Enzyme Immobilization - Science topic

Thank you .. am interested.

What would be the easiest method to separate silica particles of different particle size in large scale?

I used glutaraldehyde for attachment aptamer on the glassy carbon electrode. I hung my electrode vertically above the container with a small hole while the container filled with glutaraldehyde. The container put in the heater with a degree around 40 C. Glutaraldehyde evaporated and covered the electrode surface. Then dropcasted my aptamer on the surface of the electrode. One times that I washed my electrodes before aptamer attachment. The aptamer did not attach on the surface. You can apply other chemistry like EDC/NHS coupling.

There are too many publications that have immobilized GOx onto the CNT electrodes using drop casting or immersion methods, many of them claim to obtain DET to the electrode surface. However, this claim was finally beaten in 2018 by Philip Bartlett and I. If you are interested, have a look at the following paper: https://www.sciencedirect.com/science/article/abs/pii/S1572665717304496

Deglycosylated enzymes in which the sugar has entirely removed, especially from a glycoprotein, should be a good choice for your immobilization method.

Random oriantation as a result of the physical adsorption of an enzyme should alter its activity. This happens due to its active site being far away from the electrode surface. One solution is via the site specific approach that allows fast direct electron transfer to its redox group.

The long shelf life of an immobilized protein still a big challenge so it is not recommended to use an already immobilized enzyme. Also, to the best of my knowledge, there is no commercially available one.

Hi. Hope you are in good health.

As now I m preparing my synopsis.. Not done yet practically

Dear Soudabeh,

I had immobilized my enzyme and observed a change in the Km value. However, mine was a single substrate specific enzyme. I second the opinion of Tapan Kumar; the pore sizes, the immobilization technique used, etc., could play a role in the availability of the reactive site to your substrate.

Dear Nisha S,

You will initially have to calculate the free enzyme's activity - IU/mL (the quantity that was immobilized). Later, post immobilization, please perform the assay ( I performed the IUPAC Ghose assay). I observed a decrease in enzyme activity after immobilization within calcium alginate beads.

@Shamsher Singh Kanwar. Ashok Kumar Nadda. Thanks alot

About a year back there is an publication in RESEARCHGATE regarding this:

Use this Reference if you can:

Electrochemically Activation of Pencil Graphite Electrode for Determination of Ascorbic Acid and Uric Acids

Hello, a lot of results by below ink.

Good luck!

Hello Rafael:

There is a theory called the collision that provides the limits of an enzymatic catalysed reaction and relates the velocity constant of a bimolecular reaction with the activation energy. You can check this paper for an idea:

Catalytic Behaviors of Enzymes Attached to Nanoparticles: The Effect of Particle Mobility, BIOTECHNOLOGY AND BIOENGINEERING, VOL. 84, NO. 4, NOVEMBER 20, 2003.

regards

thank you Jules

the gold nano particles are immobilized on the electrode surface

You could use the commercially available reactive fluorescent dyes which are routinely being used for attaching fluorophores to proteins (extrinsic fluorophores). These dyes react specifically with -NH2 or -SH of -COOH groups present on the amino acid side chains. From their fluorescence, you could quantitate the number of free NH2 groups. I hope the -NH2 groups are free.

There could be some complications due to quenching, dimer formation and excimer formation. Choice of the dye is going to be critical to avoid these complications.

Dear Faterne,

I do not know, if this information is still helpful for you but since I am struggling with the same problem, I will just leave this here. I also used the paper mentioned above, however, for basically all of the proteins I investigated, this size estimate was very off and mislead me.

By now I found a Python script that calculates the dimensions from the crystal structure: https://pymolwiki.org/index.php/Draw_Protein_Dimensions

I tested the script on some proteins with well known dimensions (IgG, albumins and such) and the script appears to be somewhat reliable. And much closer to the "real" values than the ratio vs molecular weight estimate. The insturctions on the pymolwiki is easy to follow and works reliable as long s you have an somewhat high res crystal structure.

Hope that helped you or anyone else you finds this!

Best

Natalie

Amine coupling through EDC/NHS based covalent immobilization is the best option to get the best results.

If you want to compare the activities of your immobilized enzyme to that of the free enzyme, the assay should be carried out in relatively the same condition. Measure and add the CLEA already dissolved in required buffer (the dilution factor is dependent on you) as your enzyme sample needed to carry out the enzyme activity assay with relevant substrate (ABTS).

The final concentration of the immobilized enzyme (mg/ml) is dependent on your chosen dilution factor. For instance, 1 mg of immobilized protein in 100 ul of buffer gives 0.1 mg/ml of protein in solution.

