I tried to immoblize tyrosinase enzyme onto amberlite beads using Gluteraldhyde to link the Amberlite with the Enzyme , and my question is why I didn t get any activity after activite the Amberlite with the Gluteraldhyde and also in the washing buffer (the unbound enzyme ) ?
Have any one an idea about that ?
Residual glutaraldehyde could have inactivated the enzyme by causing intramolecular crosslinks. Additionally, multiple points of attachment, both covalent and ionic, of the enzyme on the beads could inhibit the enzyme.
I assume the enzyme was active just before the experiment began and that your results are reproducible. Which Amberlite resin did you select and are you following a particular protocol? Did you firstly modify the resin by aminoalkylation and then did the glutaraldehyde activation? Since there's also no activity in the wash buffer but protein is present then how are you following the physical state of the tyrosinase (from mushrooms I assume)? Perhaps you're using SDS-PAGE, SEC, etc.? The condition of the enzyme in the wash buffer will likely indicate what has occurred on the column. If there are aggregates for example then the tyrosinase would have experienced some harsh conditions on the column, perhaps residual cross-linking glutaraldehyde, to cause inactivation of protein which may also be the form in the wash buffer. Tyrosinase is a relatively robust enzyme so some step(s) in the process must not be optimal to have caused inactivation. You might want to try the experiment with BSA as a control and follow the progress with SEC and SDS-PAGE to be sure your procedure is working correctly. Each step in the process needs assay confirmation that it succeeded and you may need to do a partial factorial design in which different ratios of protein to resin to extent of bonded phase to reaction conditions are included. Investigators have succeeded in the past with this type of chemistry for immobilizing enzymes so there is likely one or more parameters that needs to be changed.
thank you DR for your answer, I fellowed this protocol
http://www.sciencedirect.com/science/article/pii/S1359511312004461
best regards
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes