Science method

Biocatalysis - Science method

Biocatalysis is the facilitation of biochemical reactions with the aid of naturally occurring catalysts such as ENZYMES.
Questions related to Biocatalysis
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In bio-catalysis reaction is going on with yeast and Chloro-Aldehyde in Lb media ,which solvent might use as mobile phase?
My mobile phase is methanol:water , can i put directly inject sample to HPLC or need any treatment.
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Maybe hexane and iso-propanol would be good flowing phase.Of course,they should be mixed in a certain proportion.Acid condition would help keep something stable.
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Hi All,
I recently joined as a Guest editor in Frontiers in Environmental Chemistry where I need to find team members as a co-editor to launch a research topic related to Biocatalysis, Bioremediation, Enzyme engineering, Microbial Enzymes, and Enzyme Immobilization. If anyone intrested to join mail me at sonal.mahajan@dypiu.ac.in.
Will mail the detailed information to that person
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Thanks alot, I am interested
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I'm interested to know if anyone in the area of biocatalysis and enzyme engineering has experience with resuspending soluble proteins (expressed in E. coli) in MS-friendly buffers, the likes of ammonium acetate, ammonium formate, ammonium bicarbonate.
The application is to use the purified protein for enzymatic assays and biotransformations and subject these reaction mixtures to LC-MS analysis.
Most people seem to stick with phosphate and Tris-HCl buffers but these are not amenable to LC-MS analysis, and it seems that the only solution is to divert the flow to waste for a few minutes at the start of the run.
Is there a particular reason that volatile buffers are not more widely used? I understand that a few of these buffers operate within the neutral pH range but I read somewhere that ammonium acetate is a non-physiological buffer, and may hamper enzymatic reactions.
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I agree with Respected William letter..
Once the protein or peptide product is obtained for analysis, dialysis or SPE tips/cartridges can often be used to remove many of the interfering salts, before diluting in mobile phase for LC-MS analysis.
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As I elute my His-tagged protein from a HisTrap HP column, I can always tell which fractions contain high concentration of protein because the eluant is pink.
The colour intensifies as the protein is concentrated, and remains even after imidazole is removed by spin or desalting column.
The storage buffer is Tris (20 mM), NaCl (100 mM), pH 8.
The protein is an NAD-dependent dehydrogenase, overexpressed in E.coli BL21(DE3) cells.
I am curious to know what chromophoric molecule or complex could be responsible for this.
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Dear Kathryn
Did you tried to add EDTA to your protein sample?
In the past i see something similar (protein eluted as orange) for a copper chaperone which was able to bind and extract the nickel from the coloumn which showed an intense thiolate-Ni(II) red shiffted charge transfer band
see the UV spectra in supplementatry Fig 8
To verify it, you can try also to repeat the purification using a different metal (eg Zinc) which is not coloured) immobilized in the IMAC coloumn and see if in this case the protein eluted is coloured or not.
If you are interested, You can find some more information of IMAC performed with different metal in the following presentation of my blog ProteoCool
best regards
ciao
Manuele
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We started a new Podcast focusing on Biocatalysis and Biotransformations. The idea is to interview all invited speakers and keynote speakers of the upcoming Biotrans 2021 in Graz.
We would be very interested in your critique, comments, questions, or suggestions.
Check it out here or at the Podcast platform of your choice: https://anchor.fm/in-the-active-site
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Next one up. This time we had a great discussion with Nick Turner
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Hello, I am try to measure the voltage and current of a solution (typically PBS or other nutrient media) with bacteria like E coli with mediators like neutral red dye or methylene blue. Typical electrodes I'll use is two carbon or copper and aluminum combination in a single compartment setup. I know E coli K-12 shouldn't conduct current compared to other electroactive bacteria, but I'm just trying to get an understanding of setup. I've been using a digital multimeter and I can usually get a voltage measurement, although it is low and varies with the electrodes I use. However, I haven't been able to get reliable current measurements since I'll get fluctuating readings of my multi meter and I don're really know what to make of it. i read papers where this simple tool is used for current and voltage so I'm not sure what I am doing wrong. Thanks.
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Hi Jaqueline,
even without knowledge of your setup but provided that your equipment itself is faultless, I guess it's safe to say that the fluctuations of the current are not caused by the multimeter.
My first suspicion is that your setup for voltage measurement causes a very low current through the solution (or, in an erraneous setup, the amount of current might have no impact at all, sorry for the imputation ;-) while during current measurements a considerable current is flowing. If so, this current could cause a change of the density of bacteria (and/or of other ingredients) in the neighborhood of the electrodes, resulting in a fluctuation of the mean conductivity.
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On the study of biocatalysis in a two-phase system, do you know what is happening (chemically) between the organic solvent and the rest of the components of the reaction?