This can then be factored to determine the specific Activity of the immobilized enzyme in U/mg.

Dear Yuan Ye,

Sodium azide is used as an antimicrobial

Best regards

It's for developing a downstream method, just enzyme immobilization

A colleague already suggested a stretavidin/biotin tag. Any other suggestions?

Yes but after very good sonication and well disribution

What do I coat onto nitrocellulose paper to immobilize antibodies on it?

Hi 

you can see this link its useful for you 

good luck

@Nisha S you can follow these article if youhave any quarry please message me.

Hope it will be helpful for your study.

regards

Iron oxide nanoparticles have intrinsic peroxidase like activity (refer to the reference below). Therefore, if your enzyme/protein is susceptible to oxidation, you may find some unexpected results.

Siva

Nat Nanotechnol. 2007 Sep;2(9):577-83. doi: 10.1038/nnano.2007.260. Epub 2007 Aug 26.

Intrinsic peroxidase-like activity of ferromagnetic nanoparticles.

Gao L1, Zhuang J, Nie L, Zhang J, Zhang Y, Gu N, Wang T, Feng J, Yang D, Perrett S, Yan X.

We usually use 100 mM CaCl2 and 1.5% alginate (Pronova UPLVG) for cell encapsulation. The particles are stable without any need of further coating. Critical points may be the lenth of alginate chains you are using (low, medium, hi viscosity), the impurities (you'd better use ultrapure alginate), the Mannuronic/Guluronic acid monomers ration (the higher content in G, the higher strength), the medium where you dissolve the alginate, the inclusion of other ions on your hardening solution... It is of great importance what Evi Lippens points out. The gellation is based on ion exchange, so if you transfere your beads to any medium or buffer w/o Ca++, your beads will desappear rapidly. Make sure also all your solutions maintain a suitable tonicity and pH.

It depends on the size of your bio-emulsifier. 

What exactly it is, a protein or a peptide or simply an organic molecule. 

Immobilization on alginate beads is normally considered entrapment and success will depend on the size of the target molecule. I am not sure how oily your medium is, definitely the viscosity would play a role in the success of the experiment

Wish u all the best .  

Thank you all for the response. I really appreciate it.

I've already check the calculation several times with my colleague and lecturer and it seems that there is no problem with it. 

Dominique, i dont get what you mean, but i express my activity in U/ml.

Mahmoud, element playing a role of activator, did you mean my support? Because im using sawdust as my support by adsorption method.

Alexander, i did not wash my support since im using sawdust. Or maybe i should try to wash it first before immmobilization. And im using 0.05M buffer. Should i try higher buffer concentration?

Regards

Suhaily

yes you can immobilize

I wonder if Fourier transform infrared spectroscopy (FTIR), which is a technique used to obtain an infrared spectrum of absorption of solids, may be utilised for this aim?

Hi. For immobilized lipase, use weighed qty of sample and use the weight in calculation as you use volume for liquid enzyme.

Paralley determine dry weight of immobilized beads and calculate the dry equivalent of enzyme in your sample to calculate activity in units on dry basis 

Mr John, thank you for your suggestion and information. it is very helpful.

- first, I will not use acid for CNT treated. I will mix only CNT and aniline monomer

- second, yes, i after that I plan to wash it using DI water and ethanol as procedure in many references

- third, after make CNT-PANI composite, I will mix it with enzyme. only mix it using stirrer

Thank you

Hi Kirian

Sodium azide and cycloheximide can also prevent both bacterial and fungal growth respectively, sodium azide at 3mM (0.02%) should be OK at this concentration but not sure if it will effect your enzyme activity or not, for xyxloheximide it is 100 microgram/mL.

Hope this help

The cellulase doesn't have to be purified to measure its specific activity. However, if the crude cellulase is a mixture of multiple enzymes, then you probably shouldn't compare its specific activity to that of another preparation containing a different mixture of enzymes.

What is the immobilization chemistry that you are using? You must know what is the concentration of enzyme in your solution to determine if dilution can be accomplished. If it is too diluted you must use ultrafiltration or gel permeation to change the buffer to the one you want or need. 

Do you want to use solar energy to make the enzyme? Or to provide any energy it might need to function? They are two different things. To answer the second one would need to know a lot, lot more about the enzyme, how it works and under what conditions it works, if it needs energy, if so what form should the energy be supplied in? Are any co-enzymes required, and if so which? And so on. Why do you want to immobilize the enzyme on a matrix? Might that stop it working?

