What are you measuring? Amount of immobilized protein or the amount of immobilizeed activity? Also have you already tested this enzyme for immobilization, or has someone else already done this (i.e. in published studies)? Perhaps your enzyme is not so stable?

Lipases have hydrophobic surface properties, so they can be easily immobilized by adsorption on hydrophobic materials , if you are going to use lipase in non aqueous solvent , immobilization by adsorption will be very useful.

check recent review by Patrick Adlcreutz , Lund university about immobilizations of lipases

Please note that acetone is not the solvent of choice when working with APTES. Better, use xylene (to be carefully removed by ethanol or isopropanol washings prior to the subsequent step) or dimethoxyethane. Pre-heating of commercial, reagent-grade glutaraldehyde is not required and can be omitted. Perhaps two hours of reaction between the lipase solution and the glutaraldehyde-activated support is too short, try to conduct the reaction for 6 - 8 hours.

Good luck!

immobilization time should be increased. You applied 2 hours and this is too short.

There are factors involved in immobilization of the enzyme/protein especially immobilization on the support. First you need to know and characterize your support very well and know if its is suitable for your immobilization study. Secondly for immobilization there are various factors such as enzyme/ protein concentration, pH of the buffer, GLU concentration..you should read the book Carrier-bound Immobilized Enzymes: Principles, Application and Design ,by Cao which explains everything very well ...you can also trying adsorbing your enzyme first on the silanized particles and then perform cross linking on the support..this could enhance the yield...check my recent paper on Laccase immobilization :)

Hi Dwi

For functionalization with glutaraldehyde on an aminated surface (your APTES modified silica), you can use a 15% (v/v) solution of glutaraldehyde in 200 mM phosphate buffer at pH 7 and leave the mixture for 16 h at RT (20-25 C). Wash thoroughly with 25 mM phosphate buffer pH 7 and perform the immobilization at RT in the same washing buffer. Good luck.

I am sorry Dwi but the data that you gave as are not enough for appropriate suggestion, so there are several points you should take into account.

First of all, the amount of crosslinker should be optimized. Since glutaraldehyde is a very versatile reagent, during your optimization of lipase immobilization on silica, different amounts of glutaraldehyde should be used to find appropriate concentration in terms of immobilization. Because in this process, the binding of additional enzyme molecules leads to changes in three-dimensional structure of the enzyme during immobilization and may also prevent access to substrate in the active site.

On the other hand, I could not see any data about your buffer pH point in your immobilization process. pH is one of the most important factors influencing not only the properties of amino acid side groups but also the solution chemistry of the insoluble support. Thus, protein–support interaction and surface properties of a protein are strongly influenced by the pH. In this aspect silica is stable till pH 7 or 7,5 but after pH 8 silica is exposed to dissociation and i thing this is not what you expected to see.

I hope these could be helpful,

Good luck..

Did you try immobilization with Na-alginate?

Hi Michael O'Donohue, using Bradford method I measured the amount of protein in lipase solution before and after the addition of silica. By measuring this, it's expected that the amount of protein is decreased that means lipase successfully immobilized on silica.

Dong Hwan Lee's "Lipase Immobilization on Silica Gel Using a Cross-Linking Method" is my reference journal for doing this.

Thank you for helping, I think I need to read more references :)

Hi Yasser Gaber, i am going to use lipase for trans-esterification that is non aqueous reaction. What about immobilization by entrapment method?

Thanks for your suggestion :)

Hi Dwi Mustika,Immobilization by entrapment would probably result in more hydrophillic microenvironment which might increase the hydrolysis action of enzyme and be less favourable for trans esterification reaction.

immobilization on synthetic polymers should be a better option.

Trupti Ashar,India

Have you checked the support loading capacity (mg of protein/gr of support)?

Are you offering more enzyme than the support loading capacity?

How can you calculated immobilization degree ?

Usually It can be find to analyz filtrate using Bradford Method to measure the amount of protein.

U can actually measure the activity on the immobilized catalyst too ..by preparing a reaction setup in a small beaker with a working volume of 10 ml and using the spectrophotometer..we did it for our laccase immobilized silica particles

Hi Mustafa Yilmaz I calculate the immobilization degree with this formula : ((Initial absorbance - finale absorbance) / Initial absorbance) * 100%

Absorbance measured in 595 nm with 25 micro liters samples + 1250 micro liters Bradford reagent.

Did I do it wrong?

Hi Rakes Nair thank you! will check your paper :)

Hi Cenk Daglioglu actually I thought to make a series of different gluteraldehyde concentration used for the immobilization process. I used Gluteraldehyde with concentration mentioned above based on my advisor's suggestion. She has done this before and she said that she has got up to 60% of immobilization degree. It makes me wonder where my mistake is.

I have just updated my question, the pH point of the phosphate buffer is 7 :)

Hi Dwi Mustika Handayani,

You may be having problems with the activation of the support since the Glutaraldehyde has the ability to polymerize with itself which impairs the binding and the enzyme immobilization on binding sites. I suggest you work with low temperature to activate it and test work with diluted glutaraldehyde solutions. This may prevent it polymerize and crosslink between themselves. Another thing would be to determine the enzymatic activity of its final wash solution, so you will know if the protein remaining matches your enzyme or is it some inibitor. Other test methods for determining protein as well. When used the Bradford, some interfering harmed resutado the end, so I started using the bicinchoninic method. I'm sending an article about the glutaraldehyde that helped me a lot in my experiments.

I'm working with Aspergillus niger lipase in esterification reactions and is works very well even on hydrophilic support.

Thank you very much Lizzy Veríssimo :)

You can try glutaraldehyde conc. below 2.5% with slightly acidic condition as it enhance the crosslinking ability of glutaraldehyde.

This is a covalently immobilization process . You know that gluteraldehyde is a very active aldehyde and it can easily react with amino groups of silica. your immobilization degree looks very low. May be two reasion:

1) The treatment of silica gel using with 3-APTES is not completed.

2) The support contains much more amino groups and amount of gluteraldehyde is not enough to react it.

I recommended you can use sol-gel encapsulation method. please check our papers. You can use same silica-based materials.

Have you check the amount of non-reacted–NH2 groups after the activation with glutaraldehyde? Usually, ninhydrin test may be used for this determination through the formation of colored complex known as Ruhemann’s purple (absorbance at 570 nm). The amount of functional aldehyde groups may be calculated from the ratio between gluraldehyde modified APTES-carrier and unmodified APTES-carrier. This percentage is strictly related to enzyme immobilization efficiency.

Have you try to reduce the percentage of APTES for carrier functionalization? Probably,

you may have an excess of amino groups on the silica surface and you should consider that as glutaraldehyde is a bifunctional molecule, each molecule of glutaraldehyde could be bound to two molecules of ethylenediamine thus becoming inactive for the enzyme immobilization.