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Perhaps you can clarify what types of experiments you are doing and what you think would be interesting. Since you are working in DMSO, you can have any possible percentage of water present, 0-99% and this is a parameter that should be explored. ( In fact, if you do not take special precautions, there will be some water present without adding any.) Other solvents which are miscible with water are acetonitrile, tetrahydrofuran, short chain alcohols (methyl-, ethyl and 2-propyl), N-methylpyrrolidinone, N,N-dimethylformamide. Reactions in all of these solvents can be directly compared with your DMSO experiments. Presumably your enzyme will not dissolve in the organic solvent, so it would be a good idea to coat it onto some inert solid support - diatomaceous earth, polymer beads, etc. This can increase activity by making more enzyme surface available. If you use a solvent which is not miscible with water, you can have enzyme dissolved in a separate aqueous phase, which is a common configuration for enzyme reactions of water-insoluble reactants.
Organic solvents often affect the conformation of proteins and can be used to misfold and inactivate enzymes (denaturation). If you are starting with an aqueous solution of enzyme, it may be a good idea to compare slow addition of solvent with gentle mixing vs dripping a small volume of concentrated aqueous enzyme into a relatively large volume of solvent. (The latter is sometimes used to precipitate proteins.) If your activity goes away, protein denaturation may be the problem, but you could also end up precipitating active enzyme. (You may need to collect precipitate and redissolve it in aqueous medium to confirm that activity survives.)
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I just read your article about Trihydroxy-10E-octadecanoate generation as a fungizide on GIT-Labor.de from 2016 (https://www.git-labor.de/forschung/lebensmittel/trihydroxyfettsaeure)
Are there any recent publications focusing on the (chemo-)enzymatic reaction cascade for the production of those TriOHs?
I would like to present this topic to my students in a lecture about industrial biocatalysis.
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Forgot to mention this one, more related to what you seek:
Best wishes,
Rogelio
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I like to collaborate with your project group on Biocatalysis and biotransformation of vegetable oils to alkyl esters. Could you please assist this collaboration by sharing essential research materials to the scope.
Regards
Mustapha
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Hi Tejo,
I am still expecting your message.
Regards,
Mustapha
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It has been known that most of the organic solvents have a detrimental effects on enzymes activity and structure but, if incubation of an enzyme in a specific organic solvent lead to an increase in the activity what could be the reason ?
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They are only similar in their functional group. The difference is established by the number of carbons. Ethanol is a little more hydrophobic than methanol.
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I am studying the effect of different organic solvents on lipase stability. To compare the wild-type enzyme with it's mutants in terms of stability, should I work at very drastic conditions like incubation in 100 % organic solvents then perform the assay or work around what is reported in literature around 30 %?
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Hi there,
There are 2 different approaches: study the effect of the presence of the solvent during the assay (ie. characterizing the immediate effect of the solvent on enzyme activity) or study the effect of the solvent during enzyme storage (ie. long term effect on protein stability). If the experiment is already documented, set your own using published protocol and compare with the published data. You may somehow extend the range of solvent concentration tested.
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I want to interpolate the amount of product formed (as concentration or % of conversion) vs reaction time in a biocatalysis process. The fitting equation should have as (y) the amount of product and as (x) the reaction time. I thought to use as the fitting equation the integrated form of the M&M but I am not able to find the correct mathematical form. Or should I use another equation?
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Without any knowledge about the experiment parameters i can only guess which equation might suit your problem.
Having the product [P] as (y) results in some pretty ugly equations most of the time. I'm assuming no inhibitory effects.
[P] = [S]° - Km * W { X }
or alternatively (for the amount of conversion)
[P]/[S]° = 1 - Km/[S]° * W { X }
where [P] is the concentration of product P and [S]° is the initial concentration of the substrate S.
W is the Lambert-W function (or prodlog). You can try
X = [S]°/Km * exp[ ([S]°-vmax*t) / Km ]
as an argument for that function. I hope your program can fit this prodlog-function.
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Lignocellulose is a renewable biomass which is widely available in nature. It contains cellulose (glucose), hemicellulose (xylose, arabinose, mannose etc.) and lignin. How one can analyse a perticular lignocellulosic (wheatbran) biomass to quantify the above to detect the proportion of each of them. Is there any specific and simple methods are available to analyse. please let me know.
Biofuels, bioenergy, carbohydrates, redusing sugars, biocatalysis, enzymology, sustainable biofuels, renewable bioenergy, bioethanol, biotechnology, pretreatment
Thank you.
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I am going to carry out SEM analysis for  my immobilized enzyme ( Alcohol dehydrogenase) on Eupergit C 250 L (epoxy-polymer). Any recommendation or precautions during sample preparation process?  
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I'm searching an enzyme assay without Pullulan or Soluble Starch like a substrate. Someone knows a method for this? Thanks!
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DNS Method.
Maltose or Glucose As A Standard
UV-540 nm
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Hello eveyone,
I am wondering if there is any biocide in order to keep fungi away from my substrate (alperujo oil, which is mainly made of organic matter, free fatty acids, chlorophylles and phenolic compounds). I am using this oil as a substrate for biodiesel production through enzymatic catalysis with lipases (ROL) immobilised in a polymethacrylate support.
For that reason, I am also searching for some biocide (sodium azide, glutaradehyde, sodium hypochloryte) compatible with both the substrate compounds and also the enzyme. If it exists, which concentration is needed in order to keep fungi away?
Thank you in advance,
Kírian Bonet
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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
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Hi,
I'm doing some development work with thionyl chloride and we are getting residues in our reaction. The round bottom containing the thionyl chloride has nothing else in it, just thionyl chloride. We are keeping this inert with nitrogen which would contain trace levels of water. We are seeing a mixture of a brown, slimy material once the vessel is emptied and a fine white salt like material. 
We're expecting the reaction between SOCL2 and H2O to give us HCl and SO2 and nothing else but this doesn't explain our brown slime or white salt. Any suggestions on what it is or what we've got?
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The residues in our thionyl chloride reaction will be S2Cl2, SO2Cl2, SO2, Ppted sulphur and HCl.
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 I want to develop a green technology for production of an active pharmaceutical intermediate ( API)  by using biocatalysts.
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Please see Chem. Commun., 2005, 1901–1903 
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Hi, I have a question about how to relate the glutamate dehydrogenase enzyme with other mitochondrial enzymes, which are directly involved in the TCA cycle. I need to know, whether changes in the GDH activity will affect the end product of TCA cycle?
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Glutamate dehydrogenase is directly linked to the TCA cycle and competes with the TCA cycle enzyme 2-oxoglutarate dehydrogenase for the same precursor: 2-oxoglutarate, an intermediate of the cycle.
A change in activity of GDH will therefore likely directly impact the flux through the cycle. This is for example important, when engineering bacteria for enhanced glutamate production, where the carbon flux is rediected from the TCA cycle towards glutamate.
Best
Christoph
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I am working  on the immobilization of an alcohol dehydrogenase on Eupergit C 250 L  . 
The activity yield falls dramatically starting from the second batch and this makes the whole process  useless as you know the aim here  is to make the enzyme reusable for many times . I was thinking that this is due to the cross-linking of the matrix extra epoxy groups with the immobilized enzyme then I followed the literature and tried to block the extra epoxy group by Glycine and BSA but , there was no significant change.  
My main reaction for the characterization process is the oxidation of Isopropyl alcohol to acetone and I am thinking  that the problem is not related to the immobilization process but due to the reaction it self .
 I think  this happen due to enzyme inhibition by high concentration (0.5 M)  of my substrate "Isopropyl alcohol"  or due to  enzyme deactivation upon product formation  as you know "acetone is used generally  in enzymes precipitation. 
Any suggestions or recommendations  
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Are you sure that all the enzyme was covalently attached to the resin during the first experiment? Perhaps half of it was associated loosely with the resin, but this half was washed off prior to the 2nd run.
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When same amount of enzyme unit both from plant and microbial sources are compared to cleavage C-O-P ester bond of organic phosphorous, it has been found that enzyme from plant sources are at least 30% more efficient in hydrolysation as compared to phosphatases from microbial sources. What are the possible reasons for that?
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I think it may be due to constitutive and inducible enzyme system, that is making the difference. Need to check
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Famous Koshland theory was developed for water-soluble enzymes (99% of all)/ Some vital enzymes, like cytochrome P450s (CYPs), are embedded into phospholipid matrix of microsomal (endoplasmic reticulum) membranes. By definition, hydrophobic substrates of CYP (mainly, medicinal drugs), has to reach CYP active center thru lipid barrier and plasticity of CYP is still a question to answer.
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Dear Ilya
This is a very interesting question but a quite complicated one to answer, unfortunately.
Obviously, for those P450 enzymes that are membrane-bound, the question is how does the substrate reach the active center. James Halpert and his colleagues on CYP2B enzymes and others on CYP3A enzymes have shown that protein plasticity is important and that Koshland theory holds since it seems that theenzyme 3D structure fits to the substrate (if I may say so).
The problem is dual: on one hand, the active center in P450 enzymes is buried within the protein core so that the substrate has to reach it through channels, on the other hand the membrane-bound P450 enzyme has to deal with highly hydrophobic substrate that have a quite high LogP values and that stickswithin the membarne bilayer instead of going freely in the surrounding cytosol.
There are a few papers on that, especially those form Michel Otyepka and his colleagues (J Phys Chem B. 2013 Oct 3;117(39):11556-11564)... and there is one from my group showing that for CYP1A enzymes what is important for P450 substrate specifciity with PAH substrates is NOT LOgP but the substrate size (Biochim Biophys Acta Gen Subjects 2015, 1850: 696-707).
