Questions related to Biocatalysis
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.
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 firstname.lastname@example.org.
Will mail the detailed information to that person
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.
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.
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
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.
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?
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.
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.
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 ?
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 %?
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?
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
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?
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,
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?
I want to develop a green technology for production of an active pharmaceutical intermediate ( API) by using biocatalysts.
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?
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
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?
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.
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,
How do I measure enzyme stability (how long the catalysis last) at preferred conditions, say at pH 7 and room temperature?
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.
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.
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?
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?
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!
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...
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?
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.
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.
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.
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?
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?
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.
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?
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?
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.
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?
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).
I am studying the activities of Laccase (EC 220.127.116.11) 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?
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?
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
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?
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.