Dear Mhetre,

Dip coating may be better in terms of uniformity.

In addition to the answer by Rafik Karaman, I would like to suggest that solar energy could be employed as a source of heat to increase the rate of the enzymatic reaction. Enzymes have optimal temperatures. Sunlight itself, or solar electric energy could be used to heat the reaction to its optimal temperature.

thnk you for your kindly help.

Dear Prof. Dedy,

Thank you very much for your nice answer and valuable reference. I'll go through this. Thank you once again as you always support me for knowledge sharing. I remember, previously, you gave me one of your paper which was so interesting. I hope to continue this bridge.

Have a nice time.

Dear Lidong,

The principal components of an immobilized enzyme system are the enzyme, the matrix and the mode of attachment. The driving forces for enzyme immobilization are the improvement of enzyme stability, increment of volume specific enzyme loading and simplification of biocatalyst recycling and downstream processing. The immobilization methods exploit the fact that proteins have amino acids with different features, whereby functional groups in side chains of these amino acids can be involved in binding to the support through various types of linkages and interactions. The enzymes can be attached by interactions ranging from reversible physical adsorption, ionic linkages and affinity binding, to the irreversible but stable covalent bonds that are present through ether, thio-ether, amide or carbamate bonds.

The recent review article contained in the following link highlights the principal factors in the development of immobilized biocatalysts which include the selection of immobilization supports, conditions and methods with respect to activity and stability of the immobilized enzymes. It also discusses the various surface analytical techniques used to quantify enzyme attachment on the surface of the carrier as well the parameters that are evaluated for the immobilized enzymes. Detail descriptions of the surface properties of immobilized enzymes are mandatory as these parameters are used to compare and assess the efficacy of the different biocatalyst preparations.

Biotechnol Biotechnol Equip. 2015 Mar 4; 29(2): 205–220.

Published online 2015 Feb 17. doi: 10.1080/13102818.2015.1008192

PMCID: PMC4434042

Nur Royhaila Mohamad, a Nur Haziqah Che Marzuki, a Nor Aziah Buang, a Fahrul Huyop, b and Roswanira Abdul Wahab

Hoping this will be helpful,

Rafik

I would try with Oxygen plasma

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

Hii

First you need to determine the Immobilization efficiency and immobilization yield.

Once you have idea of amount of protein loaded onto the particle or matrix you can find out the specific activity for the loaded protein.

Calculate the total protein of your stock enzyme and subtract the protein in supernatent or unbound protein. This will give you idea of amount of protein loaded and determine the specifc activty.

Hope it will help you.

Best luck

Dear Deepak,

It has been shown that fenugreek hydrogel-agarose matrix with gold nanoparticles is a better film than any of those you mentioned (agarose and etc.). Immobilization of acetylcholinesterase on the membrane resulted in high enzyme retention efficiency (92%) and a significantly prolonged shelf life of the enzyme (half-life, 55 days).

I suspect that your experiments may be were unsuccessful because of the instability of the immobilized enzymes on the agarose matrix.

I suggest that you use enugreek hydrogel-agarose film which consists of  2% fenugreek hydrogel and 2% agarose.

For more on fenugreek hydrogel-agarose matrix with gold nanoparticles, please use the following links:

Hoping this will be helpful,

Rafik

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.  

Dear Ashish,

The recent review article entitled "Challenges in the Discovery of Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors " by Ute F. Röhrig et al. depicts inhibitors for IDO1; among them natural products. The following text lists some references for those studies on natural products:

Natural-product-derived IDO1 inhibitors were frequently isolated from marine organisms.^{19,91,99,113−126} While providing good starting points in general for the development of new therapeutics, they also feature some drawbacks. The typical

complexity and large molecular weight of natural products make it often difficult to derive drug like compounds. In the case of IDO1, a large number of natural product inhibitors contains problematic functional groups such as quinones. Frequently, only enzymatic assay results for one or a few compounds were reported, hindering the evaluation of their potential usefulness.

Another interesting review on this topic is the one by Platten et al. entitled " Cancer immunotherapy by targeting IDO1/TDO and their downstream effectors" published in Front. Immunol., 12 January 2015 | http://dx.doi.org/10.3389/fimmu.2014.00673.

Further, the following link contains some of IDO1 and TDO inhibitors approved drugs:

For IDO2 inhibitors, please see the following link:

Front Immunol. 2014; 5: 585.

