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I attempted the crosslinking method using glutaraldehyde, which resulted in pellet formation that is challenging to dissolve, possibly due to excessive crosslinking. I'm now seeking an alternative approach.
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I've tried various 2.5% concentrations of glutaraldehyde, but all of them yield pellets that are somewhat challenging to dissolve. that's why I need an alternative approach. I performed cross-linking for 2 hours initially, and then I tried extending the time to 6 hours as well as reducing it to 1 hour. However, I didn't observe any significant changes.
( It's worth noting that I purified my cellulase protein using ammonium sulfate precipitation before starting the cross-linking process. Is it appropriate to use ammonium sulfate precipitation-purified protein for this crosslinking purpose?:
In my laboratory, two students attempted to work with xylanase and exo-cellulase, and both of them achieved success on their very first try. They didn't encounter any significant difficulties, not even when it came to dissolution.
I'm feeling unsure about what steps to take, and time is slipping away quickly. I seem to be stuck in this situation.
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Hi everyone,
sorry if this is a simple question, or if I get anything wrong here, but I'm very outside of my comfort area when it comes to enzyme activity-related calculations.
So, I need to know the concentration of enzymes that a paper used for their stock solution of Catalase.
Here is a quote from the methods: "Enzyme activities of stock solutions were 3 mM/s for GOX and 998 s-1 for CAT. To obtain a defined, stable oxygen concentration of 2% on cell surface stock solutions were diluted by 1:10,000 for GOX and 1:1,000 for CAT."
For the GOX, I think I can manage to calculate the stock, but the catalase is the issue
So, the Kcat = 998 s-1
The Vmax = 1uM/ min = 0.0166uM/ s
For for the calculation: Kcat = Vmax / E[t] , is it as simple as rearranging it to E[t] = Vmax / Kcat?
That Vmax figure is from the data sheet of the catalase, with 1 unit being equal to 1uM H2O2 processed per minute (not 100% sure this is what is meant by Vmax), hence me dividing by 60 to get the uM per second.
I also know that 1mg of Catalase = 20,000U
The issue is that I don't know how to put this all together. I am currently trying to get more familiar with enzyme kinetics, but this is taking some time. I would very much appreciate if anyone could offer some advice to help speed things up so I can start with my experiments.
If I am missing some information here please let me know.
Best regards,
Ciarán
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The value kcat is the number of product molecules formed per molecule of enzyme per second, and is reported in units of s-1. So I don't think that the description of the stock solution in terms of s-1 makes sense , nor can it be converted to an enzyme concentration. Perhaps you mis-transcribed the units in your quotation from the published account, or there was an error in the paper.
For example, let's suppose the authors meant 998 µM/s as the activity of the stock solution, since the GOX stock solution was defined in similar terms (3 mM/s).
998 µmole/(L-s) = 59,880 µmole/(L-min) = 59,880 U
(The definition you gave for a unit of catalase activity is 1 µmole/(L-min).)
The specific activity you mentioned was 20,000 U/mg.
Thus the stock solution contained 59,880 U/(20,000 U/mg) = ~3 mg.
We don't have information about the volume of the stock solution, unfortunately, so we can't calculate its concentration.
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Hello everyone it will be of great help to me as I am working on my dissertation, I have enzyme activity in mg/ml for papain, just need to calculate it in percentage.
Thank you.
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% (w/v) is grams per 100 mL.
1 mg/mL = 1 g/L = 0.1 g/100 mL = 0.1% (w/v)
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I try to measure the enzymatic activity of catalase in a symbiotic Cnidarian,
I homogenized my tissue in 0.1 M phosphate buffer by two methods
1- using fast Prep with beads
2- sonication for at amplitude 20% and even more for longer time period.
then I noticed microscopically that both of theses do not results in significant algal cell lysis
My enzymes are SOD and Catalase,
What is your suggestion. Please
given that the lysis buffer should not affect the enzymatic assays.
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you can use both beads designed for animal tissue and plant material, either combine them in a single run, or use them sequentially, following these beads selection guidance: https://www.takarabio.com/learning-centers/nucleic-acid-purification/accessory-selection-guides/sample-homogenization-beads
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I bought an enzyme with the following characteristics: 10.20 units/mg protein, 51.55 mg, and 9.70 units/mg solid. I will need 5 units in my final enzyme assay, where the final volumen will be 300 ul, and I will add 15 ul of the stock solution of the enzyme. How do I prepare the stock solution?
Thank you for your help.
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You need 5 units, to be added in 15 µl, which is 0.333 U/µl for the 20X stock solution.
(0.333 U/µl)/(9.70 U/mg) = 0.0343 mg/µl = 34.3 mg/ml
So the 20X stock solution should have a concentration of 34.3 mg of solid per ml.
0.015 ml x 34.3 mg/ml x 9.7 U/mg = 5 U
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I am a bit confused about the matter. The majority of papers and explanations I have come across indicate that the inhibited line is generally positioned above the uninhibited line, but I have observed it to be the opposite. will it still be considered as lineweaver burk plot?
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Since a L-B plot is of the reciprocals of the rates and substrate concentrations, a decrease in activity caused by the presence of an inhibitor is represented as a line that is higher up in the first quadrant than the uninhibited line. If you are seeing the opposite effect, it means that the enzyme was more active in the presence of the inhibitor than in its absence. In other words, the compound is an activator, not an inhibitor. However, it would be a good idea to share the data, since there may be other interpretations that can only be made based on seeing the data.
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I have some problems with doing the calculations for protease enzyme activity after using this protocol.
Universal Protease Activity Assay using Casein as a Substrate (sigmaaldrich.com)
Is anyone familiar with this protocol and can offer some assistance on the calculations? Any assistance would be appreciated. If i have an idea of how it's done, I will be able to complete the rest of my calculations. The explanation for doing the calculations is a bit confusing.
Thank you in advance.
Also, to those who answered my last post thank you. I was able to grasp both Amylase and Lipase enzyme activity calculations.
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are you using the assay that employs the use of 0.65% Casein? Do you establish a Standard Curve using 1 mmol/ml Tyrosine? If that is the case and you have run a set of standards from 0.05 mmol/rxn to 0.5 mmol/rxn, the calculations are relatively straight forward. See attached.
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Recently I am stuggling to improve the kinetics of an artifical enzyme. I expressed this enzyme in E.coli and then test it's kinetics properties.
I noticed that if I pick single conies for testing, there will have a variation in both reaction rate and maxium reaction. indicating in fig.1 (the y axis represent the product that have been formed and the x axis represent the time in seconds.) 4 different conies have been picked and they all contain the same plasmid that transfection at the same time and same procedures. However there is huge differeces in the reaction rate and maxium reaction.
Then I wonder if it's due to the different conies would fold the protein differently, so I did another test by add multiple conies (actually all conies on one dish) into my culture medium. And then I test this mixed enzyme with different substrate concentration to test the affinity and kinetics at the same time. fig.2 (different color represent different concentration; the dash line represent a Imaginary limitation)
The problem that makes me wonder is that: what might be the reason for this reaction have a rate limitation?
I have few hypothesis about this phenomeon:
1. based on the Imaginary rate limitation; there might have steric effects preventing the binding of the substrate. (but I don't have see enough enzymatic reaction curve that have steric effects)
2. based on the varation between conies; this artifical enzyme might have many different ways of folding (I mean this enzyme would have many different prefered structures in different bacteria cells). maybe bactria from the same coniey would prefere similar stucture? and some stucture have better enzymatic performance, others do not.
I am really appreaciarte your reading and would be very happy to receive any response.
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Since you frequently refer to colonies ("conies"), it seems that you are not growing a culture of the cells and purifying the enzyme, just extracting the enzyme from the cells in the colony. In that case, there is not much point in studying the kinetics of the enzyme because it has not been purified. The signal obtained may be due to a mixture of multiple enzymes. The amount of the enzyme obtained from a colony may depend on factors such as how many cells are in the colony, the age of the colony, and how well the enzyme were extracted from each colony.
Generally speaking, an enzyme has a single overall conformation. It will not have different structures in different colonies. It might be expressed to different levels in different colonies. Some of it may be in an insoluble form due to failure to fold properly, and the proportion of insoluble, inactive protein may differ between colonies.
If you want to study this enzyme's kinetic properties, you really should purify it.
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For the enzymatic activity assay, I have to use 4-Hydroxybenzoic acid. I have kept it in the refrigerator. In the first experiment, I saw one peak but, after 3 weeks I was 2 peaks during UV spect.
Do you have any idea or guess?
Thank you
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Thank you so much.
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I am currently doing an experiment and working on amylase, lipase and protease enzyme activity. I have followed various protocols and have done experiments. I am looking for assistance in doing the calculations for the enzyme activity. Anyone with said expertise, I would appreciate your assitance.
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THERE ARE A SERIES OF VOLUMES IN METHODS OF ENZYMOLOGY
also
TitleMethods in Enzymology Volume 61 of Enzyme Structure, Serge N. Timasheff Enzyme Structure: Part H, Serge N Timasheff Methods in Enzymology, ISSN 0076-6879 Volume 61 of Methods in enzymology: Enzyme structureEditorsC. H.W. Hirs, Serge N. TimasheffContributorsC. H.W. Hirs, Serge N. TimasheffEditionillustratedPublisherElsevier Science, 1979 and
Rossomando EF (1990) Measurement of enzyme activity. Methods in Enzymology 182: 38–49
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I have collagenase from sigma product code c9891 100mg,Type IA, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid, For general use
I do not understant from the product data sheet how many units are per mg (U/mg)....
How can I make a 30U/mL solution? How much buffer should I add to what quantity of powder to obtain 100mL at 30U/mL solution?
I would appreciate your experience. Thank you!
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Thank you very much!
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I basically have normalized fluorescence data generated from a series of protein dilutions. I need to calculate K, K should be the same in all dilutions, thus the need for a global fitting approach. I have been using XLfit, but would like to move into a script format. Thanks in advance! Thus far, I found the package renz, but to my understanding, it does not accomodate for global fitting. Thanks in advance!
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COPASI: Biochemical System Simulator.
free download from copasi.org
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Hi everyone,
I am measuring the activity of some mitochondrial enzymes, by measuring the oxidation/reduction of relevant substrate using spectrophotometer. However, after analysis, my enzymatic activity (nmol/min/mg) becomes a negative value (not always, but most of times).
Is this because my analytes are being used (thus decreased) so their absorbances decrease?
Another question; if the extinction cofficient of my analyte in 340 nm is 6.22 mM-1.cm-1, and the length of cuvette is 0.6 cm, and the concentration of my analyte is 0.1 mM, how should I calculate my enzymatic activity in nmol/min/mg?
Big thanks in advance
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If you are measuring the conversion of NAD(P)H to NAD(P)+ at 340 nm, the absorbance decreases as the reaction progresses. You should perform your calculation using the absolute value (i.e. positive value) of the negative absorbance change.
Use Beer's Law to calculate the change in concentration of the analyte based on the absorbance change, where the absolute value of the absorbance decrease divided by the path length (0.6 cm) and divided by the extinction coefficient (6.22 mM-1cm-1), give the concentration change of the analyte in mM units.
The calculation of specific activity from this result requires knowing the volume of the sample that was measured and the concentration of protein that was present during the reaction.
specific activity = nanomolar concentration of product formed or substrate consumed divided by concentration of protein in mg/liter and divided by time of reaction.
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I have the commercial cellulase product (100mg) with 13000 U/g. I need to know how to convert this U/g units into FPU unit for saccharification process with lignocellulosic biomass. Thanks in advance.
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You have 13000 U/g. Is that dry powder or liquid? If it is powder you need to dissolve it in a known final volume of liquid. As you have 100mg, you have 1300 units of enzyme and the units per ml value is determined by the volume. If you have 100mg/1300 units dissolved in 10ml, i.e., 10mg/ml, that’s 130 units per ml. But if you had 1mg/ml enzyme (100ml), you would have 13units/ml.
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I know that metal ions are present in enzymes, and that this affects their stability and function.
However, when producing or engineering enzymes, I wonder if metal ions have a negative effect.
Metal ions are interacting with cysteine and histidine in proteins such as zinc finger and ring domain.
Are there cases where cysteine and histidine are replaced with other amino acids to remove metal ions and maintain the structure?
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Metals when present in proteins are there for an evolved reason; usually structural and/or catalytic function. Both HEW and T4 lysozyme have an extensive history of the impact of various metals in their structure and function, so maybe review that literature. The only "problem" I could see from metals might be partial or mixed residency and this giving a mixed population of enzymatic activities, complicating analyses. Many enzymes have been extensively studied by chelating out one metals and then adding back another, see this paper and refs therin.
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I'm working with simulated digestive fluids.
Due to the low concentration of pepsin that I need for my work, it is difficult for me to weigh very small amounts of the reagent each time I need to use it (in fresh). That is why I decided to prepare a stock using 50 mg, from which I can take aliquots whenever I need it. But I don't know if it affects the fact of defrosting it continuously every time I need to use it.
I diluted the powder of Pepsin in HCl 0.01 N. I didn't find info about the appropriate storage temperature.
Also, does anybody know if its correct to store the solution in HCl 0.01N?
I emphasize on this because a found a protocol that says: "A stock solution of Pepsin is dissolved in 10 mM Tris buffer, 150 mM NaCl, pH 6.5. The stock solution has to be stored on ice or refrigerated at 4°C. Just before the assay, the concentrations of pepsin in 10 mM HCl has to be prepared".
From this, I understood 2 points:
A) The pH is adjusted to 6.5 to limit proteolytic activity
2) Storage on ice limits proteolytic degradation.
But, as I mentioned before, weighing small amounts increases the systematic error. That is why it is more practical for me to have a stock and keep it in storage.
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Avoid freeze thaw cycles. Make your stock and dispense into 50 or 100μl aliquots (single use is even better) which you keep on ice until you freeze the batch. The colder you store your stock at the longer it will last. At -80°C pepsin suspended in 0.01M HCl lasts for a year. Pepsin is more soluble in HCl than it is in Tris so more concentrated stock solutions can be made.
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In B.subtilis spore surface display cot partner with native promoter is fused in frame with a passenger protein via a linker like GGGGS. The assumption here is that a fusion mRNA is formed, and translation starts from the cot partner START codon and ends at the stop CODON of the passenger protein. The linker permits the independent folding of the two proteins. My question is will omitting the N terminal START codon of the passenger protein (enzymes in my case) affect its stability and activity? I'm having multiple enzymes fail with multiple cot partners like cotB,CotC and CotG. I have not included the START codon in the passenger protein in my constructs. I have been checking the literature and so far in every study they have included the start codon of the passenger protein. Any thoughts about its impact?
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The answer is that it depends. Most often the N-terminal Met is not needed in the protein, but there are some cases where it is. So I would think that while usually you would not need to include it, but on the other hand there is no strong reason not to include.
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I have to conduct enzyme assay of around 200 samples to check their kinetic rate.
Traditionally, for small number of samples we carry out cuvette based spectrophotometry assay. But for such large number of samples carrying out individual spectro analysis will be time consuming and chances of error. It will be helpful if any alternative and parellel analysis technique/protocol suggested.
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Do you have access to an absorbance plate reader? You could use one to make the measurements in 96-well or even 384-well plates. It is highly beneficial to also use a multichannel pipettor (8 or 12 channel) to put reagents into the wells. A plate shaker improves mixing of the samples in the wells. Some plate readers can also perform the plate shaking function. If the absorbance measurements are in the far UV (<340 nm), it would require a plate reader designed for such measurements, and special UV-transparent plates.
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From the practical experience I have observed, when I have added extra amount of tissue homogenate (more than recommended) to study any enzyme activity (for low expressed enzyme assay), the initial absorbance starts with very high value (some times nearer to 1) and never gets satisfactory result. Moreover, excessive amount of tissue homogenate makes the rection mixture colored (similar to tissue homogenate color).
So, what should the balanced amount of homogenate should I use for any enzyme activity assay?
How do the excessive amount of homogenate affect the reaction?
Thanks in advance.
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It varies depending upon the specific protocol of any author you are following, reagents you are using and the abundance of that specific enzyme in your tissue. However to know the best enzyme volume you can perform serial dilution.
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Dear all,
I got results after performing an enzyme kinetics with 4 different substrate the kcat of B and D is respectively two times lower than A and C. However, I am seeing the higher Km of A i.e 174 nM as compared to B i.e 54 nM. Can anyone please suggest me how to rationalize this result ? The Kd value however, is always doubled for A and C when compared to B and D respectively. Below are the data attached for your reference. How could it be explained the research paper ?
Thank you
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The different kinetic constants for the different substrates reflect the chemical differences between the substrates, including the strengths and types of their interactions with the enzyme active site and differences in the activation energy of the reaction. The differences in the kinetic constants between the substrates in this case are actually quite minor, and could easily be within the range expected for experimental variation.
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I am preparing gastrointestinal fluids to test yeast survivability in-vitro.
The protocol requires me to prepare two solutions, gastric juice and intestinal fluid, requiring me to add pepsin and pancreatin respectively after the autoclaving.
There are MCE, PTFE and PVDF filters available in the lab, and my concern is possible loss of enzymatic activity due to using unsuitable filter. Which one, or another, do you suggest for preparing such solutions?
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10/20/22
Dear Emre,
I can't tell you which filter is the best one to use. However, this is something that you can easily test for yourself. You can try running small batches of the pepsin & pancreatin separately through each type of filter and measure the enzymatic activity of filtered vs. unfiltered enzymes. One thing to keep in mind is that membrane filters are not always "benign". Sometimes they bind some of the material (in your case, protein) that you are trying to filter. If you look in the catalogs of companies that sell membrane filters, they often describe a particular membrane as exhibiting "Low protein binding". If you follow the above suggestion and find less enzyme activity in the filtrate of one or more of the membranes, it could be that some of the enzyme protein was bound by the filter. To check this, it would be a good idea to measure not only the enzyme activity in the filtered- and unfiltered preparation, but also the protein concentration in the filtered- and unfiltered sample.
Something you mentioned in the first part of your question: Exactly what are you going to autoclave? Autoclaving pepsin, pancreatin, gastric juice and intestinal fluid will denature enzymes and other proteins that they contain.
I hope this information helps you.
Bill Colonna Iowa State University, Ames, Iowa, USA wcolonna@iastate.edu
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Does the stability change with stock concentration?
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Hello,
This might be a very ridiculous general question but here we go:
I am trying to find kinetic parameters for the metabolism of a drug by the enzyme CYP3A4. The problem is, all literature Vmax values are specific activities either in pmol/min/mg or pmol/min/pmol CYP3A4, but I need Vmax values to be in the form: mol/h or mol/(h*L) ( for a systems biology application that uses these units in rate laws)
I am not an expert on enzyme kinetics, so I am not sure how one makes the conversion to fit the Vmax data into a this format. Do we need the enzyme concentration (in g/L or in M), as well as the incubation volume?
Some insight would be highly appreciated.
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Vmax depends upon and is, ideally, directly proportional to the concentration of the enzyme. Specific activity divides Vmax by the amount of protein present (typically in mg, as in µmol/min/mg). Specific activity can be used to describe the level of activity of any enzyme-containing preparation, without requiring that the enzyme be purified.
kcat divides Vmax (in concentration units) by the enzyme concentration to get s-1. Purified enzyme is normally used for measuring this parameter.
You can express the enzyme activity in whatever way suits your needs, but you should be explicit about how the calculation is made, the source of the enzyme, what protein concentration was used, and the specific activity of the preparation so that others can repeat the work.
If you keep the enzyme concentration constant throughout all experiments, and its activity remains constant throughout, then, for the sake of your calculations, you can leave Vmax in the form of moles of product formed per unit of time or, dividing by the volume of the reaction, as concentration of product formed per unit of time.
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Do we need to test the enzymatic activity of the supernatant and the wash too? and what is the differences between recovery and residual activity?
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Please go through the following reference:
Boudrant J, Woodley JM, Fernandez-Lafuente R. 2020. Parameters necessary to define an immobilized enzyme preparation. Process Biochem. 90:66-80.
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In a hypothetical scenario, researchers discovered a protein that is responsible for lactose intolerance in cats. The results of the multiple sequence alignment for these two proteins are as follows:
Different parts of the protein were cloned and expressed in vivo in a mammalian cell. The enzymatic activities for different parts of the protein are summarised in the table below.
a. Which part of the amino acid segment most likely contains the active site? [1 mark]
Your answer here.
b. Which construct will you generate to produce more than 85% enzymatic activity?
Justify you answer. [3 marks]
Explanation in bullet points (25-50 words).
c. Describe the protein engineering strategy/strategies and steps to generate this new construct. [3 marks]
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Thank you very much for the clarification Annemarie Honegger
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I am working towards to design a study to asses the efficacy of different LRRK2 inhibitor compounds in neuron. I have been meaning to ask if there is any gold standard to check LRRK2 enzymatic activity.
Thank you
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You may consider the detection of phosphorylation of LRRK2 substrates via immunoblotting:
1. Autophosphorylation at S1292
2. T73 Rab10 phosphorylation
3. to a lesser extent, T72 Rab8 phosphorylation
You may also consider Phostag assay for Rab10 phosphorylation, as published by the lab of Alessi doi: 10.1042/BCJ20160557
Good luck
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Hi
The question is: Determining hexokinase activity in rat brain where 10 µl of a 10% (100 g/L) brain homogenate (containing hexokinase) is mixed with 1990 µL of reaction medium (containing the substrate glucose and the coenzymes ATP and NADP+ and the auxiliary enzyme glucose-6-phosphate dehydrogenase). The reaction mixture is placed in a cuvette (light path = 1 cm) in a spectrophotometer at 37°C, where the hexokinase reaction can be seen as an increase in absorbance at 340 nm. The reaction proceeds at a constant rate during the measurement period, and during 10 min a total increase in the light absorption at 340 nm of 0.10 is recorded. The absorption coefficient of NADPH = 6300 x M-1 x cm-1
I need to find the enzyme activity of hexokinase in µmol/min/g. I don't understand how I need to interpret the first line (10 µL of a 10 % (100 g/L) brain homogenate) - and how should I use these numbers?
Thanks in advance!
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10 µL of a 100 g/L (= 100 µg/µL) homogenate contains 1000 µg = 1 mg = 0.001 g.
That will be the number in the denominator when you divide by the number of g to calculate µmol/min/g.
The change in absorbance of 0.1 unit is converted to the concentration of NADPH formed using the extinction coefficient of 6300 M-1cm-1 and the Beer-Lambert Law: absorbance = extinction coefficient x pathlength x concentration.
An absorbance change of 0.1 for NADPH with an extinction coefficient of 6300 M-1cm-1 in a 1-cm pathlength cuvette corresponds to a NADPH concentration of 0.1/(6300 x 1) = 1.587 x 10-5 M = 15.87 µM.
This amount of NADPH was formed during 10 minutes, so the rate of NADPH formation was 1.587 µM/min. This occurred on a reaction volume of 2 mL, so it can also be expressed as 1.587 µmoles/(L-min) x 0.002 L = 0.00317 µmoles/min.
This happened when you used 0.001 g of material, so the specific activity was (0.00317 µmoles/min)/(0.001 g) = 3.17 µmoles/min/g.
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I am working in laccase enzyme production by bacteria. In that i have a problem with quantitative estimation of laccase enzyme. I have confirmed the laccase production with plate assay using guaiacol. But i face the problem with quantitative method of laccase enzyme. So kindly suggest me some tips and methods for the laccase enzyme assay. Thanks in advance:)
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Dear I am published two paper from the phD thesis to my Student in (2020):
1. Doaa Khalid Mezaal, Essam Fadel Al-Jumaili and Ahmed Majeed Al-Shammari (2020). Production and Purification of Laccase Enzyme by Klebsiellapneumoniae K7 . IISTE.
Chemistry and Materials Research . Vol. 10, No.5, PP:17-23.
2. Doaa Khalid Mezaal, Essam Fadel Al-Jumaili and Ahmed Majeed Al-Shammari (2021). Study of Production and Characterization of Laccase Enzyme from Klebsiella pneumoniae K7 Isolate, Medico-legal Update , Jan- March, Vol,21 No.1 pp: 216- 223.
if you don't get these papers i can sent to you by your e-mail.
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I found the dns method to measure the amylase activity, but I cannot get the dns reagent. Therefore, could you suggest me an alternative method or alternative reagent to dns reagent?
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You can Use Nelson- Somogy Method.
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Sometimes the enzyme may not be activated or may not function properly unless the it is co-expressed with other protein for post-translational modification or assembly into a functional catalytic complex.
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Does a better docking score or stronger binding energy between a mutant enzyme with its substrate always translate into an improved enzymatic activity?
How should RMSD, RMSF, radius of gyration, solvent-accessible surface area (SASA), etc. from molecular dynamics simulation be used to guide enzyme engineering with the aim of improved product synthesis?
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You may want to Google "simulation of enzymatic catalysis"
Some interesting hits:
Two Sides of Quantum-Based Modeling of Enzyme-Catalyzed Reactions: Mechanistic and Electronic Structure Aspects of the Hydrolysis by Glutamate Carboxypeptidase
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Hi, Iinserted gene of Reverse transcriptase into pPLc245 plazmid. If I'm right, this plazmid doesn't code cI857 represor. For expression od reverse transcriptase from this plasmid I used E. coli DH10B. RT was expressed after increase of cultivation temperature to 37 °C. Before this thermo induction I cultivated E. coli DH10B with pPLc245 at 28 °C. But at this temperature RT was not expressed. Why is this possible? Is E. coli DH10B coding cI represor or pPLc245 could contain gene for this represor?
Thank you for all responses.
Bohuš
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If your plasmid really is pPLc245 (and not some derivative of it) then it should not carry the lambda repressor gene and you should get constitutive expression at any temperature. So I can't really explain your result unless RT does not fold properly to have activity at RT or 28.
I'm actually a bit surprised this worked at all for you though, generally plasmids with superstrong promoters like lambda pL are not very stable under conditions where there is no repressor.
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Hi, every body
I am investigating the enzyme profile of a white-rot basidiomycete fungus. In the enzyme assays, we detect enzymatic activity by color change. However, my fungus produces pigments that do not precipitate with centrifugation, so it causes errors in the results. Does anyone have a solution? Thanks.
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Dear Khajezade,
Greetings and have a nice time.
You can remove the pigment easily by using acetone or chloroform. Any one of them is useful to dissolve the pigment well. You can try any one.
With regards.
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I would like to ask you, when I measured the enzyme activity of papain by the national standard method, but the sample tube and the blank tube became zero and negative values when subtracted from each other. The blank tube was added with TCA first, which means that it was caused by the casein itself and showed a light blue color.
How can I adjust the OD value of the diluted papain, which is also light blue, to 0? Is there a problem there?
There is no air bubbles in the measurement, and the instrument operation and personnel operation have been excluded.
Translated with www.DeepL.com/Translator (free version)
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Hello, I agree with Engelbert Buxbaum, apparently there should be no problems following that methodology, I would also suggest that you review some aspects that may be affecting the analysis, such as:
-preparation of reagents and buffers (verify pH, ionic strength, etc.)
repeat the procedures in the same way for the blank and the sample, eg the incubation time, the same centrifugation time to remove the pellet
check the detection range of your spectrophotometer and the wavelength selected for analysis.
You can also do a quick test and use the reaction blank without enzyme (control -) as the autozero and use the reaction medium plus the enzyme plus the TCA as the blank to inactivate it before the incubation process, after this time centrifuge and then that it would be your positive control to correct any color that could be caused by amino acid residues and then read your sample
I hope I have helped you in some way
regards!!!
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I am about to calculate alpha-amylase activity for some fungal samples. I followed the protocol of Sigma-Aldrich (https://www.sigmaaldrich.com/DE/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/enzymatic-assay-of-a-amylase) and incubated my samples in 0.2 % starch solution for 3 minutes.
According to the protocol, the equation for the U/ml value is:
U/ml = (mg of maltose released * dilution factor) / (ml of enzyme applied)
I think I also have to consider the incubation time of 3 minutes, and divide my result from the equation by 3?
Thanks in advance for help.
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This is a case where a non-standard unit definition is being used. According to the Sigma document the unit is:
Unit Definition: One unit will liberate 1.0 mg of maltose from starch in 3 minutes at pH 6.9 at 20 °C.
The 3 minutes is already accounted for by the definition, so you don't have to divide by it.
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I have been failing to express a very large trypanosome (160kDa) protein using various expression systems. The protein is a chimera of 3 enzyme activities and I can express truncated forms containing at least one of these enzyme activities. So, I was wondering if I can express and purify these enzyme 'domains' individually and somehow get them to associate, in order to perform enzymatic assays assessing their potentially interactive (regulatory) roles.
For instance can an inducible di-cre recombinase-type system be used? Ideally any recombination domains (or tags) that I may fuse to my recombinant construct would be as small as possible.
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Look, unless your project is to design methods for expressing protein domains, I'd forget your idea of making it in pieces and hoping they will somehow combine properly. It is 1) unlikely to work and 2) very time-consuming. And 3) you'd need to have the entire protein as a comparison anyway!
Try finding a better way to express the native protein. Have you tried putting it in yeast? E. coli strains that are optimized for large proteins? Under an inducible promotor? Lots of good protocols already exist.
Good luck!
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I have diluted my chrysin in DMSO, however when run an enzymatic assay in phosphate buffer pH 7.4 (50 mM) and pH 7.6(10mM) it formed precipitation? do you have any suggestion to stop it but still using the same buffer? Thank you
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The buffer will have be different or the precipitation will happen again ; ) Some have used cyclodextrins to encapsulate compounds with limited solubility, like chrysin, but you'll need controls to be sure its still accessible to your enzyme. Some nonionic surfactants may help, often bellow their CMC, but controls will need to be done to be sure the assay is still functional. Enzymes that catalyze membrane bound or insoluble substrates are a real challenge, good luck!
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The Vigna radiata plants were subjected to Cd stress ranging from 50-200 ppm. Why does the enzymatic activity (SOD, APX, and CAT) increase under increasing metal stress despite the decrease in protein content?
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And what about TAS??
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Hi, I'd like to ask whether it is necessary to precipitate MMLV Reverse transcriptase before affinity chromatography purification (Äkta)? My colleague must do this step with his Taq DNA polymerase. He use (NH4)2SO4 or Na2SO4 + PEG. Without this precipitation is polymerase inactive.
Thank you for your responses.
Bohuš
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the so called Pluthero method to purify Taq is quite old (1993) and while simple it can result in low yields and DNA contaminants (see "A simple and efficient method for extraction of Taq DNA polymerase" 2015)
I don't think the ammonium sulphate precipitation is required for activity (please correct me if I' wrong) but it may be worth a try on MMLV RT, as they are very different proteins. Yields are sometimes not very important and Am sulph precipitation can be a very useful method to isolate/purify and concentrate protein preps. I might just use a modern MMLV RT prep like this https://www.protocols.io/view/recombinant-protein-expression-of-mmlv-rt-h-yxmvmxmw9l3p/v1?version_warning=no
iff your constructs are similar, good luck!
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How to know whether a particular protein is an auto cleaving protein or not (i.e. Self cleaving) ?
Also suppose a particular protein is degrading immediately so How to know whether this is due to Auto cleaving nature of the protein or due to some other proteins (i.e proteases) in the supernatant.
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If you want to know if the protein is cleaved during its biosynthesis in a cell or following secretion from the cell (either autolytic or by some other protease), you could follow its mass over time following induction of the gene. This could be done using 35S-methionine labeling, immunoprecipitation and SDS-PAGE or by Western blot, both requiring a specific antiserum or antibody.
I think it is difficult to establish whether the cleavage is autolytic or not when the protein is expressed in its native setting. It might help to express it heterologously, so that whatever activating enzyme would normally be present would instead be absent. Then if the protein is expressed in a soluble form but is not cleaved, you would have evidence that cleavage is not autolytic.
Another approach would be to overexpress the protein in bacteria such that it formed inclusion bodies (which often happens but is not predictable). Then, the protein would be unlikely to be cleaved because it would have precipitated before folding. Refolding the protein from inclusion bodies would be necessary to render it in a soluble form. Because proteins in inclusion bodies can be purified before refolding, you could find out whether the pure protein cleaves itself once it refolds by SDS-PAGE or protein mass spectrometry or N-terminal sequencing.
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Hi, has anybody experience with addition of ammonium ferric citrate to expression media? I decided to ide this because of its perfect solubility in water.
Thanks for your responses!
Bohus
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When I want to express proteins with Fe-S clusters and purify cluster loaded protein (holo-protein) I add 0.2mM (final concentration) to my E.coli culture. I induce with lactose and also add L-cysteine as S source. As I grow my cells in LB, I don't think the extra nitrogen I am adding is doing much. I don't think by adding ammonium ferric citrate will impair E.coli growth or protein production, for sure it will not harm the induction experiment under normal conditions. If you could specify a bit more your objective, we might give you more insight.
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I am trying to assess the activity of PFK enzyme in mESC lines. Recently in the metabolomics analysis, I discovered that although the cells show an increased uptake of glucose 6-P and fructose 6-P, there is a major drop in the levels of downstream metabolites starting from fructose-1,6-bisP all the way up to pyruvate. (except glyceraldehyde-3-P, 1,3-bisPglycerate and 2-phosphoglycerate)
There is also an increase in the levels of glucosamine-6-P, mannose, mannose-6-P and of 6-P gluconate and ribose 5-P. These levels could be increased as G 6-P and F 6-P could be shunted into the pentoseP pathways.
Therefore, I am planning to check if there is a blockage between the F 6-P and fructose 1,6-P by detecting the activity of the PFKinase enzyme.
So far the kits I've found to do this, are based on the calorimetric assay where PFK activity is determined by a coupled enzyme assay, in which fructose-6-phosphate and ATP is converted to fructose1,6-diphosphate and ADP by PFK. The ADP is converted by the enzyme mix to AMP and NADH. The resulting NADH reduces a colorless probe resulting in a colorimetric (450 nm) product proportional to the PFK activity present. One unit of PFK is the amount of enzyme that will generate 1.0 mmole of NADH per minute at pH 7.4 at 37 °C.
I was wondering if there is an in cell method, more precise for cell extracts.. I am not sure how sensitive or specific this calorimetric assay would be.. Any comments/ suggestions would be much appreciated.
Many thanks.
Best,
Pooja.
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Did you solve your problem?
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Hi everyone, I have been trying to calculate the Km value of a recombinant HCV NS3/4A protease using a FRET substrate, the cleavage of which can be detected at 500 nm using excitation wavelength of 355 nm. I have a RFU vs. time graph of different concentration of the substrate (attached). Next, I am calculating the initial rate which is the slope of the linear initial part of the progress curve, with the ultimate goal to plot the 1/[S] vs. 1/[V] graph and calculate the Km from the Lineweaver-Burk equation. However, if I calculate the slope from this graph for the time range between 0-10 minutes which is the initial linear part, the intercept for the 1/[S] vs. 1/[V] graph is negative but Km value can not be negative. I was wondering if anyone can guide me on how to calculate the initial velocity correctly from the graph I have? Thank you so much!
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I plotted your data (extracted using webplot, Rohatgi) using graphPad and obtained values very close to what you got, for single y data. Normally I usually plot replicated data, side by side column or enter Mean. SD and N. replicated data usually gives narrower 95%CI data
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Can I say for a given same enzyme, more number of substrate A is changing into the product than substrate B per second ? Please make this this this clear to me. I know Kcat/Km characterized high efficiency. However, I am getting into this situation somehow. Is it possible ? How I would justify or rationalize this in the paper for publication ?
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As described in Cornish-Bowden Fundamentals of Enzyme Kinetics, if the substrates are both mixed together in the reaction with the enzyme, the ratio of the velocity of A to the velocity of B is (kcat/Km*[A])/(kcat/Km*[B]), so if the samples are mixed and the substrate concentrations are equal, A will be converted faster at all concentration, thus the name specificity constant for kcat/Km. However, if the reactions are separate and the concentrations are near saturating, the kcat will determine which rate is faster.
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I am a student from a relatively developing country and the commercial enzyme is expensive for us. My graduation thesis is desperately in need of an available standard curve for my AchE assay so that I can calculate my results using it. Please is there anyone who currently doing the AchE assay who can share it with me or where can I find it, are there papers or textbooks that I can find it in?
Your help would be very much appreciated.
Thank you
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A standard curve must be prepared under the identical conditions as the assay. It is not likely that one that you did not prepare yourself will be accurate.
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I can only start with raw feather from chicken/duck, but not feather powder.
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I'm having the same problem! Any update?
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I'm trying to run an assay for Caspase 3/7 activity to visualize apoptotic cells in flash frozen, unfixed mouse skin sections. The assay involves a non-fluorescent reagent that is processed into green fluorescence by endogenous, active Caspase 3 or 7 in apoptotic cells.
After thawing the slides for 3 minutes and washing once in PBS to remove OCT, I added the reagent to the unfixed tissue (I've tried 1 hour at room temp, or 30 mins at 37C). After the incubation, I post-fixed the sections with 4% paraformaldehyde, added DAPI mounting medium then visualized. 100% of the cells had green fluorescence, and the nuclei appeared enlarged so it looks like the cells are bursting or autolysing during the incubation. I'm wondering if I should fix the tissue immediately after thawing to stop the damage to the cells.
What would be the best method for post-fixing flash frozen mouse skin so that endogenous enzymatic activity is maintained? Does anyone have experience specifically with using the CellEvent Caspase 3/7 assay from Invitrogen in frozen tissue sections?
PS. I have also tried a TUNEL assay which generated no signal.
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I would suggest you to carry out ELISA or WB analysis of Caspases 3/7 for the detection of apoptosis in flash frogen tissue. If you fix the tissue, the enzyme activity will be lost, hence you can homogenize the tissue and take the sup to anayse capase-3/7 activity using ELISA and level by WB.
As you mentioned in your content, you can go ahead with the TUNEL Assay that is very sensitive ,(For example R & D Systems USA) and you can analyse DNA fragmentation in a single cell of you sectioned tissue.
Best
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Most people say that imidazole doesnt affect the majority of downstream applications for purified proteins but it is a chelator, so you would think that it might chelate the Mg2+ in solution and inhibit Mg2+ dependent reactions. Anyone seen any papers that discuss what concentration Imidazole will chelate magnesium or manganese ions in solution?
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Not only ligand concentration but pH is also an important factor of chelation .
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Hey guys,
I'm using (for me new cells) E. coli Arctic express for protein expression (reverse transcriptase). I tried expression at 20°C and 10°C. At 20°C solubility of my proteine was 50% and at 10°C it was about 57%. I'd like to know some your experiences and tips how to use these cells (the best media for them, optimal temperature for night culture or growth up to induction and after induction, how to eliminate chaperonins after expression, how long should lasts expression, some supplements to media, concentration of antibiotics in night culture, concentration of IPTG ... )?
Thank you for all advices!
Bohus
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I've not used Arctic Express directly but have some experience of overnight protein expression systems.
We would grow the starter cultures up to whatever the required OD600 density was (this took some optimisation for each construct so no fixed answer on this point), then turned off the heat and propped the incubator lid open to the ambient temperature (somewhere in the teens). This was using Autoinduction medium containing glucose, glycerol and lactose (there are various recipes for this online), so it only starts induction by metabolising the lactose when really high OD after the glucose is exhausted and derepresses the promoter.
Key things we found improved expression levels were adequate aeration - using 3 litre plastic baffled flasks containing at most 1L of culture would give a very aerated culture, and selecting "good" colonies - I engineered a cleavable fusion GFP reporter upstream of the reading frame so you could identify high expression clones by putting the plate on a blue light box, as well as being able to see where the protein was during purification stages. Sometimes it was necessary to go for colonies that didn't express the highest fluorescence to maximise solubility.
Although it costs more, carbenacillin beats ampicillin hands-down for the same plasmid - it breaks down more slowly and is more stable in acid pH that can arise as cultures are prolonged and denser.
Elimination of chaperonins after culture - there is a protocol to wash some of these off your protein on column by addition of ions and ATP - it is a number of years since I've done this so I can't remember the precise details. Again though, this is published data and can be found by searching for this.
The final answer is 'try try try'. Every protein is different, so asking questions here and doing literature searches, then trialling your findings in your own laboratory is often the only way to optimise the system in your hands - what works for one person may not be completely applicable in another laboratory.
Very best of luck!
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Hello everyone,
I'm working in my doctoral thesis in the purification of a lipolytic enzyme from an halophile archea. Currently, I'm trying to purificate the recombinant enzyme using Haloferax volcanii as an expression system. The problem is that suddenly when filtering my crude extract through a 0.45 um nitrocellulose membrane I lose 90 % of the enzymatic activity, when that did not happen before and at most I lost 20 % of the activity.
Has the same thing happened to someone else? Or do you have any ideas that could help me? Thanks!
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Yep, nitrocellulose is a poor choice
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We are used the protocol for urease extration adaptated (to C. neoformans) from Amin et al.,2013:
Briefly, the broth cultures were subjected to centrifugation (5,000 × g, 4 °C) and the recovered mass was washed twice using phosphate-buffered saline (pH 7.4) and then stored at -80 °C. Subsequently, was thawed to ambient (room) temperature, followed by mixing with 3 mL of distilled water and protease inhibitors (TLCK, Tosyl-L-lysyl-chloromethane hydrochloride) and sonication for 60 s. After centrifugation (15,000 × g, 4 °C), the supernatant was desalted by eluting through SephadexG-25 column. The resultant crude urease solution was mixed with an equal volume of glycerol and then preserved under refrigerator (4 °C) for further uses.
Okay, we ran the suggested protocol, but after 2 days, enzyme activity was no longer observed.
Then, we tried to exclude the TLCK from the protocol, again the activity was observed on the day of extraction, but after it lost the activity.
We also tried to activate urease with sodium bicarbonate and Ni solution, without success.
In another procedure, we repeated the protocol and lyophilized the solution resulting in a white solid. So we tested it on this day, we prepared a urease solution (10 mg / mL) and the activity was good, but after a few days, again the activity was lost.
We always work with refrigeration, and we are careful to keep the solution on ice when we handle it.
Please any suggestions?
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Something I would have done differently is to have used a pH-buffered solution during the procedure to maintain the pH at the optimal pH of the enzyme.
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Hi, I did heterologous expression of reverse transcriptase in different strains of E. coli at 3 temperatures, 37 °C, 28 °C and 20 °C. As I expected, BL21 provided the best solubility at 20 °C and the worst at 37 °C. On the other hand strain MC4100 had the best solubility at 37°C and the worst at 20 °C. Has anybody similar experience with E. coli MC4100?
Thanks for responses!
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What expression system are you using? The typical pET based vectors used in BL21(DE3) strains do not work in MC4100 as it lacks the T7 RNA Pol gene required.
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As I am characterizing an 3' exonuclease and calculated the apparent Km and Kcat for two different substrates. What I observed is little bizarre. A substrate (S1) which looks like (qualitatively) degraded slowly by the enzyme has Km lower (~60 nM) and lower Kcat (5.1 sec-1) while other substrate (S2) apparently degraded faster, has higher Km (~175 nM) and but slightly higher Kcat (~8.2 sec-1). Now I have difficulty in understanding why is this happening. One more thing I observed is that S1 gets inhibited after 150 nM substrate concentration. While S2 continue to maintain it's saturation plateau even at 8000 nM. I am little perplexed how to rationalize this result. Any help will be appreciated much in this regard. Need an explanation about preferential degradation of the substrate by the enzyme.
Thank you in advance.
Prem
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I've edited this answer, hopefully a little clearer now, esp. with an example uploaded.
I think the issue is perhaps that you are not taking into account the fact that S1 displays substrate inhibition. Because S2 does not show this effect you are not comparing like with like. Effectively the enzyme is operating in the presence of an inhibitor with S1, whereas with S2 it is not.
However, even ignoring this effect, the Kcat/Km ratio (or Vmax/Km ratio) does not actually predict which is the better substrate in a real assay situation. You need to consider [S] too.
To see the effect of varying S you would need to plug numbers into the equation v = Kcat [E] S/(Km + S). I think you will find that at low S, S1 has the edge and at higher concentrations S2 has a higher rate. This is actually too simplistic for S1 and Adam B Shapiro has given a more complex equation which is relevant to the situation with an inhibitor in the system.
I have now uploaded data for two hypothetical substrates S1 and S2, neither of which show substrate inhibition, using the standard equation v = Vmax[S]/(Km + S). The tabulated data show that there are conditions in which you can get more product with a substrate with a LOWER Vmax/Km ratio. In fact, you can get almost any rates you like by varying S, thus Vmax/Km (or Kcat/Km) ratios alone do not tell you which is the better substrate.