Best reagrds,
Philippe Urban
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Hello Every one,
I am working on saccharification of biomass with crude enzyme. In this case I need to select  certain concentration of enzyme (eg: 500 U/ ml), If suppose my enzyme source concentration is 2000 U/ ml . Can I simply dilute the crude source with sterile distilled water up to 500 U/ ml or something else? and what is the best concentration of crude enzyme for saccharification of biomass?
Thanking you all,
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Thank you All
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How do I measure enzyme stability (how long the catalysis last) at preferred conditions, say at pH 7 and room temperature?
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Prepare a stock solution of the enzyme at each set of conditions you want to test. Then, at various times, withdraw a portion of the solution and test its activity in the enzyme assay.
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I am looking a practical method of biocatalytic oxidation of octanol to octanoic acid, so I want some practical biocatalysis methods which will be efficient for large scale synthesis as well.
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For making vinegar from wine Acetobacter is used. I don't know if it would work on octanol.
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Glycerol is a green solvent but it is too viscous i.e. the real problem for whole cell biocatalysis. We can use it as a medium for pure enzyme catalysis but I don't know whether it work for whole cells or not. 
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Agreed with David. Moreover, if you increase the temperature for biocatalaysis, then bear in mind temperature optimum for your enzyme. Also miscibility of solvent with enzyme cell will be less resulting in slow reaction.
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ACC oxidase is an enzyme which converts ACC into ethylene in plants/fruits
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ok thanks for ur information
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In case of using crude enzyme samples, which we knew the properties of the crude enzyme is not pure and heterogenous. Is it better to use the DNS (with rochelle salt before) or use the DNS reagent first and rochelle salt at the end of the reaction?
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You have the answer, You can include sodium-potasium tartrate to increase stability of the DNS reagent.
HNS
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As everyone knows: 
Vmax = Kcat x [E]t 
with [E]t is total enzyme CONCENTRATION
Vmax unit eg umol.min-1 (Im sorry, I mistyped this before)
Kcat unit should be min-1 or s-1
So my question is, if the [E]t is really a concentration, we can never get the unit for Kcat as min-1 or s-1 but there always the volume unit appears along with the time unit, such as ml.ml-1. 
However, if you use the unit mol for [E], you will get the unit for Kcat as s-1 or min-1.
This raises to me a question that do people really mean [E] as the concentration with the unit e.g. mol.l-1 or do they mean [E] as the AMOUNT of enzyme with the unit of mole only?
Thank you.
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Much of the misunderstanding about units employed for describing enzyme activity derives from the practice (which has a long history) of expressing activity in terms of AMOUNT of product formed per unit time (e.g. micromole/min) instead of using the RATE of product formation (e.g. micromole/ml per min). In chemical reaction kinetics, reaction rates are always considered. Why enzymology still uses amount/min is difficult to explain,.but it is probably too late to change things now. As long as authors are aware of the paradox there is no cause to worry. Some confusion can arise however when authors use 'mol' as an abbreviation when they probably mean mol/litre
Peter
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I'm looking for any study that found fungal biotransformation of terpenes by the secretions of a fungi grown in agar petri dishes (or any solid substrate). That is, some fungus capable of biotranfsorming terpenes without "touching" them with the hyphae, but biotransform the terpene by secreting some substance (exoenzymes or similar). If you don't know about any study like this on terpenes, may you know about some with other substances? or with bacteria or any other organism?
I've been quite extensively searching about that and by now I have not been able to spot any article like this.
Thank you very much!
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Hello Ander, the diversity of terpenes imply that there are multiple degradation pathways involved. You may want to have a look at this paper which is specifically looking at limonene degradation:
Wang, Y., Lim, L., Madilao, L., Lah, L., Bohlmann, J., & Breuil, C. (2014). Gene discovery for enzymes involved in limonene modification or utilization by the mountain pine beetle-associated pathogen Grosmannia clavigera. Applied and environmental microbiology, AEM-00670.
I am currently pursuing this work by doing the functional characterization of genes putatively involved in the initial steps of the proposed pathway.
Salutations
Philippe
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chemical synthesis 
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Hi Mohammed
I work for the last almost 25 years in the biocatalysis of a big chemical company...I would take the words of Obama " Yes, we can!" The exeptance is much better today, you have a lot of bioprocessing and a lot of bioconversions that work far better than traditional synthesis, solvent free, low temperature and low  pressure settings that you can switch to almost any location worldwide.  It  is also not just a special favour that this big companies give to this technique; their likes base on hard economical facts, so you can be sure that if there is a preference for bioctalysis method, it needs to be better. New echnics like the construction of catalytical enzyme "antibodies" or the usage of new, non-natural amino acids to create new proteins offer you a far wider range in the future
I can give you an example from a field on which I worked for 10 years:   the kinetic racemic separation of chiral molecules. There is no direct synthetic acess, because besides of a few very complex asymetric hydrogenisations, that have no practical value for comercial production, everything is done by cristallization. There are companies that offer the classical Pasteur's tartaric acid cristallisation in big industrial scale.
First, you need to do big material efforts to realize and recycle the chemicals. this can be ecomomically and logistically difficult... and second:
you are fixed on the cristall eutectic point of your mixture....this parameter fixes the lowest energy level to a specific concentration, what means that you cannot purify your product over this value, even if you cristallize until the end of days.
Mostly you try to use other cristallisation helpers with high chiral purity, but as your organic synthesis reaction always ends in racemic molecules, you need to get this from a "natural" ,means biochemical ,way
So you get in trouble if this value is f.E.  ee=97,0% and your customer asks you for a high pure prodct, ee>99,5%. This is not obtainable with a "classic" method.
Nevertheless your kinetic racemic separation gives you the chance, even if the Quality of the reaction is not perfect, to purify up too the given purity.
In case of your desired enantiomer does not react with the enzyme, the wrong one diseapears in a higer number than 1:1 from the reaction you will always get almost 100% chiral purity. you can predict this, as soon as you see any kind of ciral activity between enzyme and substrate.
Other, fermenation based reactions can work with extremly cheap incredients or extremely high conversion rates
kind regars
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Hi there,
for a continuous enzymatic reaction (membrane reactor with immobilized enzymes), I am looking for a buffering substance that is not or only slowly degraded by thermic or other processes. pKs should be around neutral.
Phosphate would be the best choice, but I cannot use it due to its chelating properties for divalent cations which are a vital component of my solution.
Tris would be the next option, but it is quickly degraded over time as I recall correctly...
I do not want to let it all fail because the buffering substance gives up even before the enzyme dies...
Any ideas?
best regards,
peter
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It would be helpful to know what pH you would like to maintain. Here is a web site about buffers that you may find useful.
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We need a non-aqueous system for dissolving glucose. But, we found DMSO had a bad effect on the biocatalyzer-enyzme. Is there any other solvent for glucose can be used in biocatalysis?
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Glucose is very hydrophilic and dissolves readily in water. If you can work with very low concentration of glucose in your system, it may be possible to look into other solvents with lower dielectric constants than water. Go through the list of various solvents (with decreasing dielectric constants) and look up for solubility of glucose.
You seem to be using enzyme(s), which generally speaking, work in aqueous medium. Despite the fact that some regions or pockets can be very hydrophobic but to maintain normal structure (and function), the medium should still be aqueous. 
Seems like a tough situation... good luck!
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I am going to carry out research in this area. I would like to apply these materials as biocatalysts for biodiesel production. I am trying to get challenges in this field so if you have related materials and ideas, please provide me some details.
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There is a paper not exactly with enzymes but with sugars from honey as capping agents for gold and silver nano particles and pd and fe nanoparticles.   Honey Mediated Green Synthesis of Pd Nanoparticles for Suzuki Coupling and Hydrogenation of Conjugated Olefins, Nanosci. Nanotechnol. Lett. 2012, 4, 420-425. S. M. Reddy, K. K. R. Datta, Ch. Sreelakshmi, M. Eswaramoorthy and B. V. Subba Reddy. 
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How does one quantify total cellulose, hemicellulose, and lignin in biomass before pre-treatment of lignocellulosic biomass?
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Try this.
Relux the biomass sample at 80 C with 0.5 M NaOH for 4 h, (100 ml/ g of biomass) filter and wash the residue with plentiful of distillled water until PH 7- this should give you hemicellulose.
Soak another sample with at room temperature in concentrated H2SO4 (30ml/g of biomass) for 24 h, and then reflux at 100 C for 1h. Filter and wash the residue with distilled water until PH-7 - this should give lignin.
You can deduce cellulose by difference.
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We are a new group at Fiocruz (Brasil) that wants to try to produce recombinant transglutaminase from Streptomyces sp. for biocatalysis. However, I am really surprised that even when there are a lot of microbial transglutaminases ( http://www.ncbi.nlm.nih.gov/protein/?term=transglutaminase), industry and research groups mainly use that from Streptomyces. Could anyone help me or advise why this happens? I am really curious because maybe it could be possible to try to express another mTG more easily or with different specificities and properties.
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But researchers only use mTG from Streptomyces and no one more use another or tried to used. This is really weird.
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Generally enzyme unit is defined as, "amount of enzyme required to release μM of product per min or require to catalyze uM of substrate per min." But I am using 50 ul of purified enzyme (0.01 μM concentration) with 50 ul of substrate (total reaction mixture is 100 ul now) followed by incubation for 5 min and estimation of reducing sugars by DNS method. Now I am able to get mg of sugar released per min from standard curve but I am confused about how to define the unit in this case as I am using fixed/ known concentration of enzyme.
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you have mentioned that you use the purified enzyme for your calibration curve, if the purity of the enzyme is close to 95-100% then you can recalculate your sample enzyme activity per mg of the enzyme in the sample.
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When we are going to calculate the acid value of oil, we perform various titration, What is the exact mechanism involved?
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What M. B. Deshmukh said is correct defination of acid value of any material may be oil, polymer resin, etc.  In this case you titrate KOH against carboxylic group present in material. Until last carboxylic group is not get react added KOH is consumed for that. First drop of KOH which do not have carboxylic group for reactions makes reaction mixture basic and solution turns into pink colour due to already present indicator.
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I have assayed cellulose activity, by DNS method, I got released reducing sugar in mg/ml for this. How do I calculate enzyme activity and express in IU?
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First, convert the amount of reducing sugars from mg/mL to micromol/mL. Then multiply by your assay volume to find out how many micromoles of reducing sugars were released. Divide by the time of the assay and you will get the IU. One IU is defined as the amount of enzyme that releases one micromole of redicing sugar per minute in your reaction conditions.
Also remember that it is more common to express activities per weight of enzyme present in your experiment (i.e.: IU/mg of enzyme).
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I tried to figure out the overpotential term that exists in MECs. Going through the literature to find a sensible meaning, I understood that it should be defined based on the specific electrochemical cell that we are working on it. Does this mean the potential difference between the theoretical and actual potential that is needed to form Hydrogen? Should it be calculated for each half cell separately? What are the sources of these overpotentials? Do you choose a metal catalyst because of the low activation overpotential for gas products (H2) evolution reaction? How is it possible to discuss an overpotential when the metal catalyst is replaced with a biocatalyst?
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Some of my workmates are working on hydrogen production in alkaline media by means of electrodeposited electrodes. This is one of their papers and I hope that it will help you:
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For example pancreatin includes lipase, amilase and protease. I want to know their assay results and also a quantitative results, because each enzyme activity result doesn't show me the percentage of pancreatin powder.
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I want to seperate the enzymes in pancreatin powder and assay them individually
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I presented a compound to a microorganism for some time after that I extracted metabolites (the transformed product) and tested it on TLC card. On TLC there was two different spots other than the original compound. I tried many methods to separate them by HPLC: two different gradient methods and on isocratic method, but every time I got only the original compound peak but nothing else. Do you have any suggestions to separate the products?
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Basically the HPLC method you need to use depends on several things. One important thing that you should follow is that you need to use the HPLC method that can separate the compounds that were separated with TLC card. Or at least similar class of compounds. For example, if TLC method you used was for detection small acidic water-soluble compounds you do not have a real chance to get separated on reverse phase HPLC designed for separation hydrocarbons.
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Need liquid catalase.
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Naomi is right. I would add thta concentration should not be higher that 1 mg/ml and then you can dilute if necessary. Catalase has a high turnover number
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I am working on lipase immobilization on silica gel. First, I did a pretreatment for silica gel using 15% 3-APTES in acetone and stirred in 50 degree Celsius for 2 hours. I used gluteraldehyde 2% as a cross-linker and it had been modified by heat in 65 degree Celsius for 25 minutes. Then the modified gluteraldehyde was mixed with treated silica gel in 20 degree Celsius, 2 hours, 300 rpm to activate the silica gel matrixes. Immobilization process was done by mixing the lipase solution in phosphate buffer pH 7 (0,1g/ml) with the activated silica gel, with constant mixing at 300 rpm in 20 degree Celsius for 2 hours. A lipase solution before and after the addition of silica (the supernatant) was analyzed using Bradford Method to measure the amount of protein. By measuring this, it's expected that the amount of protein in the solution is decreased so I can conclude that the enzyme successfully immobilized on silica with a certain immobilization degree. After the calculation, I found my immobilization degree very low (about 2,46%). What should I do to enhance the immobilization degree?
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You can try glutaraldehyde conc. below 2.5% with slightly acidic condition as it enhance the crosslinking ability of glutaraldehyde.
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Should I use IC50 or Kd, or something else?
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It seems that you expect that your compound may increase the activity of the enzyme, which would make it an "activator" not an an "agonist," a term used for receptors. It would be useful to measure the maximal -fold increase in enzyme activity, and the concentration that produces half of the maximal effect (EC50) when comparing the effects of multiple compounds. The Kd (dissociation constant), a measure of the affinity of the compound for the enzyme, would also be of interest but might be more difficult to obtain.
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I need a procedure that does not contain veratryl alcohol in the case of lignin peroxidase and is not at all in the UV range.
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I have tried plate inhibitory assay for the identification of inhibitors. After adding one particular compound, the zone size was increasing three fold than control. What is the mechanism behind that?
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Interesting.....seems like "hormesis" effect.....
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I am having some problems to find out the linearity with hydrogen peroxide using the Kishimoto and Takahashi method.
Kishimoto, M;. Takahashi, T. A Spectrophotometric Microplate Assay for L-Amino Acid Oxidase. Analytical Biochemistry 298, 136 - 139 (2001).
