Enzymatic Assay of Laccase (EC 1.10.3.2)

Laccase (EC 1.10.3.2) 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

1. Objective

To standardize a procedure for the enzymatic assay of laccase

2. Scope

The scope of this procedure is all products that have a specification for laccase activity.

3. Definitions

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.

4. Discussion

Syringaldazine + O₂ ^Laccase > Oxidized Syringaldazine + 2H₂O.

5. Responsibilities

It is the responsibility of Analytical Services personnel to follow this protocol as written.

6. Safety

Refer to Safety Data Sheets (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS:
T = 30 °C, pH = 6.5, A_530nm, Light path = 1 cm

7.2 METHOD:
Continuous Spectrophotometric Rate Determination

7.3 REAGENTS:

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):