I immobilize enzyme on chitosan macrobead after activating them with glutaraldehyde. I want to set up optimum condition for emobilized enzyme. Optimum pH shift from6.5 to 8?! Is there any reason for it?!
Maybe it has to do with the the fact that chitosan has many primary amino groups. At the lower pH, these will nearly all be protonated, giving the particle a high positive charge. The charge may repel the substrate of the enzyme from the particle, or adversely affect the function of the enzyme. At the higher pH, the positive charge of the particle will be lower because of deprotonation of the amino groups.
The pH is one of the major parameters capable of shifting enzyme activities in reaction mixture. Immobilization usually results in shift of optimum pH due to conformational changes in enzymes. This shift in optimum pH could be resulted from the change in acidic and basic amino acid side chain ionization in the microenvironment around its active site.
Hi Parastou, I agree with the above comments in that the reactivity of chitosan amine groups are very much dependent on pH.
Dr Shapiro has pointed in the right direction for the shift in optimum pH. Because of the charged groups on the chitosan, the 'localised' pH at the surface of the medium will differ from that in the bulk solution and it is the latter that will be measured by a pH meter. Thus the optimim will have appeared to change. Ephraim Katchalski, in the 1990s, published many articles on the properties of immobilised enzymes and included discussion of the likely effects of localised pH on catalytic activity. You may find that chasing up some of these articles will provide the solution to your problems.
Peter
I do agree with Adam B Shapirar and Peter J Butterworth . Keep in mind , the matrices employed for enzyme immobilization play an important role in optimum pH of the immobilized enzyme.
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