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I agree with Jay Fox
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Malate thiokinase active site residues
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I did a quick search in the main enzyme reaction databases (BRENDA, KEGG, CSA, MACiE, SFLD, EzCatDB) and I found some literature references that may have relevant information in BRENDA: http://www.brenda-enzymes.info/php/result_flat.php4?ecno=6.2.1.9 Good luck with your search.
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I am studying the activities of Laccase (EC 1.10.3.2) enzyme in soil. Using ABTS (2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid). I cannot observe change in absorbance at 420nm or near about it (410-420nm) activities. I am using protocol, copy is attached here and can also be accessed online via link www.eeescience.utoledo.edu/faculty/weintraub/Laccase_protocol.doc‎. I tried in many different ways with all possible options. But I cannot make any attempt successful. Should I change the substrate other than ABTS?
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The ABTS is from the best or the best substrate of laccase, it is widely used to this enzyme activity. The substrate is very soluble in water then it is very easy to use with the enzymatic protein and the product is very stable and very easy to oxidase in 1 min if you have good activity. I have work a lot with the substrate for laccase activity, the product is green and highly detectable at 420 nm. I have just to remarks:
1- The concentration that you use is good (0.4 mM) but you can increase until 1 mM if you want to screen the little activity.
2- It is not necessary to add H2O2 is not a peroxydase that you test, the seconde substrate of laccase is the oxygen and you not need H2O2.
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Directed Evolution is very complex and many techniques were used, and it is not easy to just list them here shortly. Basically you need a mutagenesis technique and afterwards an assay to identify your desired variant. Enzyme production and cloning methods are also involved.
- mutagenesis, random: many techniques: most popular epPCR and uncountable derivates, shuffling and again uncountable derivates
- screening / selection: always a new methods has to be designed, depending on the enzyme activity and task
You can check a lot of very nice papers form the groups of Frances Arnold, Manfred Reetz & Karl E. Jäger, Uwe Bornscheuer and many many more.
Here are some new articles of my old group:
Bornscheuer, U.T. (2013), Protein engineering as a tool for the development of novel bioproduction systems, Adv. Biochem. Eng. / Biotechnol.,
Davids, T., Schmidt, M., Böttcher, D., Bornscheuer, U.T. (2013) Strategies for the discovery and engineering of enzymes for Biocatalysis, Curr. Opin. Chem. Biol.,17, 215-220
Bornscheuer, U.T., Huisman, G., Kazlauskas, R.J., Lutz, S., Moore, J., Robins, K. (2012) Engineering the third wave in biocatalysis, Nature, 485, 185-194
If you need more information, please come back to me, Marlen
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Recently I tested my kinase activity by using phos-tag, and I need a control to confirm the kit is working. Do anybody know where can I by the cheap kinase and the substrate together?
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Jianbo, I understand your dilemma, but an unrelated kinase is not a very useful positive control. If you just want to see if Phos-tag acrylamide can separate a phosphorylated protein from an unphosphorylated protein you can use casein and alkaline phosphatase. Both reagents are cheap and come in solid form and they are what the Kinoshita group used used to validate the technology when they developed it.
Even if you find that your phos-tag gel does separate phosphorylated from unphosphorylated proteins it doesn't tell you whether your kinase is functional or not. Phos-tag just might not wok for your protein substrate, or only a very small fraction of your total substrate is phosphorylated. Since you don't have much information about the activity of your kinase your best bet is to monitor kinase activity directly, I would suggest using 32P-ATP as a nucleotide-substrate and seeing if you get any 32P transfer to your protein-substrate. With this method you can tell if even a small fraction of your protein-substrate is being phosphorylated.
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I'm interested in screening lipases for a lactonization reaction. The substrate contains a secondary alcohol and a a carboxylic ester, so effectively looking for a transesterifcation reaction. The desired lactone would form a nine-membered ring. I'm aware of a number of kits available for screening immobilized lipases, but wondering if there is an obvious 1st choice for this type of reaction in an organic solvent. I have only experience using PPL, but this was to make alpha-omega lactones from omega-OH fatty acids(eg. C15-C18), not for internal lactones with substiutuents "beyond" the two reacting groups. Any advice appreciated
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I would use the same protocol you used for the omega-OH experiments. A problem you should foresee is that cyclization of medieum size rings (8-10 atoms) are very difficult because of the transannular esteric effects. But your goal is a very interesting one. Keep us posted!
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I've attached my enzyme covalently on carbon nanotube, I wonder what kind of structural characterization do I need to prove that my enzymes have already attached?
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Maybe try electrophoresis?
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I have brought four different mutation/replacement at one important (catalytic) active site residue of an enzyme. I found that the mutant reflects no activity at the normal identical assay condition provided for wild enzyme. However, when the substrate cofactor and enzyme concentration is being increased in an assay, the active site residue mutants reflect measurable activity, although less compared to wild enzyme. The mutant enzymes have demonstrated less affinity for substrate and cofators since the kinetic parameters like Km and Kc values have increased by several fold. I wonder what structural and fucntional impact the higher substrate and cofactor would bring on a mutant that leads inactive enzyme (no activity) to active enzyme (as shows activity). The mutations were like Glu to Ala, Asp, Gln, Asn.
I do not know at the moment how I should support this fact with explanation and with further experiments. Would it be appropriate to compare the kinetic results of mutant with wild enzyme? The assay conditions vary in each case.
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Dear Irshad,
This question has two interesting layers.
The first one is somewhat trivial (even though interesting because very few people usually think in these terms), the chemical activity is commensurate with time. In another words if you wait long enough (sometimes longer than the age of the entire universe) the chemical reaction would proceed without catalysis (in gas or liquid phase). What it means is that everything (including your "inactive" mutants have "some" activity) and has some rate (sometimes infinitesimally small). So there is no qualitative difference between your "active" and "inactive" enzymes and your duty as a researcher is to establish the level of activity and differentaite it from the base reactivity (without a catalyst). The two simplest variables to change are protein and substrates concentrations. To illustrate this point I am citing two papers from my past where the activity was knocked down by multi-thousands fold (J. Mol. Biol.. 277(1998)647, Acta Cryst D 61(2005)1072).
The second layer is much more exciting. This is a question about the details of a catalytic process and what happens in the case of a mutant. In order to answer this question we have to have a rudimentary understanding how a particular enzyme (and as a matter of fact, every enzyme) is working. Without the detailed knowledge of the enzyme I cannot help you in details but I can point you to the important factors. Biological catalysts (that includes proteins, nucleic acids or even lipids) work by accelerating the reaction taking advantage of several physical processes that are contributing to what is commonly described as a transition state stabilization, or equivalently, lowering of the crossing thermodynamic barrier. These processes are:
1) Detailed three dimensional alignments of the incoming reactive atoms of the substrate and the aiding groups of the catalyst (residues of the protein). That applies to the angles, distances, and planes as the orbitals must be disturbed to achieve the final electronic transition leading to the formation or breakage of the bonds.
2) There must be detailed alignments of the charges (or charged groups of the substrate and the catalyst) to facilitate the electron or proton transfer responsible for the realization of the chemical rearrangement. Therefore the active site groups and pH play a crucial role in activity.
3) There must be substantial change in the electrostatic field at the active site to promote the reaction. This is why most of the active sites are located deeply in the protein matrix. By closing active site loops and expelling solvent (water) the dielectric of the medium changes by a factor of 20 making any electrostatic interactions stronger by the same factor thus facilitating the chemical rearrangement which is nothing else but changes in electron distributions.
4) What directly follows from the last two points is that enzymes usually must isolate the active sites from the aqueous environment by closing the loops and expelling mobile waters.
5) The act of catalysis itself is not static and definitely not an equilibrium event (as considered as an individual act), therefore, it is a dynamic nonequilibrium event. The height of the barrier preventing spontaneous chemical transformation varies all the time as well as the velocity of incoming substrate. Therefore, the protein dynamics plays a crucial role in enhancing the fluctuations leading directly to the enhanced rate of crossing the barrier. Almost precisely like a stronger gun promotes shooting through the shielding.
There are several other factors influencing activity but let's focus on the presented above.
In answering your question why your "inactive" mutants are active you have to determine which of these factors are changed, and which are preserved in your mutant, and which are playing a crucial role in the acceleration of the rate. It is obvious that almost everything stays the same, with the exception of the positioning of the crucial oxygen atom that was located at the Glu residue of the WT enzyme. So the answer is probably covered by the experiments that I cited. When you exchanged Glu for Asp you deviated the distance between the substrate and the crucial oxygen by at least 1 Å. Such changes usually lead to a change of the rate by 100 fold. In changing Glu to Gln or Asp you probably changed the polarizability of the oxygen in question (pKa) by at least several pH units which leads to changes on the scale between 100-1000 fold. In Ala mutation probably a spurious water molecule took over the role of a reactive oxygen and lowered the rate by more than 1000 times.
In conclusion there is nothing surprising that your mutants have activity and that you were forced to raise significantly the concentration to see it. Concentration is usually a proxy for changing the time or energy state of your system to access the areas that are not available under "normal conditions". This is like rate of nucleosynthesis that is not normally observed (it would constitute a medieval transmutation in alchemy): you have to have much higher pressure and temperature to get to force the nucleons to coalesce and form new atoms. So we can quite easily transmutate nowdays the elements into each other in nuclear reactors.
I hope the answer was not too long so I do not loose the audience but I think that many similar questions are posed on the Research Gate and do not get in depth response.
To provide a support for your results for the publishing purposes you just simply search the literature and find similar cases. More mutations or different assays might but not necessarily elucidate any possible answer.