Dear Steny,

You might use the following procedure:

1-Enzymatic Assay of Laccase (EC 1.10.3.2)

http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-laccase.html

7. Procedure

7.1 CONDITIONS:

T = 37°C, pH = 6.5, A530nm, 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 37°C (Buffer)

(Prepare 100 mL in deionized water using Potassium Phosphate, Monobasic, Anhydrous, Sigma-Aldrich Product Number P-5379. Adjust to pH 6.5 at 37°C with 1 M KOH.)

7.3.2    0.216 mM Syringaldazine Solution (SYR)

(Prepare 3 mL in absolute methanol using Syringaldazine, Sigma 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 37°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. References & Attachments

Ride, J.P. (1980) Physiological Plant Pathology, 16, 187-196.

2-Isolation, Molecular Characterization and Reactivity with 2,6 Dichlorophenol of a Laccase and Isolation of Laccase Gene Specific Sequences from Lignin Degrading Basidiomycete Phanerochaete chrysosporium (TL 1)

http://scialert.net/fulltext/?doi=biotech.2006.522.529&org=11

Enzyme characterization: Estimates of the laccase optimum pH were obtained by using 50 mM acetate buffer (3.6 to 5.5) and 50 mM citrate phosphate buffer (pH 2.6 to 7) and the pH stability was assayed in the pH range of 2.0 to 6 using 50 mM citrate phosphate borate buffer. The optimum temperature and stability was determined between 50 to 80°C using citrate buffer.

Standardization of laccase assay: Laccase assay was standardized and performed with different substrates viz., guaiacol, 2,6 dimethoxy phenol (DMP), ABTS (2,2'-azinobis-3ethylbenzthiazoline-6-sulfonate) and standardized. This was qualitatively explored by changes in the optical absorbance spectra of the reaction mixtures which contained 0.5 mM of ABTS (Sigma Chemical Co., St. Louis, Mo.) in 0.1 M sodium acetate buffer (pH 6.0). On addition of enzyme source oxidation of ABTS was monitored by determining the increase in A420 (A420 = 3.6x104 M-1 cm-1), guaiacol (A465 = 1.2x104 M-1 cm-1) and DMP (A468 = 4.96x104 M-1 cm-1) in a spectrophotometer (EC India Ltd.,). Heat killed enzyme served as the control. One unit of enzyme activity is defined in μmol of ABTS oxidized min-1. Kinetic studies were performed at 25°C by measuring the initial velocity in 3 mL glass cuvettes with 1 cm path length Inhibitor studies were carried out using guaiacol in 10 mM citrate-Na2HPO4 buffer (pH 6.0).

Hoping this will be helpful,

Rafik

Why do you use guaiacol ? It is a volatile molecule and relatively toxic. I would prefer ABTS which is, if I remember well, more sensitive due to its higher molar extinction coefficient.

The guaiacol assay is very accurate and easy method to estimate laccase.You may try the method of Arora and Sandhu as under:

Laccase (E.C.1.10.3.2)

Laccase activity was measured according to Arora and Sandhu (1985). The reaction

mixture containing 3.8ml acetate buffer (10mM, pH 5.0), 1ml guaiacol (2mM) and

0.2ml of enzyme extract was incubated at 25°C for 2h. The absorbance was read at

450nm. Laccase activity has been expressed at colorimetric units ml-1 (CU ml-1).

I am enclosing the paper for your reference also. Let me know if any clarity is needed.

Regards

Mukesh

Thankyou so much.. I did the assay with 1ml culture supernatant , 1ml Guaiacol and 3ml of 10mM sodium acetate buffer.  But there was no red color formation during the reaction.. 

The method is for 0.2mL of enzyme extract, I do not have protocol standardized for 1mL. You may proceed with 0.2mL extract. the colour upon incubation is light brown to dark brown , depending upon the laccase activity of culture. Follow the method as detailed in https://www.researchgate.net/profile/Dr_Mukesh_Chander/post/How_to_standardize_Laccase_activity_assay_by_Guaiacol/attachment/59d6277b79197b8077985c3f/AS:325980372652032@1454731185337/download/Mukesh+Chander+IJCMAS+2015.pdf

I did the assay with 1ml culture supernatant , 1ml Guaiacol and 3ml of 10mM sodium acetate buffer. But there was no red color formation during the reaction....