Biotransformation - Science method

A simple analysis to confirm if biotransformation of mercury has occurred includes:

1. Total Mercury vs. Methylmercury Analysis:

• Measure total mercury (Hg) and methylmercury (MeHg) concentrations using techniques such as cold vapor atomic absorption spectroscopy (CV-AAS) or inductively coupled plasma mass spectrometry (ICP-MS).

• An increase in the proportion of methylmercury relative to total mercury suggests microbial methylation.

2. Speciation Analysis Using Chromatography:

• Gas Chromatography (GC) coupled with Mass Spectrometry (MS) or Atomic Fluorescence Spectroscopy (AFS) can be used to differentiate inorganic mercury (Hg²⁺) from organic mercury species (MeHg, ethylmercury, etc.).

• If methylmercury or other organic forms are detected, biotransformation has likely occurred.

3. Volatile Mercury Detection:

• Gold amalgamation with CV-AAS or GC-MS can detect elemental mercury (Hg⁰) in the headspace of microbial cultures, indicating microbial reduction of Hg²⁺ to Hg⁰.

4. Colorimetric or Fluorescence-Based Mercury Sensors:

• Some biosensors and chemical assays can detect specific mercury species, providing qualitative or semi-quantitative confirmation of biotransformation.

These methods allow for a straightforward confirmation of mercury biotransformation in environmental or laboratory settings.

Hello,

Your analytical approach covers a broad spectrum, and the techniques you've proposed are highly effective for understanding different aspects of mercury bioremediation.

Here are a few additional methods to complement your research:

These methods can provide a deeper understanding of how mercury interacts with bacterial cells and the transformation processes it undergoes.

We really do not have enough information to provide specific suggestions for analysis. Does your sample have mild to strong chromophores ? If so, use a scanning diode-array detector inline for detection. Change the column to one that has a particle size of 5 microns for better resolution. Your isocratic mobile phase is VERY weak. To improve resolution of actual sample peaks, proper HPLC method development must be undertaken. Generic advice may include: Use a gradient HPLC method starting at 5% organic increasing to 95% organic (i.e. ACN) over time. Hold at 95% organic to insure all samples have eluted off the column. Review the results. Is retention found for the compound(s)? Does the K prime of the sample show adequate retention? Is the peak shape Gaussian? HOw many peaks were detected? What do their UV/VIS spectra look like? Etc. Do this following good chromatography fundamentals Please contact a local, experienced chromatographer to assist you.

To accelerate the bioconversion of rice husk into organic fertilizer in less than two months, strategies include mechanical processing to increase surface area, microbial inoculation with lignocellulolytic fungi, nutrient additives to support microbial growth, vermicomposting to enhance decomposition, and maintaining optimal moisture, temperature, and aeration conditions during composting.

Danial Bahroz Tahir It's not my field at all, but I found several papers on your Q1 and a few on Q2 with a simple Google search. I presume you have looked at these?

In most of the biotransformation, it is NOT necessary when using an unprocessed as a whole biocatalyst, while using isolated enzymes during biotransformation delignification may be preferable

Dimethyl formamide (DMF) is a good solvent to dissolve most organic compounds. I suggest you make a stock solution of 100 mg/mL in DMF then add 20 to 50 uL (i.e. 2-5 mg) into the 50 mL grown microbial culture for its biotransformation. Try this hope you will get it biotransformed.

Good luck,

Faheem Khan

I suggest ethanol or DMSO.

There's probably no one recipe for all drugs. I think it's pretty consistent that an amorphous form, if you can obtain it, is more rapidly soluble, and that smaller particles dissolve more quickly (due to the larger surface area), but the other factors will vary from one drug to the next. You don't specify if you're relying on low (slow) solubility for the sustained release effect.

Great question, thanks for asking.

