Using 0.3% glutaraldehyde solution is fine. Run the cross-linking step for around 3 hours at pH = 6.5 and  25°C and after that wash the beads with distilled water until no residual glutaraldehyde is detected in the supernatant. Use around 10 ml enzyme solution of higher concentration (1 mg/ml) for 1 g cross-linked Chitosan and incubate at 25°C for 2 hours and after that filter the beads and wash them with distilled water until no free enzyme could be detected in the filtrate.

Ok, i do that!another thing؛ how can i assay from these beads?do you know article about assay method from macrobead?! i should calculate enzyme distrbution on each bead for enzymatic assay?

I would use an enzyme assay instead of a Bradford assay to calculate the quantity of immobilized enzyme. The bradford test is sensitive to many interferences.

You should be sure chitosan is not protonated in the immobilization step. As I remeber the pKa of amino groups of chitosan is about 6.5.

ok Bastine,thanks.after bead formation i should be sure no residual NaOH remain,so, after beads treatment with Glutaraldehyde,i must be sure there isn't any gltaraldehyde.how can be sure about these?! another thing,i must say how much enzyme(mg) there is on each bead surface according to bradford test and assay from these?! i get confused!can you suggest an article about assay methode for emobilized enzyme?

Dear friends

I have done a comprehensive study on immobilization some sorts of enzymes on electrospun fibers. First, I should say that that the procedure you choose is true, since chitosan is redolent with free amine groups in the backbone of the polymer chains meaning that they are prone to form bond with aldehyde containing materials such as glutaraldehyde (GA). The chemistry of this reaction (amine and aldehyde) is known as Schiff base reaction that results in formation of imine. It is necessary to denote that the reaction is formidable and you can possibly reach to high degrees of conversion even in ambient temperature. However, one should be sure that the abundant amine groups on the surface of the beads are saturated with aldehyde prior to immobilization of enzymes.

Something look ambiguous in the proposed question since you said Bradford test revealed no immobilization. May I ask how? let me guess some options and you later tell me which one was your intention. ok?

1. you possibly poured the first solution after immobilization (called filtrate) followed by further washing with PBS. After several times of vigorous washings you added Bradford reagent (a brown colour liquid) to all liquids (filtrate and stepwise washing residuals) and surprisingly found out that the solutions have changed colour to deep blue, hence you compared it with previously developed standard BSA curve and found that almost nothing has been immobilized. Is that so? 

If yes, I have to say there are some sources of mistakes. Firstly, DO NOT PREPARE YOUR STANDARD CURVE WITH BSA, JUST DEVELOP IT WITH THE ENZYME YOU WANT IMMOBILIZE! Sounds bizarre? I personally have an experience in this field an I say BSA is not a reliable source to compare your enzyme with it. It always results in egregious blander. It is strongly probable that you have immobilized a negligible amount of enzyme but since the method is not sensitive you cannot measure it. 

2. Secondly, I can simply say you have not used appropriate amount of GA and it could not saturate amine contents. In my own study I found that immobilization was optimum when GA concentration was 5 wt%. further increase in GA concentration resulted in drastic lose of enzymatic activity. SO MAY I SUGGEST TO JUST SIMPLY INCREASE THE GA CONTENT?

dear Aref,thanks for your answer.yes,guess1 is true!but i compare free enzyme,beads before and after enzyme incubation.then compare them with BSA standard curve.i produce enzyme in bacteria and i can't make different concentration of it!i only have 0.5mg/ml enzyme than i want immobilized it on chitosan bead!i think i do immobilize nearly,but i should optimize that.why you say don't compare with BSA?!it's a protein that i can make different concentration of that and draw standard curve for it,what is wrong when compare enzyme before and after immobilization with standard curve?!

i  use 0.3% glutaraldehyde based on literature as optimum concentration.

I have some experience with enzyme purification although it is NEITHER my field of research NOR my interest. By the way, I understand what you say. Regarding your answer, I believe that you are proceeding in wrong !

BSA test (better said Bradford test) is to measure  HOW MUCH ENZYME has been immobilized (mg protein/g support). Meaning that you develop a curve (call it standard) with known concentrations of BSA and the Bradford reagent is the colourant reagent that reacts with amines groups of BSA as the standard source of protein to change colour from light brown to dark blue. Then, you normally collect the washing liquids after immobilization process and then again react them with Bradford on the grounds that if there is any protein in it, it must react with Bradford. OK?  You normally interpret the intensity of the colour developed after the addition of the Bradford in a spectrophotometer and compare it with standard curve you previously developed. Only then you will be able to measure how much enzyme leached (not immobilized on the support). Further subtraction of the unbound enzyme from the initial content of the first solution you utilized for immobilization will tell you how much exactly has been immobilized. THIS IS AN OVER ALL AND NORMAL PROCEDURE.

However, as far as I am concerned the sources of proteins are different from each other and the affinity of Bradford to a typical enzyme (or any other protein source) is strongly dependent on its 3-D structure and availability of the amine groups.  IT IS PROBABLE THAT BRADFORD IS NOT SENSITIVE ENOUGH TO YOUR ENZYME HENCE, IT WILL NOT DETECT THE PROTEIN IN VERY DILUTE SOLUTIONS . 

ok,i think understand what you say,by the way;i have another question!for litlle amount enzyme concentrate,what is the best ratio for enzyme to support for immobilization?and what is your idea about pH6.5 for immobilization?

ok Shaghayegh,but i don't understand your mean,how i can compute enzyme to support ratio?!can you expain more?!!thanks.

Dear Parastou

It turned out to me that your questions are serious and my explanation was not satisfactory. For further information please feel free to contact me through my e-mail adress: [email protected],

or simply just message me in my face book. (Aref Daneshfar)

Regards

Aref

For the estimation of immobilization efficiency, you can use an enzyme activity assay by the following equation:

EF(%) = (Ai/Af) x 100

where Ai is the specific activity of immobilized enzyme which is equal to the difference between the specific activity of free enzyme solution (Af) and the specific activity of the unbound enzyme in the washing buffer.

thank Daniel,I try it.