Hopefully, you can now see some possible explanations why your S2 substrate was better than you expected.
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Hi, I'd like to try expression of reverse transcriptase with low temperature, 15 °C, due to solubility. Should I use some special plasmids for cold expression? I ordered cells for this purpose - E.coli Arctic express. Is it enough or is it better to combine these cells with plasmids for cold expression? Thank you all!
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you can try the pcold vecotrs of takara
i used in some year ago for a protein fusion that was degraded with expression at 20-25-30°C in pet vectors and performing the expression at 17°C, I obtained good results.
you can see an example of it at minute 6' 10'' of the following video
present in my blog: ProteoCool
Sincerely in other cases, with more stable proteins they provided lower expression than vectors based on T7 promoter therefore i suggest to you to test it in parallel with T7 and not replace it.
good luck
Manuele
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Hi guys,
Is there anybody who adds betaine (trimethylglycine) to media for protein expression? I read that betaine is able to increase solubility of proteins. If you use it for this purpose which form is the best? Is there any proven concentration?
Thank you for all answers!
Bohus
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Dear Bohuš Kubala please have a look at the following original research article in which the addition of osmolytes such as betaine has been studied in detail:
Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol–overexpressed molecular chaperones
Fortunately this paper is freely available as public full text. I hope it helps.
Good luck with your research!
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Hi, I'm trying to produce reverse transcriptase. I need to increase its solubility. I would be grateful for some experiences and advices. Thanks so much.
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1. Try adjusting the temperature as you have, even trying at 30 could generate some soluble protein,
2. Adjust the amount of IPTG that you use to induce the expressions
3. Adjust the time you carry out the expression for, if you get soluble proteins at 20 C then leave over night to generate more protein.
4. If there is any information out about you protein of interest, does it require a chaperone protein to assist its folding, if so this will need to be co-expressed with the protein,
5. Check, double check, and get someone else to check that in you sequenced clone your gene of interest is in frame with the His Tag, you will be surprised how often the cloning is done incorrectly.
6.a Change the tag or place it at the c- terminus in the construct, it may be that your tag is affecting the folding of the protein, but less likely here as you have tried to express fragments and they are still stubborn. I would try to express the protein with no tag to see if it is soluble in the cells you are using.
6.b If I have an insoluble protein then MBP (maltose binding protein) is one of the best bets, a bulky soluble protein that can be easily cleaved off in many cases.
7.Change the cells you are using to express, as it is a viral protein, the codon bias may be restricting the accurate synthesis of your protein, therefore it may be worth trying Codonplus BL21, or other derivatives such as Rosetta gami strains
8. Another option is to purify the inclusion bodies of you insoluble proteins, and refold them, this can be done either by refolding on a his-trap or sequential dialysis in decreasing urea concentrations
I hope that offers you some avenues to try out!
9. For the non specific bands contaminating your sample, that is normal - always try to extend the washing step on the purification column to remove non specific binders, the other option there is to identify the concentration of imidazole that your protein of interest elutes at, then create a gradient and collect very small fractions to minimize contaminations. APS cuts before this step also reduce the amount of protein you load onto a column, overloading can generate many non specific interactions!
Good luck!!
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Hello!
I am looking for examples of industrial processes which are catalyzed by enzymes (either natural or recombinant). Particularly, I am interested in processes that end up with enzyme removal or inactivation (thermal or chemical), and I am trying to find examples from different areas - the food industry, textiles, pharmaceutics, fuel, etc.
Could you help, please?
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Also, Enzymes are used in industrial processes, such as baking, brewing, detergents, fermented products, pharmaceuticals, textiles, leather processing. Here are a range of processes showing how enzymes are used. See the RG link:
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First of all, I apologize for my poor English.
I would like to measure the enzymatic activity of COMT, referring to the following reference, but is it theoretically possible to substitute norepinephrine bitartrate hydrate?
Thank you very much.
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It is just a salt form of NE. I haven't done this assay but Googling NE + bitartrate + COMT + assay produces results that show this substrate form has been used by others.
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Anyone know how to measure catalase activity in brain homogenates in terms of μmol/min/g of brain. Reaction was stopped by addition of dichromate and absorbance was measures after 1 min at 570 nm. Controls are without H2O2 for each samples.
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There are several tests available to measure the catalase activity in brain homogenates. Hope you are trying to analyze enzyme activity using colorimetric assay kit. We have used the sensitive kit purchased from BioVision, Inc., CA that is very sensitive reproducible. If you are using the colorimetric assay kit, follow our paper published in Brain Research Bulletin 95 (2013) 54– 60. For more detail, see our research paper. Follow the kit mannual for calculating the catalase activity. All the best.
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Can you plzz provide some references for effect of Mn fertilization on soil enzymatic activities
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Please have a look at some enclosed PDFs...
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Hi!
A special primer has to be used with only 19 bp poly dT (Tm 44.9) instead of 30 bp in original study (Tm 55.2). Will this induce failure of mRNA capture? Will this cause RNA detachment in Reverse transcription using Superscript II (@42 ℃)? If this is a problem, how about start reverse transcription @ 37℃ or 40℃ for 20min, then rise it to 42℃? Will superscript II be completely block at lower temperature?
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19T-V is better than 19T in targeting the beginning of the polyA site, yes. I believe 19T-VN just gives a slightly tighter clamp.
As to the reaction conditions, yes: heat your RNA + oligodT to 65 for 5 mins, crash cool on ice, then add the buffer/dNTPs/enzyme, mix well, spin down briefly, leave at RT for 10mins, then over to 42 for 25mins.
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I have Laccase from trametes versicolor with 0.5U/mg activity.
I need enzymatic activity of 50U/g in solution. What should be the amount of enzyme I add to obtain enzymatic activity of the above said?
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If you have a solution with 0.5 U/mg is the same of 500 U/g, so if you wish 50 U/g you must dilute 10 times that solution in the appropriate solvent or activity buffer.
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I am working with erythroid progenitor cells in culture and want to assess the activity of an intracellular (cytoplasmic) enzyme. I have a protocol from the literature for the activity assay, but I can't seem to find any detailed info on the lysis buffer and methods used prior to the assay. For westerns I lyse with RIPA on ice and then sonicate, but I know I can't use RIPA for this, so my two questions are:
1. What lysis buffer recipe do you recommend to extract cytoplasmic proteins for an activity assay?
2. What lysis method do you recommend in conjunction with this buffer? Details would be appreciated!
I know there are many lysis buffer options and I'm just not sure how to figure out which one is both easy to make and will work for this purpose, and if one lysis method (freeze/thaw vs sonication, etc.) is more/less suitable for retaining enzyme function for downstream testing?
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You may also have a look for this option;
Thermo M-PER™ Mammalian Protein Extraction Reagent for RIPA like non-denaturing lysis buffer...
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A full-term female infant failed to gain weight and showed metabolic acidosis in the neonatal period. A physical examination at 6 months showed failure to thrive, hypotonia, small muscle mass, severe head lag, and a persistent acidosis (pH 7.0 to 7.2). Blood lactate, pyruvate, and alanine were greatly elevated. Treatment with thiamine did not alleviate the lactic acidosis. Which of the following enzymes is most likely deficient in this patient?
a) Alanine amino transferase
b) Phosphoenolpyruvate carboxy kinase
c) Pyruvate carboxylase
d) Pyruvate dehydrogenase
e) Pyruvate kinase
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Pyruvate carboxylase
Rationale: A biotin-dependent enzyme belonging to the ligase family that catalyzes the addition of CARBON DIOXIDE to pyruvate. It occurs in both plants and animals. Deficiency of this enzyme causes severe psychomotor retardation and ACIDOSIS, LACTIC in infants.