I would like to add to previous answers that salts containing sodium, potassium, magnesium and calcium are the major minerals found in biomass ash, these elements are part of the alkali and alkaline earth metals (AAEM). For example, during pyrolysis, these minerals, especially AAEMs, act as catalysts and possess a great potential to alter the resulting bio-oil composition. The feedstock could be pretreated prior to pyrolysis to reduce its ash content.

https://pubs.acs.org/doi/10.1021/acs.chemrestox.1c00078?__cf_chl_jschl_tk__=pmd_Chw4x0B5k6_htcGALIVeJ4LLe.dNhLbEhumaIefhf14-1634841073-0-gqNtZGzNAjujcnBszQjR

Please check the attached manuscript. You can use the equation to calculate the % yield mentioned in the attached file.

Next one up. This time we had a great discussion with Nick Turner

First better to detect quantitatively any accumulation of Arsenic than perform analysis. For detection you need to use about 20-80mg of cells digestion in HNO3.

Also check https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155122

Drinking-water containg Arsenic increases the risks of cancer in the skin, lungs, bladder and kidney, as well as other skin changes such as hyperkeratosis and pigmentation changes.The effects on environments include death, inhibition of growth, photosynthesis and reproduction, and behavioral effects. Environments contaminated with arsenic contain only a few species and fewer numbers within species. If levels of arsenate are high enough, only resistant organisms, such as certain microbes, may be present. If arsenic poisoning occurs over a brief period of time, symptoms may include vomiting, abdominal pain, encephalopathy, and watery diarrhea that contains blood. Long-term exposure can result in thickening of the skin, darker skin, abdominal pain, diarrhea, heart disease, numbness, and cancer.

Have a look at this useful RG link.

I also have the same question. Besides, what is difference between NaOH and NaClO2 in removing lignin?

I hope this link help you

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987086/

Hi Nelson,

You are right, the biodegradation of PET and PU is much better established. This is largely due to the presence of ester bonds in their chemical structure, which makes them substrates of different types of esterases. Despite this, they are still difficult substrates for biological degradation. Polyethylene, polypropylene and PVC are even more recalcitrant. Their biological degradation is more difficult compared to the others and therefore not a realistic goal of P4SB.

Hope that helps.

Best regards,

Jose

hi

i dont know this answer help you or not, but according to my research , the enzymatic depend on concentration of substrate and enzyme , while temperature decreases , because cell is following osmotic pressure , for low temperature the concentration of substrate and enzyme increases , maybe your answer lie down in temperature dependent .

@Muna please do share them. Thanks in advance

Hi Tejo,

I am still expecting your message.

Regards,

Mustapha

The body produces a steady stream of metabolic waste products (ammonia, bilirubin, urea, lactic acid, etc.)

along with toxic byproducts called – exotoxins - as a result of microbial activity in the human intestine. Continual exposure to toxins originating from both within the body and outside the body gives credence to one of the body’s most vital functions – hepatic biotransformation. The sole purpose of the biotransformation process is to convert toxic compounds into non-toxic, water-soluble compounds which can easily be eliminated. The liver (hepatic) is the key player

in this process.

Detoxification - for many years the term “detox” referred to breaking free of alcohol or drug addiction. Nowadays,

detox means removing all toxins from the body; not just poisons from substance abuse, but also heavy metals, chemical additives, and other toxins found in our food, water and air. As well as the metabolic waste products produced by the body.

Biotransformation - is defined as a metabolic process whereby chemical modifications or alterations are made by the body of a specific chemical compound, usually by means of enzymatic activity. As mentioned these chemical compounds originate either from within the body, usually the result of metabolic waste and are called endogenous compounds or they come from outside the body and are called exogenous compounds.

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.

Dear Santosh,

Ozone is not "present in the uppermost layer of the atmosphere": its concentration peaks in the stratosphere, which is indeed higher up than the troposphere, but there are other atmospheric layers at even higher altitudes. For an introduction to the structure of the atmosphere, see https://en.wikipedia.org/wiki/Atmosphere_of_Earth#Principal_layers, and for an initial perspective on the vertical distribution of ozone in the atmosphere, see http://paoc.mit.edu/labweb/notes/chap3.pdf.

Inert chemical compounds do indeed exhibit vertical profiles that smoothly decrease with height, like CO2, for instance. However, ozone is a chemically active substance: it is continuously 'created' and 'destroyed' in the stratosphere, due to a set of chemical reactions between the molecules present in that layer and stimulated by ultraviolet solar radiation. The observed vertical profile is the net result of these chemical reactions, combined with the specifics of the stratospheric circulation of the atmosphere, which is rather decoupled from that of the troposphere.

