The ABTS is from the best or the best substrate of laccase, it is widely used to this enzyme activity. The substrate is very soluble in water then it is very easy to use with the enzymatic protein and the product is very stable and very easy to oxidase in 1 min if you have good activity. I have work a lot with the substrate for laccase activity, the product is green and highly detectable at 420 nm. I have just to remarks:

1- The concentration that you use is good (0.4 mM) but you can increase until 1 mM if you want to screen the little activity.

2- It is not necessary to add H2O2 is not a peroxydase that you test, the seconde substrate of laccase is the oxygen and you not need H2O2.

ABTS oxidation is dependent on pHand sometimes the oxidized product is not stable. Other possible substrates are syringaldazine and 2,6-dimetoxiphenol. I like 2-6-DMP. See FEMS microbiol letters, 204, 175-181, 2001 for more details

I used n-naphthol instead of ABTS. The absorbance of the product of reaction is 530 nm.

Pointing S.B., Qualitative metyhods for the determination of lignocellulolytic nzyme production by tropical fungi. Fungal Diversity, 1999 2: 17-33

Kluge M.G., Spectrophotometric assay for detection of aromatic hydroxylation catalyzed by fungal haloperoxydase-peroxygenase. Appl. Microbiol. Biotechnol.

2007 75: 1473-1478

The ABTS is from the best or the best substrate of laccase, it is widely used to this enzyme activity. The substrate is very soluble in water then it is very easy to use with the enzymatic protein and the product is very stable and very easy to oxidase in 1 min if you have good activity. I have work a lot with the substrate for laccase activity, the product is green and highly detectable at 420 nm. I have just to remarks:

1- The concentration that you use is good (0.4 mM) but you can increase until 1 mM if you want to screen the little activity.

2- It is not necessary to add H2O2 is not a peroxydase that you test, the seconde substrate of laccase is the oxygen and you not need H2O2.

Is it an instrumentation limitation which is giving you problems at 417 nm? If it is then I suggest you move to 645 nm. The molar absorptivity is still quite high then. 13400 M-1cm-1 I believe. If that's not it then I recommend just making all new solutions. I have wasted a lot of time trying to figure out which solution was giving me problems in my work to no avail and end up making new solutions anyway. I also recommend just running a test. Throw in some water H2O2 and ABTS.

Thanks every one. I used a stock of 25mM ABTS. To my observations, I should add H2O2 and using ABTS in the range 33uM to 400uM as final concentration in total reaction volume of 360ul gives in all soil samples a postive test and reaction signal was noticed just after addition of 10ul of 0.3% H2O2. A clear peak was noticed at 420nm. When working with soil it is better to use buffer and soil solution togather to establish baseline.

ABTS is the best substrate for laccase activity at 525nm, with pH around 3-4. If the activity appear with you after adding H2O2, I think the enzyme is peroxidase and not laccase. Laccase activity didnot need H2O2, only O2 from the surrounding. I think the problem is " There is a shortage in activity or no laccase activity, you should work with another established laccase.

Dear Zahid: Do you know your basic problem? It is that you have taken for granted that laccase will be present in the soil samples you are testing, in detectable quantities (activity wise). This is the cause for losing hopes when you get negative results. Don't do this. First of all let me know why do you feel laccase should be present in all soil samples that you have gathered.

Dear Jai Ghosh: You are very right. Laccase should suppose to be present as I have found lignin peroxidase and manganese peroxidase activities in my soil samples in a bio-degradation experiment. I think along with these enzyme, laccase must have been produced by microorganism or naturally available in soil.

I have gone through many standard protocols they reported the addition of H2O2 in ABTS assay for laccase. However, without H2O2 addition I have tried the assay but couldn't get any activity even after 24 hours incubation. Should or should not add H2O2 is still unclear to me.

Protocol is attached.

Hi

1-do not use hydrogen peroxide

2-check soil sample for heavy metals concentration

3-change pH and test temperature because different laccases have different opima

4-if required add cuso4

We do not recommend to use H2O2 in laccase activity tests with ABTS as a substrate.

Sometimes, we faced with a negative influence of chloride ions of a sample on the activity when their concentration was high, while laccase activity of the tested sample was relativly low.

I have a question about the ABTS laccase activity assay. In our hands, a 0.4 mM ABTS slution reading at 420nm is out-of-scale... how can you interpret correctly the results, without diluting down to an acceptable reading?

I think the problem here may be the source of your Laccase. ABTS is simply the best method and most reliable method for assaying Laccase activity at 420nm. You can try other substrate s such as Syringaldazine, 2, 6 Dimethoxy phenol, guaiacol etc. if it laccase activity is still not detected, then your source does not express the enzyme. Also, you can induce laccase production using CUSO4 and then check the enzyme acivity