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Hi every one,
I am extracting enzymatic extract from plant (50 mM Sodium phosphate buffer (pH 7.8), 0.1 mM EDTA, 0.1 % (v/v) Triton; 1 mM PMSF). I mesure the protein content and enzymatic activity fom this extract.
So I want and I need to know if the enzymes extract can be conserved for further enzymatic activity measurement (SOD, CAT, APX, ...).
I really need your help, thank you very much
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I have been successfully storing at -80 °C, making a separate working solution for other immediate assays, to avoid multiple freeze-thaw.
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If I isolate/enrich lysosomes from mammalian cells through either differential centrifugation or using commercial kits, what is the best method of storage to preserve enzymatic activity? Is there a specific buffer that would be the most appropriate? Organelles should be intact at this point so would slow freezing as with cells be more appropriate than snap freezing with liquid nitrogen?
NB: structural integrity of lysosomes is less important than enzymatic activity.
Thank you,
Sam
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Thank you for your suggestion Maurice Ekpenyong . Although I’m hoping to look at enzyme activity rather than any cytochemistry. Reading the paper, it sounds like DMSO and snap freezing with LN2 similar to Freezing live cells would be enough. I may have to run a comparison.
best wishes.
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I'm studying digestive enzyme supplements and a large proportion of them contain a wide variety of proteases (Serratiopeptidase, DPPIV-peptidase, pepsin and others) as well as some other, mainly carbohydrate-digesting, enzymes. These are all often contained within the same non-enterically-coated capsule so liquid can leach inside once the capsule is consumed. Would it be expected that the proteases start digesting each other as well as the other enzymes, and would this then reduce the effectiveness of the supplement?
Thank you for any answers
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Hi. Yes . It breakd down proteins into amino acids. lipase enzymes break down lipids (fats and oils) into fatty acids and glycerol.
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How to measure the percentage activity of LDH (0.5 ug/ml) using 2 mM of NADH and 10 mM of pyruvate ( 25 °C, pH 7.00 ) and observing oxidation of NADH at 340 nm? Termination of reaction is necessary while measuring the activity?
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Thanks @M Angeles Zorrila Lopez- Perea
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For some reason, we need to have sodium chloride (NaCl, 0.05-0.1M) in our system. Will this affect the reverse transcriptase and block the SMART reverse transcript?
Thanks
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1. Km much, much lower than the prevailing substrate concentration ([S] >> Km)?
2. Km around the prevailing substrate concentration ([S] ≅Km)?
3. Km much, much higher than the prevailing substrate concentration ([S] <<Km)?
Please give examples too. Thank you!
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Hi there,
I think the best example to illustrate your question is the hexokinase/glucokinase which are in charge of the first step of glycolysis. Hexokinase exhibits a low Km compared to glucose concentration because it has the role to incorporate glucose into glycolysis in order to produce ATP at the end. As the cell needs ATP at any time hexokinase exhibit a low Km compared to glucose concentration in cell. Then its activity is more or less constant and near maximal capacity whatever the situation. Glucokinase has the same activity as hexokinase but only at the hepatic level (whereas hexokinase acts in any cell). Its role is to sense glucose level in order to regulate insulin/glucagon balance therefore glucokinase exhibits a higher Km so that glucose concentration variation will induce also a variation in glucokinase activity which will initiate a cellular response in order to optimize glucose metabolism. So basically the same reaction is used in 2 different ways by 2 different enzymes belonging to the same enzyme family: their properties are in accordance with their respective roles in the cell.
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I was wondering if the methods of enzyme immobilisation reduce enzymatic activity. I am aware that everything dependes on enzyme kinetics and the type of immobilisation, ¿but do the enzymes immobilised via covalent or ionic bonding experience a reduction in their catalytic activity?
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  • The equilibrium constants that describe binding of it substrate to the enzyme and dissociation from (k1 and k2 and hence Km) it will change, simply because the substrate can no longer approach the enzyme from all angles. One way to look at this is that the effective substrate concentration seen by the enzyme will be lower than the molar concentration.
  • The electrical charges of the protein and the counterions (some of them on the matrix) will create an electrical double layer which charged substrates have difficulty crossing.
  • Some enzyme reactions produce or consume protons, creating a pH gradient between the surface and the bulk medium.
  • Binding of the enzyme to the matrix may change its conformation and/or its flexibility, which would affect kcat and hence Vmax.
  • The velocity of the enzyme inside a matrix will increase with [S] only until diffusion of substrate and product through the pores of the matrix become rate limiting, this lowers apparent Vmax. Increasing [E] beyond an optimal value will increase enzyme cost w/o the benefit of increased reaction rates. This effect depends on the Sherwood number Sh of the system.
For all these reasons (and may be some more) the measured enzymatic activity will be lower by a stationary efficiency factor ηs = turnover of immobilised enzyme / turnover by free enzyme ≤ 1, or by the operational efficiency ηo = τf / τi ≤ 1 (where τ is the relaxation constant, the time for [S] to drop to 1/e ≈ 37% of the starting value). Note that the proper way to determine τf is after destruction of the matrix by ultrasound.
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I want to study the activity of a mannosidase against a substrate using a 96-well plate.
Following the protocol, the reaction lasts for 1h and I will end up with a absorvance value at 450nm.
How can I convert that to enzymatic activity (umol/min/mg)?
I know I have to create a standard curve but, should I create it using known concentrations of the product formed (mannose) or would be okay to do it using known concentrations of the enzyme?
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Knowing you are wanting to measure the Enzymatic Activity of a Mannosidase, you should be using a p-Nitrophenyl Mannopyranoside as a substrate, where the product you measure in the reaction is the P-Nitrophenol, which has a specific Extinction Coefficient. You can then simply measure the Activity of your mannosidase in the appropriate buffer system and using the appropriate Stop reagent, to obtain a Delta A405 of a test sample verus a Blank Sample and with Volume of reaction known, dilution of the Mannosidase known, Time of reaction known, and the Extinction Coefficient, obtain your desired umol/min/mg. See example in Attachment.
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I have a batch of fish liver samples than I would like to preserve for internal controls during EROD assays. Would lyophilizing the fish liver samples prolong the length of time that I can keep them stored -80C without affecting EROD activity?
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Juan C Pérez-Casanova - I would not recommend freeze drying the basic liver samples - that's a complex tissue to freeze dry and preserve the enzyme. Solid tissues, even small samples, generally take days to freeze dry, and then the challenge is to reduce the residual moister to <2%. Freeze drying can preserve enzymes well, when properly prepared, most PCR reagents and enzymes are freeze dryed, for example. I suggest that you extract your enzyme, aiquot in buffer and freeze dry the aliquots, then store in the freezer. Do watch out though, even freezing can affect activity as there can be a pH shift in buffer, when you freeze.
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I'm preparing enzyme-responsive polymeric nanosystems and according to the manufacturer's information, the enzyme is in
-» "50 mM sodium acetate buffer, 1 mM EDTA, pH 5.0.";
-»"≥10 units/mg protein" and
-» unit definition "One unit is defined as the amount of enzyme that will hydrolyze 1.0 µmol of compound x per min at 40°C, using 100 mM Na+/K+ pH 6.0, with 1.33 mM EDTA and 2 mM DTT as the activation buffer."
The flask has 50 micrograms of enzyme.
Concentration: 0.451 mg/mL.
416.0 U/mgP
I wanted to prepare 0.5 UN/ mL solution.
Thanks very much
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@Marco: you are definitly right! I made a slight calculation error... ;)
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