There is a large literature on the dynamics and chemistry of the atmosphere: you will find many relevant sources by searching for these keywords on the Internet.

Good luck in your investigations, Michel.

Hi Sonika,

The simplest way would be to use Baker's yeast to convert sugar to ethanol.

Good luck,

Jim

Hi David,

Thank you very much for your answer. The paper is a good one. A big question in metabolism prediction is at one point to be able to predict when a compound is phase II-ready or even whether it needs phase II (which most xenobiotics need). I guess at this point, most of these questions need to be answered by a collection of machine learning models.

Thanks,

Yannick

This paper claims that neither 3-HC nor 3-HC glucoronide have been observed as products of human liver microsomal activity on 7-ethoxycoumarin.

please check phenylalanine ammonialyase, cinncamyl aldehyde dehrogenase, peroxidase

then phenol and lignin levels

I am not very familiar with that issue, but for other reasons I am more familar with serratia and I have a reference that might be useful for you... good luck 

Exp Ther Med. 2015 Mar;9(3):795-800. Epub 2014 Dec 18.

Cloning and sequence analysis demonstrate the chromate reduction ability of a novel chromate reductase gene from Serratia sp.

Deng P1, Tan X2, Wu Y1, Bai Q1, Jia Y1, Xiao H1. 

Hi Shengwen,

Replying to your question regarding CYP panel on chemotherapy, it's very important to keep in mind that even within an FDA-approved panel one must operate with pre-assessment of, say, each individual intra-tumor expression of CYP and its monooxygenase activity towards the drug used. The latter pattern is major source of variability in drug pharmacokinetics and response. For instance, early studies by Patterson et al. (1995) identifying  P450R as a key player in tirapazamine bioactivation. This evidence came from studying the in vitro sensitivity of breast tumor cell lines to tirapazamine that exhibited an inherent 6-fold range in the CYP activity. Using short exposures (3 h) to tirapazamine, the level of P450R correlated strongly with the extent of tirapazamine toxicity in hypoxic conditions. However, aerobic sensitivity was also measured following both short-term (3 h) and prolonged (96 h) exposures to tirapazamine and was also shown to be dependent on P450R activity.

Besy wishes,

Ilya

Yes, sure! TLC would work. Try cyclohexane: ethylacetate (9:1) as mobile phase on silicaagel TLC F 254 plates and use iodine vapor or even H2SO4 spray to get the two product separated and even quantified- should be perfect for separation of an alcohol and a ketone! Thanks, Biswa

Hi Olga.

Unfortunately you did not specify the name of the drug, or even designated the route of its administration, So, its impossible to help you with excretion data analysis and interpretation. In the given circumstances, your drug’s data looks similar to those characterizing the excretion of octoclophelin. To make comparisons (and take some useful information), please see the results published by Z. Franc et al. in Biochemical Pharmacology, Volume 19, Issue 4, April 1970, Pages 1443-1448. In a nutshell, Octoclothepin is absorbed well from the intestinal tract of mice; while in rats, absorption is slow. If given intravenously, highest concentrations of the drug were found in the kidneys and in the lungs and the lowest in the spleen, skin and muscle. Elimination is slow and depends on the route of application and on the animal species. The urine of rats treated with 35S-octoclothepin contained 35S-octoclothepin, 35S-octoclothepin sulphoxide, 35S-sulphate, 35S-sulphate esters and unidentified metabolites. Twenty per cent of the intravenously administered 14C-(methyl)-octoclothepin was eliminated as 14CO2.

Best wishes,

Ilya

Yes, Dr. Shapiro, there is almost perfect accuracy in approximation of octanol:water partition coefficient (logP) to distribution of a lipophilic P450 substrate between water compartment of the cell and its phospholipid-enriched endoplasmic reticulum membrane.  It has been, alongside with others, shown, for a example,  in our paper published in The Biochemical Journal,  160:75-83, 1976, in our survey:  “Role of microsomal phospholipids in organizing and regulating the activity of a monooxygenase system” (Biological Reviews, 84:3-21, 1977), as well as in my (with Dr. Lyachovich) monograph entitled “Structural Aspects of Monooxygenases Biochemistry” (Science Press, NAUKA, 337 pp, 1978).

Since then, many data were published describing the crucial role of microsomal phospholipids in a substrate binding and metabolism at P450 (CYP) active site.  Thus in  Toxicol In Vitro.18(1):89-97, 2004, D. Lewis published a paper “Quantitative structure-activity relationships (QSARs) for substrates of human cytochromes P450 CYP2 family enzymes”, where substrate lipophilicity was determined of key importance to P450 binding affinity and enzyme selectivity. From an extensive compilation of log P values for P450 substrates, and by analysis of relationships between partitioning energy and substrate-binding free energy, the relevance of lipophilicity and other factors pertaining to P450 binding affinity was explained, leading to the formulation of lipophilicity relationships within substrates of each human P450 enzyme involved in drug metabolism. Furthermore, log P values for P450 substrates appear to represent markers for enzyme selectivity. Together with the important roles of hydrogen bonding and pi-pi stacking interaction energies, the desolvation of the P450 active site makes a major contribution to the overall substrate-binding energy.

LogP values become even of greater importance after revelation and dramatic developments (both in academic research and clinically-applied areas) of drug-drug interactions at CYP active site. Thus, among many recent data of others, Yi-Ting Zhou et al. presented a paper “Pharmacokinetic drug–drug interactions between 1,4-dihydropyridine calcium channel blockers and statins: factors determining interaction strength and relevant clinical risk management”, published in “Ther Clin Risk Manag. 10: 17–26, 2014). They studied phisico-chemical and enzyme kinetic parameters of coadministration of 1,4-dihydropyridine calcium channel blockers (DHP-CCBs) with statins reductase inhibitors, which is common for patients with hypercholesterolemia and hypertension. Authors investigated the inhibitory effects of 13 kinds of DHP-CCBs on human CYP-isoenzyme-dependent reactions using microsomes from human B-lymphoblast cells expressing CYP. Interestingly, lipophilicity was found to be an important factor in the strength of the pharmacokinetic interactions of DHP-CCBs. The logarithm of the molecular 1-octanol-water partition coefficient (logP) values indicated significant positive correlations with the interaction strength.

Thank you, for your nice inputs!

Karim sir, how do you inactivate it? Can I just autoclave it if it has to be sterilized for further use?

Just a query, if the bacteria was spread all over the plate or streaked ? Or bacteria was put into holes. I mean, did the bacteria come in contact with whole of the lignin-plate and the entire plate turned black ? Lignin must be brown, and further browning (dark) means oxidation of phenolic moieties in lignin making them brown- thus the bacteria must have "oxidase" activity. Alternatively, the lignin was degraded by bacteria (with "ligninase" activity) thus breaking the ligning into monomeric monolignols/phenolics which are autoxidised in presence of "air" only. To check the later event, the same bacteria if fed with only simple phenolics (sinpic acid etc.) must not yield darkening thus indicating absence of oxidase activity and only ligninase activity. Another very distant possibility would be that the bactrial cells are lysed (in contact with lignin) to release some chromophores which are bacterial pigments, or simply bacterial pigments oxidizing to dark ones. I would be interested to know the outcome of further results too. Best wishes. 

Hello, you mention the concentration of glucose is 30-50 g per liter or the total culture medium prepare. You can dissolve the glucose in 150 ml sterile water and filter through a millipore membrane, and that is the medium and add to a sterile laminar flow hood or directly the bioreactor with a peristaltic pump . The second way is : 150 or 200 ml autoclave sterilization at 15 lb / cm2 for 15 min and adicionas to the culture medium . The fact of putting glucose together with the other components of the medium, is primarily nitrogen source as it reacts with compounds forming glucasa inihiben microbial growth ( Maillard reactions ) if your medium does not have high concentrations of amino acids , proteins , ammonium salts put no problem glucose culture medium and esterilices together .

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

try this too

There are several possibilities and several questions:

Despite all that there are several possibilities:

To the best of my knowledge there is a (direct) route of polycondensation (but limits in view of the final molecular weight) and/or as already mentioned by Marc the ring opening polymerization starting from dilactide... see e.g. "Current Progress on Bio-based Polymers and Their Future Trends By SpecialChem - August 8th, 2013"

Best regards and Happy 2015, Joachim

Hi Daniel,

 Look out for enzymes responsible for your mentioned transformations and then check which microbe produce that enzyme of your interest. OR

You can screen for microbes that produces your enzyme of interest using enzyme screening assays. Most of the enzyme screening assays are already deduced so you can easily find an organism producing your enzyme with all the available infrastructure of your lab. Its all chemistry behind transformations so if you understand that chemistry then you can play with it anyhow. 

Good luck

Dear Clement,

here you have links to the other two publications studying plant tissues with EXAFS.

Happy reading,

Ana

Dear Vladimira

You may get some information on metabolites products and major mecchanism of Thymol and Carvacrol in this attachments.

Thanking you

I have worked with bioconversion of Progesterone to 11-alpha-hydroxyprogesterone using fungal strains. I used a low concentration of solvent that was tolerated by fungal strains to improve substrate solubility and used a magnetic stirrer to keep the substrate dispersed (I used a 5 Lt Schott bottle as a mini bioreactor in absence of actual bioreactor). Using micronized substrate powder also helped. If you are using a small bioreactor, there should be no problem of keeping substrate dispersed. Uptake will definitely be an issue as others have already indicated, but dispersing or emulsifying agents can help. The challenges could be also in analytical measurements to obtain representative samples. Type of microbial strain you need to use for bioconversion will also be an important issue. I would prefer to use a small bioreactor than shake flasks, if you can avail the facility though. Give it a go, and I am sure you will work it out as I did.

On of our students made this on E.Coli by inserting genes and blocking some pathways.I'll check if I can find the thesis. 

Thyme leaves contain thymol that increases  xenobiotic-metabolizing enzymes. sulfotransferase b,  that might show significantly higher ECOD, GST, and QR activities. 

Dear Rathinam, This is vague question, you know. You perform extensive literature review on this topic.

So, this is a very complicated system that you're looking at, particularly depending on the composition of the soils that you're using. If you're not controlling some of the factors in some way (i.e. - using a buffer to control pH), then one of those factors could be inter-related with the others.

The observed redox potential of a simple system where you're looking at just one metal (in your case, arsenate and arsenite) is dependent on the concentrations of the two and the equilibrium between them, which is also related back to the standard reduction potential of the system through the Nernst equation. In your case, if you were just looking at arsenate and arsenite, as you increase the arsenite concentration (and increase the ratio of reduced to oxidized), the redox potential would become more negative. In addition, if any of the equilibria that are present in the system involve protic species (which is also true for arsenate and arsenite), then protonation constants would come into play in the redox equilibria. There have been a number of studies that have looked at the relationship between Eh (system redox potential) and pH in the past, and they have produced detailed plots of the system speciation when both Eh and pH are varied. These plots are called either Pourbaix diagrams or just "Eh vs pH" diagrams.

Your system may also be slightly more complex since you will have all of the soil components, many of which can have an effect on all three of the system characteristics that you mentioned in your question.

I hope this was helpful!

Regardless of the problem of logP-calculation, I'd like to advice you to rethink whether you really require this value. It has become obvious for quite some time now, that the results of Laane et al., to which you obviously refer, have only a very limited application range. There actually seems to be a relation between enzyme behaviour and log P as long as you compare solvents of the same type. When comparing solvents with essentially different functional groups logP is not really an option to estimate the enzyme behaviour.

Yes through this method you would be able to utilize extracellular enzymes. You can also apply a different method. Grow the fungus. Once it is fully grown. Sonicate the culture in the flask. This may burst the cells and intracellular enzyme come out of cells into the medium, which may catalyze the transformation of your compounds.

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!

Correction to the above: A coupling enzyme is required. It is S-adenosylhomocysteine hydrolase (SahH). It can be cloned from the genome of the bacterium Pseudomonas aeruginosa and expressed in E. coli for purification.