Enzymatic Assay: 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):

	Test	Blank
Deionized water (Reagent 7.3.2)	-------	0.50
Buffer (Reagent 7.3.2)	2.20	2.20
Laccase (Reagent 7.3.2)	0.50	-------

Equilibrate to 30 °C .Monitor the A530nm until constant using a suitably thermostatted spectrophotometer.Then add:

	Test	Blank
SYR	0.30	0.30

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.

7.5 CALCULATIONS

Units/ml enzyme =	ΔA530nm Sample = A530nm/min Test - A530nm/min Blank) (df)
	(0.001)(0.5)

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

7.5.2.4	Units/mg solid =	Units/mL enzyme
		mg solid/mL enzyme

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.

8. Approval

	Print Name	Sign Name	Title	Date
Prepared by:	Gene McNaughton	Gene McNaughton	Originator	09/02/03
Approved by:	David Lintz	David Lintz	Supervisor	09/03/03
Approved by:	Jeff D. Heiland	Jeff D. Heiland	Quality Assurance	09/03/03

References

1. Sheikhi F, Roayaei Ardakani M, Enayatizamir N, Rodriguez-Couto S. 2012. The Determination of Assay for Laccase of Bacillus subtilis WPI with Two Classes of Chemical Compounds as Substrates. Indian J Microbiol. 52(4):701-707. https://doi.org/10.1007/s12088-012-0298-3

2. Ride J. 1980. The effect of induced lignification on the resistance of wheat cell walls to fungal degradation. Physiological Plant Pathology. 16(2):187-196. https://doi.org/10.1016/0048-4059(80)90033-8