pass through the homogenizer, 100g/L, the cell disruption can reach 90% above and the recovery was 110%.Aqueous two-phase system was employed for the extraction of acid urease from disrupted cells. The effects of different factors such as, polyethyleneglycol molecular weight, the concentration of PEG and potassium phosphate, pH and the presence of sodium chloride on acid urease partitioning were analyzed. Aqueous two-phase formed by 15% PEG 4000 and 10% potassium phosphate, pH value 6.0 and 4% NaCl showed the best separation capability.Under these conditions, the activity yield was up to 83% and the purification factor was 4.5 . The crude enzyme was purified to electrophoretic homogeneity using two successive steps including superdex 200 and Mono Q with a yield and purification-fold of 42% and 9.18 respectively. The N-terminal sequence of acid urease was Ser-Phe-Lys-Met-Asp-Arg-Lys-Gln.The molecular weight of the enzyme was estimated to be 430,000Da by gel filtration and 72,000Da by SDS-PAGE, suggesting that it's a hexamer comprising of six identical subunits. The purified enzyme was optimally active at 35℃and at pH4.5. The activity was stable between a pH range of 4.0 and 6.0. It was stable when up to 55℃, but stability of acid urease decrease rapidly above 65℃. The enzyme exhibited an apparent Km of 19.5μmol/L and a Vmax of 109μmol urea/mg·min at 35℃and pH4.5. The activity can be activated by Na+, Mn2+, tartaric acid and succinic acid but inhibited by Ca2+, Zn2+, oxalic acid and acetic acid. 83% and 79% of urea in two kinds of Chinese rice wine was decomposed by incubating with the enzyme (0.05 U/mL) at 37℃for 4 days.