URICASE from Bacillus sp


Appearance White amorphous powder, lyophilized
Activity GradeⅡ 1.5U/mg-solid or more
Contaminant Catalase ≤1.0%
Stabilizers Borate, EDTA, nonionic detergents
Stability Stable at -20°C for at least One year (Fig.1)
Molecular weight approx.150,000
Structure 4 subunits per molecule
Isoelectric point 4.7
Michaelis constant 1.36×10⁻⁵M (Uric acid)
Inhibitors Ag⁺,Hg⁺⁺
Optimum pH 8.5(Fig.4)
Optimum temperature 45°C(Fig.5)
pH Stability pH 6.0-9.5 (25°C, 20hr)(Fig.6)
Thermal stability below 60°C (pH 8.0, 10min)(Fig.7)
Effect of various chemicals (Table 1)


This enzyme is useful for enzymatic determination of uric acid in clinical analysis.




Uric acid+O₂+2H₂O                                                   Allantoin+H₂O₂+CO₂

The disappearance of uric acid is measured at 290nm by spectrophotometry.

Unit definition:

One unit causes the oxidation of one micromole of uric acid per minute under the conditions described below.


A. Uric acid solution 0.001%[Dilute the stock solution (0.01%) to 10-fold volume with 50mM borate buffer containing 0.001% Triton X-100 and 1.0mM EDTA, pH 8.0](Should be prepared fresh) Stock solution : 10.0mg uric acid/100ml of above buffer (Store at 0-5°C)
B. KOH solution 20%
C. Enzyme diluent 50mM borate buffer containing 0.001% Triton X-100 and 1.0mM EDTA, pH 8.0


Concentration in assay mixture
Borate buffer 42 mM
Uric acid 40 µM
EDTA 0.83 mM
Triton X-100 0.00083%

1. Prepare the following reaction mixture in a test tube and equilibrate at 37°C for about 5 minutes.

2.0ml Uric acid solution (A)
0.5ml Distilled water

2. Add 0.5ml of the enzyme solution* and mix by gentle inversion.

3. After exactly 5 minutes at 37°C, add 0.2ml of 20% KOH solution (B) to stop the reaction and measure the optical density at 290nm against water (OD test).
At the same time, prepare the blank by first mixing the reaction mixture with 0.2ml of KOH solution after 5min-incubation at 37°C, followed by the addition of the enzyme solution (OD blank).

* Dissolve the enzyme preparation in ice-cold enzyme diluent (C) and dilute to 0.01−0.02U/ml with the same buffer and store on ice.


Activity can be calculated by using the following formula :

ΔOD (OD blank-OD test)×Vt×df

Volume activity (U/ml) =                                                               = ΔOD×0.105×df

12.2 ×1.0× t × Vs

Weight activity (U/mg)=(U/ml)×1/C

: Total volume (3.2ml)
: Sample volume (0.5ml)
: Millimolar extinction coefficient of uric acid (㎠/micromole)
: Reaction time (5 minutes)
: Light path length (cm)
: Dilution factor
: Enzyme concentration in dissolution (c mg/ml)


  1. S.Asano, K.Yamamoto, S.Teshima, T.Kikuchi and Y.Kawamura; Rinsho Kagaku, 23 (3), 214 (1994)
  2. K.Yamamoto, Y.Kojima, T.Kikuchi, T.Shigyo, K.Sugihara, M.Takashio and S.Emi; J.Biochem. 119, 80 (1996)
Table 1. Effect of Various Chemicals on Uricase
[This enzyme dissolved in 50mM borate buffer, pH 8.0 (5U/ml) was incubated with each chemical at 25°C for 1hr.]
Chemical Concn.(mM) Residual
Chemical Concn.(mM) Residual
None 100 EDTA 5.0 104
Metal salt
2.0   o-Phenanthroline 2.0 91
MgCl₂   89
α,α′-Dipyridyl 1.0 89
  91 Borate 50 102
Ba(OAc)₂   118
IAA 2.0 93
FeCl₃   95 NEM 2.0 107
CoCl₂   91 Hydroxylamine 2.0 91
MnCl₂   93 2-Mercaptoethanol 2.0 104
ZnSO₄   91 Triton X-100 0.10% 93
Cd(OAc)₂   87 Brij 35 0.10% 93
NiCl₂   89 Tween 20 0.10% 98
CuSO₂   78 Span 20 0.10% 103
Pb(OAc)₂   91 Na-cholate 0.10% 98
AgNO₃   44 SDS
0.05% 91
HgCl₂   46 DAC 0.05% 89
PCMB 2.0 96
MIA 2.0 91
NaF 2.0 91
NaN 2.0 91

Ac, CH₃CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; IAA, Iodoacetamide; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.

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