Laccase (EC 22.214.171.124) is a cuproenzyme that oxidizes various types of phenols and similar aromatic compounds aromatic amines with the reduction of molecular oxygen to water, therefore, is used as a biocatalyst. Spectrophotometric methods for assaying the laccase activity are based on the formation of product(s) resulting from the enzymatic and inevitable succeeding chemical reactions.1
To standardize a procedure for the enzymatic assay of laccase
The scope of this procedure is all products that have a specification for laccase activity.
One unit will produce a DA530nm of 0.001 per minute at pH 6.5 at 30 °C in a 3 mL reaction volume using Syringaldazine as substrate.
Syringaldazine + O2 Laccase > Oxidized Syringaldazine + 2H2O.
It is the responsibility of Analytical Services personnel to follow this protocol as written.
Refer to Safety Data Sheets (SDS) for hazards and appropriate handling precautions.
T = 30 °C, pH = 6.5, A530nm, Light path = 1 cm
Continuous Spectrophotometric Rate Determination
7.3.1 100 mM Potassium Phosphate buffer, pH 6.5 at 30 °C (Buffer)
(Prepare 100 mL in deionized water using Potassium Phosphate, Monobasic, Anhydrous, Product Number P-5379. Adjust to pH 6.5 at 30 °C with 1 M KOH.)
7.3.2 0.216 mM Syringaldazine Solution (SYR)
(Prepare 3 mL in absolute methanol using Syringaldazine, Product Number S7896.)
7.3.3 Laccase Enzyme Solution (Laccase)
(Immediately before use, prepare a solution containing 25-50 units/mL of Laccase in cold deionized water.)
7.4 TEST METHOD
Pipette the following reagents into suitable vials (in milliliters):
Equilibrate to 30 °C .Monitor the A530nm until constant using a suitably thermostatted spectrophotometer.Then add:
Mix by inversion and record the increase in A530nm for approximately 10 minutes. Obtain the DA530nm/min. using the maximum linear rate for both the Test and Blank.
df = dilution factor
0.001 = the change in A530nm/min. per unit of laccase at pH 6.5 at 30oC in a 3 mL reaction mix
0.5 = volume (in milliliters) of enzyme used
7.6 FINAL ASSAY CONCENTRATION:
In a 3.00 mL reaction mix, the final concentrations are 73 mM potassium phosphate, 0.02 mM syringaldazine, 10% methanol, and 12.5 to 25.0 units laccase.
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