Published online 2014 Nov 20. doi: 10.3389/fimmu.2014.00585

PMCID: PMC4238401

IDO2 in Immunomodulation and Autoimmune Disease

George C. Prendergast,1,2,3,* Richard Metz,4 Alexander J. Muller,1,5 Lauren M. F. Merlo,1 and Laura Mandik-Nayak1,5

Regarding inhibitors specificity: the answer is yes; there are specific inhibitors for IDO1 and IDO2 (see the mentioned links and attached review).

Hoping this will be helpful,

Rafik

The immobilization protocol and support will bring changes in configuration of enzyme, it will effect the inactivation energy.  

Ooooh sorry, 

Yes im using MWCNT 

I functionalize only using glutaraldehyde

enzyme:candida lipase rugosa

I tried the same method as b-glucosidase f-MWCNT by glutaraldehyde base on link article

Hi there,

I suggest to monitor protein concentration at 205nm which is very sensitive as you detect peptide bond absorption at this wavelength. For info:

thank you

I will try and tell you the result

Hi Elaheh, you should to dissolve both enzymes in a buffer with a pH value below the pI of MSN and above the pI of your enzymes. You can try the mixture in equal molar ratios or equal activity ratio.

I think that it depending on the method of activity determination, but you can try this:

1) Use a definite weight of the support containing the immobilized enzyme.

2) Use a definite volume of your substrate within your reaction mixture to calculate the dilution factor exactly.

3) Use the equation of:

Unit /g immobilized enzyme= (Change in absorbance/ extinction coefficient of substrate x 1000) x (total reaction mixture (ml) / weight of used support (g) ) .

You can treat it additionally by vapour of glutaraldehyde in order to cross-link. An example in attachement.

Good luck my brother.

Hi Mihammad,

I have used DNSA reagent to do Xylanase Activity measurements, see publication attached. HoweverI think the composition for alpha amylase activity might be the same , see link. I hope that might help you.

Good luck

Immobilized enzyme activity and Immobilization efficiency are two different things. 

Immobilization efficiency is the protein binding capacity of a particular matrix/support that you are using. The percent of the total protein bound onto the matrix indicate its immobilization efficiency. 

Activity of immobilized enzyme can be calculated as U/mg of your immobilized biocatalyst.

Good luck

Hi Tiago,

From your question, I guess that you want to link unmodified DNA with a nanoparticle in a way that ensures that your structure is not disturbed.

I'm not sure if this is feasible. You could extend one of the strands of your origami and have a freely floating sticky end in solution. You could then take magnetic nanoparticles with the complement of the sticky end attached to it. This could be done by using thiolated DNA and gold nanoparticles. The sticky ends on the nanoparticle and the origami should hybridize, connecting both.

This issue is complicated by the size of the origami and the nano-particles; whether they are too big to be effectively connected by dsDNA. I'm aware that sticky ended cohesion can be used to link gold nanoparticles together. Another issue is that this interaction is not permamanent.

On a related note, you could extend one of the strands in the origami and place a thiolated base at the end of the same.

Regards,

Atul

Instead of Lineweaver-Burk plot, you can calculate Michaelis-Menten constant for immobilized enzyme using hyperbolic function. The kinetic parameters: maximal current under saturated substrate conditions (Imax) (corresponds to the maximal rate of enzymatic reaction Vmax, according to Michaelis–Menten kinetics) and apparent Michaelis constant (Km) are the corresponding 'a' and 'b' parameters of hyperbolic function y=ax⁄(b+x). 

Dr Pratap

The comment by Colonna is fine.

My question why you do not determine immobilized  enzyme amount throughout its activity assay?  This is a simple approach. If your immobilized enzyme is pure (or nearly pure),  you can establish as standard: Enzyme Unit  (EU) = f (enzymatic protein concentration). Then, it is possible to deduce the immobilized enzyme amount once it is assayed and expressed in EU term. A correction factor  is probably required, because enzyme immobilization is not occurring at 100% due to loss, denaturation and change in the first order rate constant of  the enzyme itself.

You have to make three sets of reaction mixture, one of control and one blank. In blank only water and Nessler's reagent were added. In control and reaction mix add substrate, water, buffer and enzyme but in control first add TCA to stop the reaction. Incubate all (control and reaction mix). In reaction mix add TCA after incubation. Finally add Nessler's reagent in all wait for 5 min and read at spectrophotometer. Surly you will get positive results.

