I'm helping in a project that aims to produce and immobilize the laccase enzyme from P. ostreatus, but we have not got a good immobilization protocol yet ( adequate concentration of glutaraldehyde and good response surface). Perhaps anybody who has done this kind of immobilization by CLEAS?
Hello Alejandro,
if you wish to use the CLEAS technique, please refer for example to the paper: Preparation and characterization of cross-linked laccase aggregates and their application to the elimination of endocrine disrupting chemicals, by Cabana et al., in Journal of Biotechnology 2007. CLEAS are not always suitable for applications, where a strong, inert support is required. Why not to try an inorganic support such glass beads, porous silica, or so on?
Hi Alejandro,
For the immobilization of laccase using the CLEA methodology, two steps are required aggregation (using salt, organic solvent...) and crosslinking, and so glutaraldehyde is not always the best choice, it might cause some inhibition. You might try coupling of chitosane with a carbodiimide such as EDAC, as a crosslinker it gave globally good results, choice of the buffer might also be crucial, as the type of buffer, its concentration and its pH. Meanwhile, could you give more information about the protocole you're actually using? I recommend that you check this paper, "Laccase-Based CLEAs: Chitosan as aNovel Cross-Linking Agent" by Arsenault et al. (2011) for chitosan as a crosslinking agent or this one "Parameters in preparation and characterization of cross linked enzyme aggregates (CLEAs)" by Talekar et al. (2013).
Keep me updated if you need more information.
Imad
Enrico Sanjust, thank you so much for your answer, this week we've just gotten some good results immobilizing the laccase, but we didn't get good results measuring the surface response from the laccase already immobilized, I think that as we're using a spectrophotometer to measure the activity, so I think that maybe the CLEAS formed is interfiring with the light beam to measure the change of color.
Imad Eddine Touahar thank you so much for your answer, as you can read above, we've just gotten good results immobilizing laccase by CLEAS, but we're having some problems measuring the response activity from the laccase already immobilized. Thank you for your suggestion, the next week we're going to try to immobilize with chitosan.
As for the interference with spectrophotometric readings, it might occur for bigger particles, but, according to my experience, it is possible to overcome that by two means, first is to shake your plate or cuvette enough before reading and rerun your readings 2 or 3 times, the other way is to use the classical kinetics by taking spectrophotometric readings on a regular time basis (every 1 min or 30 sec) and then draw your absorbance vs time, calculate the slope and so on. Hope this might help, good luck with the immobilization.
Hi Alejandro, you could try a blank containing the same CLEAS preparation but obviously without your chromogenic substrate. Alternatively, you could abandon the direct kinetic measurements, and build up an assay where the CLEAS suspension is gently stirred with your chromogenic substrate for a fixed time. Then the reaction could be stopped (for example by addition of a suitable amount of a strong acid) or the suspension quickly filtered or centrifuged to measure the absorbance increase in the resulting clear solution...
Thank you Enrico Sanjust and Imad Eddine Touahar for your answers, we will try some things (including some of your suggestions) to break through this problem.
Thank you.
First, prepare a fresh raw enzyme solution ...Precipitate dropwise with cold acetone or propanol at 4°C under constant agitation. add glutaraldehyde (25%) at a final concentration of 2% W/vol. Contact time: 1 to 2hrs....
First, prepare a fresh raw enzyme solution ...Precipitate dropwise with cold acetone or propanol at 4°C under constant agitation. add glutaraldehyde (25%) at a final concentration of 2% W/vol. Contact time: 1 to 2hrs....
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology