I tried to immobilized tyrosinase onto amberlite and without using Gluteraldyde
Gluteraldyhde, and i have got 100 percent of bound (immoblize enzyme), aim the problem is there is no activity and i think That Amberlite inactivate Tyrosinase. Does anyone have some idea about this one?
Dear Harir,
Tyrosinase is a copper-containing enzyme present in plant and animal tissues that catalyzes the production of melanin and other pigments from tyrosine by oxidation, as in the blackening of a peeled or sliced potato exposed to air. The optimal activity of tyrosinase is at pH =6 and pH =7. On the other hand, amberlite lr 120 is sulfonic strong acid cation resin. Therefore, it is expected that the activity of the enzyme tyrosinase to be diminished due to one or both effects: the acid environment that the resin make and its ability to exchange the copper contained in the enzyme.
Hoping this will be helpful,
Rafik
I didn t understand Pr Jihon H t luong THERE ARE TWO TYPE OF AMBERLITE ACID AND BASIC ?!
Let's assume one is controlling pH by a buffer, so that the immobilisation by the ion-pairing mechanism is achieved. 1) The optimum pH for retention of tyrosinase can be far from the optimum for catalysis, 2) The system is dynamic thus the tyrosinase ion will be replaced with another ion depending on the concentration in the sample. This paper may be useful: http://pubs.acs.org/doi/abs/10.1021/la900751w
Thank 's a lot Professors for your answers ,
Eupergic is suitable as support for tyrosinase immobilization??
Glutaraldehyde is very harsh and toxic to the protein which must be used at lower concentration to functionalize the matric or support.
In your case you can try 0.5 to 1 M of Glutaraldehyde and incubate the matrix for 2 h. Wash the glutarldehyde treated matric for 3-4 time to remove the extra free Glutarldehyde carefully. Then you can use the same matrix for your enyzme immobilization.
Best Luck,
Regards,
AKN
Yes at higher concentration Glutarldehyde denature the protein and some enzyme are very sensitive towards it.
It is widely used as a crosslinking linking agent but at lower concnentration only. It can be used as a cross linker for tyrosinase immobilization and my suggestion is only to use the same at low concentration and wash the matrix thoroughly before mixing the enzyme.
Thanks
Thank you very much for your answer ,
in your opinion 1 percent is enough ?
best regards
- not 1%
- 0.5-1 M glutaraldehyde to be used.
- generally glutaraldehyde in lab is 25 or 50% you can calculate on the basis of its mol. weight
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