Dear Parastou,

 The short answer is YES. Just recently I’ve reviewed a paper where poly(acrylic acid) and PAA/multi-walled carbon nanotube nanofibrous membranes were fabricated by electrospinning to immobilize acetylcholinesterase. The structure of the membrane was studied by scanning electron microscopy, transform infrared spectroscopy, thermogravimetric (TGA) and mechanical analyses. The tensile strength and modulus of the nanofibrous membranes increased by 2 to 4-fold. The immobilized enzyme showed excellent reusability even after several cycles of washing. Moreover, the pH and thermal stability of the immobilized enzyme was improved compared to the free enzyme. The results demonstrated that the membrane could be counted as a suitable support for immobilization of acetylcholinesterase.

 Best wishes,

 Ilya

Usually is the amino terminus of the enzyme. To interact with the NH₂ of Lysine you need to work in a pH above the pKa, that is above 10. Since most of enzymes don't resist at such pH, most immobilizations are carried out at pH close to neutrality and the Lysine is little prone to interact with glutaraldehyde.

If you are able to work with a high pH you can attach the enzyme to several Lysine residues making a very stable ligation.

You welcome!

Enrico

thank you Professor and sorry for delay.... after synthesis of graphene quantum dot , i add  enzyme in it and wait I wait to be immobilized... enzyme will be immobilized by covalent bonding of carboxylic group of quantum dot and amine group of enzyme.this is my idea

I'm assuming you are using the glutaraldehyde to couple the amylase to the carrier that has an amine function on it.  For this to work best, treat the carrier with glutaraldehyde, and then add the protein.  Bad things happen to  solutions of proteins treated with glutaraldehyde (cross linking and precipitation, for example), which is why glutaraldehyde is an effective disinfectant.  

A more important question, is what is the planned substrate for the amylase.  High molecular weight substrates diffuse slowly, and may not be accessible to active sites of immobilized enzymes.  An example from the 1980s was the immobilization of A. niger amyloglucosidase.  Conversions of starch hydrolyzates by this immobilized enzyme gave significantly lower dextrose values than the free enzyme.  The slower diffusion of the large substrate, coupled with the high concentration of product at the active site, resulted in a high rate of reversion reaction and lower overall yield.   So, think about what you're doing on a molecular level, and see if it makes sense.

you may i) use one of the several commercially available colorimetric assays methods (BCA, Bradford, Lowry...), ii)measure absorbance at 280nm, iii)measure enzyme activity of the obtained solution, iv) if the sample is too diluted you may try to concentrate it through freeze drying and then proceed with one of the proposed strategies.  

 Read attached file.

Best regards

Esam H. Mansour

Thank you very much!

Thank you for your answer!

Focus on tracing of the amide I band (1600–1700 cm-1) of your FTIR spectra, and using OPUS run 2nd derivative procedure.   From 2nd derivative tracing you can evaluate secondary structure of your protein using empirical protein structure-frequency relationships in the amide I region:  Alpha helix 1648 – 1657;  Beta sheet 1623 – 1641, & 1674 – 1695; Beta Turns 1674 – 1695; Random coil 1642 – 1657;  Aggregates 1610 – 1630.   Good Luck.

I used tributyrin as a substrate and NS10086 enzyme.

Accidentally found this, I think it suit all the requirement you mentioned above

Similar to the answers above:

You use x Units of the enzyme for a given amount of support material. After the immobilization procedure, you can first measure the amount of Units not bound (after separation from the immobilized enzyme). Then you measure the activity (Units) bound to the support. In an ideal case, you would bind 100% of the Units used to the carrier in an active form. This is in reality unlikely. From the measurements given above you can at least estimate how much was bound (Units used - units in supernatant, Units bound) so you can estimate how much activity was lost due to immobilization (inactivation of the enzyme). From all this data, you can have a balance of your method. Most important is activity bound, not amount of protein! The second important feature is the activity (U/mg carrier) of the immobilized enzyme and especially the long-term stability after e.g. several batches of reaction (that is why one is doing the immobilization usually). I hope this helps.

Hi Miroslav Pohanka !

Just follow the link:

Cheers...

because ethanol is a non-solvent for chitosan and facilitates the solidification of

chitosan beads;)

I would guess that the porosity of the particles depends on the procedure used to make them. If they are not porous, then external surface area alone determines the amount of enzyme that can be immobilized on them. I see from the literature, however, that there are ways of making them porous.

At first, the optimal pH and temperature of enzyme activity must be determined. For pH stability, enzyme solution was pre-incubated at different pH in the range of 3.0–9.0 for 30 min at optimum temperature for enzyme. The residual enzyme activity was determined.

Read the attached papers