Urease from Canavalia ensiformis (Jack bean), CAS No.9002-13-5

CAS: 9002-13-5 Cat. No.: U128713 EC Number: 232-656-0

GRADE & PURITY EnzymoPure™, ≥45 units/mg dry weight

Overview

Jespersen (1975) reports that ammonium carbamate is produced in citrate and Tris buffer.
Urease occurs in many bacteria, several species of yeast and a number of higher plants. Varner (1960) has reviewed it. Two of the best sources are: Jack beans (Canavlia ensiformis) from which it has been crystallized and thoroughly studied, and Bacillus pasteurii.
The enzyme is important in assaying for urea. See Guilbault and Montalvo (1970). Its immobilization has been reported: et al. (1974), James and Pring (1975), Messing (1974), Nakamoto et al. (1975), Sundaram (1973) and Tran-Minh and Broun (1975).
Characteristics of Urease from Jack Bean:
Km: 1.3mM in Tris⋅HCl (Cesareo and Langton, 1992).
Specificity
Urease is specific for urea and hydroxyurea (Fishbein and Carbone 1965). See also Sundaram and Laidler (1970).
Composition
Monomeric (a)urease can polymerize to form six unit polymers of about three million daltons. (Fishbein et al. 1970; Fishbein and Nagarajan 1972a). Andrews and Reithel (1970) report on the sulfhydryl groups. Contaxis and Reithel (1971) indicate the molecule can be split in half with no loss of activity. See also Contaxis and Reithel (1972), Fishbein and Nagarajan (1972b), Lynn (1970), and Bailey and Boulter (1969).
Optimal pH：7.4 
Stabilizers
EDTA in concentrations of 1 X 10-3 M. 50% glycerol solutions protect urease crystalline suspension for several months at 4°C.
Inhibitors
Heavy metals. NH4+ ions formed. See also Fishbein and Carbone (1965). Sodium and potassium ions are inhibitors (Cesareo and Langton, 1992).
Urease Assay
Method
Worthington has adopted an assay method where the hydrolysis of urea is measured by coupling ammonia production to a glutamate dehydrogenase reaction.
formulas
One unit results in the oxidation of one micromole of NADH per minute at 25°C and pH 7.6 under the specified conditions. In addition to increased sensitivity, the assay method possesses the advantage that it can be manipulated to permit quantitation of urea.
Reagents
0.1 M Potassium phosphate buffer, pH 7.6
0.023 M Adenosine-5'-diphosphate (ADP) in phosphate buffer
0.0072 M NADH in phosphate buffer
0.026 M a-Ketoglutarate in phosphate buffer
1.8 M Urea in phosphate buffer
Glutamate Dehydrogenase: Dilute to approximately 500 units/ml in 50% glycerol or phosphate buffer. Store cold during use.
Enzyme
Dissolve enzyme at one mg/ml in 0.1 M phosphate buffer, pH 7.6. Immediately prior to use, dilute further in buffer to obtain a rate of 0.02-0.04 ΔA/minute.
Procedure
Adjust spectrophotometer to 340 nm and 25°C. Pipette into each cuvette as follows:

0.10 M Phosphate buffer, pH 7.6 2.4 ml

0.023 M ADP 0.1 ml

0.0072 M NADH 0.1 ml

0.026 M α-Ketoglutarate 0.1 ml

1.8 M Urea 0.1 ml

GLDH (500 units/ml) 0.1 ml

Incubate in spectrophotometer at 25°C for 5-10 minutes to achieve temperature equilibration and establish blank rate, if any. A slight change in absorbance may be observed due to trace ammonia in reagents. Upon obtaining a zero change in absorbance, add 0.1 ml appropriately diluted enzyme. Record decrease in A340 for 8-10 minutes. Determine ΔA340/minute from the linear portion of the curve. A slight lag may occur.

Calculation 

Specifications

Product Name

Urease from Canavalia ensiformis (Jack bean), CAS No.9002-13-5

Synonyms

Urea amidohydrolase

Grade

EnzymoPure™

Specifications & Purity

EnzymoPure™, ≥45 units/mg dry weight

Biochemical and Physiological Mechanisms

Urease catalyzes the hydrolysis of urea into carbon dioxide and ammonia. Urease is involved in nitrogen metabolism and urea degradation. Urease from Canavalia ensiformis binds 2 nickel ions per subunit.

CAS

9002-13-5

Enzyme Commission Number

3.5.1.5

Molecule Type

Enzyme

Storage and Shipping

Concentration

≥45 units/mg dry weight

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Unit definition

One Unit oxidizes one micromole of NADH per minute at 25°C, pH 7.6. The hydrolysis of urea is measured by coupling ammonia production to a glutamate dehydrogenase reaction.

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