I am working on laccase enzyme from a bacteria which is very active in low temperature. While running the crude enzyme on native PAGE for zymogram analysis using ABTS, active bands are visible below the dye (BPB) front.
Even during crude concentration by ammonium sulfate precipitation activity is retained in the supernatent even after 95 % saturation.
Any suggestions please.
Lignin derived alcohols,aromatic aldehyde, ketones as well as organic acids mall ate etc. could transform ABTS radicals into its reductive form and thus check your sample for those and H2O2 production . Good luck
Yes, ABTS IS NO SPECIFIC AT ALL. Other compounds can oxidixe this agent. Other substrates such as Syringaldazine are better.
ABTS it is not a good discriminatory substrate for laccase since can be also oxidized by cathecol oxidades, peroxidades and tyrosinases. I will try with Syringaldazine or 2,6-dimethoxyphenol (DMP). Pay attention, differently from ABTS, these substrates are pH sensitive.
Moreover, ABTS is a laccase mediator that oxidize low molecular substrates such as non-phenolic lignin components with high redox potentials (veratrylalcohol and 1-(3,4-dimethoxyphenyl)-2-(2-methox-yphenoxy)propane-1,3-diol) and lignin dimers such as 1-(3,4-dimethoxyphenyl)-2-phenoxyethane-1,2-diol.
Many enzymes can oxidize ABTS: peroxydases, other multi-copper oxidases, etc.
Dear All,
I have enzymatic crud extract from Actinobacteria , I d like to extract and purify Tyrosinase, and when I use L dopa i found a heigh activity and for 2,6 DMP substrat there were no activity but for syrngaldazine there were Low activity , how do you interpret this results
best regrads
The laccase activity could be determined spectrophotometrically by measuring the increase in absorbance at 525 nm caused by the oxidation of syringaldazine in 0.05 M phosphate buffer pH of optimal activity (ex. pH 5.5 for the laccase from Trametes sp,) using a spectrophotometer. One unit of enzyme (U) is defined as the amount of enzyme required to oxidize 1 µmol of syringaldazine per min using an extinction coefficient ε525 65 mM-1 cm-1 (Harkin and Obst, 1973, Mustafa et al., 2005).
Dear Harir Mohamed, you've produced a peroxidase-tyrosinase extract. It is normal since almost all reported tyrosinase-producing microbial strains also produce other polyphenol oxidases such as peroxidase and laccase concomitantly.
Dear Dr Andrea Zille ,
thank u for your valuable answer, However , in soem case they produce only tyrosinase as reported in this two Publication :
1/http://www.jmb.or.kr/journal/viewJournal.html?doi=JMB022-02-08
2/https://www.researchgate.net/publication/283536228_Partial_purification_and_characterisation_of_two_actinomycete_tyrosinases_and_their_application_in_cross-linking_reactions
Other than the ABTS there are more substrate are available for laccase enzyme such as syringaldazine, 1- naphthol, and 4- and 5-hydroxyindoles.
ABTS is not a standard reagent to assay laccase. chances are it may give false positives with enzymes other than laccase. A variety of blue copper oxidases, phenol oxidases and some peroxidases also give positive results with ABTS. It is better to use some other substrate such Guaiacol (Methoxy phenol) to verify your results.
Regards
Mukesh
You may try more specific substrate like Syringaldazine. Changing the gel percentage may also resolve your native band on PAGE.
Good luck
ABTS is one of the best substrate for laccase, being transformed by oxidation to the green radical-cation. It is possible to have other enzymes in your extract like peroxidases which also reacts with this substrate.
I have tried using Guaiacol which is giving positive results. I am getting highest activity at low pH (2.5) using ABTS as well as guaiacol.
Using Syrinzaldazine, activity was not found in the crude filtrate. Might be, something wrong going with my syrinzaldazine assay.
There are eight isomers of laccase enzyme described. Some are very resistant to temperature, they are also active in a large range of conditions. However proteins should precipitate with ammonium sulfate.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes