Extracellular Enzymes - Science topic

To get the amount of enzyme synthesized you could use an immunoassay approach, but if you want to measure the active enzyme (which is not necessarily the same as the amount of protein synthesized) you need and assay in which a substrate is converted into a measurable product (e.g., by absorbance or fluorescence).

If you construct a standard curve of signal vs concentration using pure product you can convert the values obtained with real samples (i.e. substrate being converted into product) into umoles produced per unit time. 1 unit of enzyme is normally defined as 1 umol substrate converted/product formed per min-1. You can then express enzyme in your samples as umol/min/ml (activity) if you take account of the volume of sample used in the test, and any dilution of the sample prior to assay.

Without any specific assay details it is impossible to give an exact formula to use but the general principles above can be applied to any assay. You just need to incorporate your specific times, volumes, and dilutions in order to determine the activity.

Dear Tori,

I recommend you to check:

Handbook of detection of enzymes on electrophoretic gels.

Gennady P. Manchenko 2nd Ed.

CRC Press

ISBN 0-8493-1257-4

You can find a lot of protocols to detect enzymes by zymogram.

Best regards

Внеклеточная ферментная активность сохраняется только при благоприятных условиях среды, при которой микроорганизмы непрерывно выделяют их в окружающую среду для получения питательных элементов для своего роста и развития. При наступлении неблагоприятных условий, микроорганизмы теряют активность и перестают выделять ферменты.

Try this website protocols

http;//www.BioDataCenter.ir

Hey Noelle, not sure if you have an answer already, but if you divide EEA by microbial biomass and multiply by 1000, you get units in umol activity/mg SMB/h or mmol activity/g SMB/h. Hope all is well! See below:

umol/g soil/h * kg soil /mg SMB * 1000 g soil/kg soil

Because pKa of HCN is 9.31 (20 °C)/ 9.22 (25 °C) you can use NaHCO₃ (aq) as acid ( pK₁ for carbonic acid: 6.35 ).

Hello Haseena,

An activity in the cell free supernatant could be because of any extracellular secretion of the organism. Hence you need to go further with the purification of the crude supernatant for determining the agent responsible. Bioactive organic lead can be determined through liquid-liquid solvent extraction (polar or non-polar) and for enzyme or bioactive peptides proceed with protein purification. Thereafter, you can confirm the bioactivity in these partially purified fractions.

The size difference you mentioned (~10 kDa) is sufficient to separate the two proteins by SEC unless they stick to each other or some other proteins in your samples. Sometimes PAGE gives us a wrong idea about the actual state of the protein of interest. Keep us informed about the progress.

I used Isopropanol for bacterial bioflocculant

The answers above are great, and I agree that your compounds are likely still good for use.

Still: Buy new ones.

The time you/your student work on establishing that they're still good will cost more that re-ordering the labelled substrates, and it's one less issue that could come up during review when you get your results published.

IN GENERAL, FOR YOUR PURPOSE, YOU HAVE TO DO THE FOLLOWINGS: 1，UTILYZE A ROOT SPECIFIC EXPRESSION PROMOTER AS OTHER PEOPLE SUGGESTED ABOVE;  2， CHECK THE DIFFERENCE IN THE CODONS OF THE PLANT SPECIES YOU WANT TO EXPRESS AND THOSE OF BACTERIUM'S. IF POSSIBLE , CHANGE THE BACTERIAL CONDONS TO PLANT PREFERRED ONES IN THE GENE; 3, CHOOSE A SIGNAL PEPTIDE OF A PLANT GENE TO BE FUSED WITH YOUR GENE AT N-TERMINUS SO THAT THE GENE COULD BE EXPRESSED AND TARGETED TO APOPLAST. THERE ARE MANY PLANT PROTEINS SECRETED OUT OF CYTOPLASTS. JUST SELECT AN IDENTIFIED EXTRACELLUALER PROTEIN AND ITS SIGNAL PEPTIDE SEQUENCE AND PCR FUSE IT TO YOUR GENE.

Thank you Dr Reyes for your valuable input.

It depends how you obtain fungal colonies. Do you start from one spore or just a "loopful" of mycelium? in this second case the colony diameter or area is not indicative of growth. Thus I would suggest to measure only the decoloration halo diameter excluding the diameter of the colony

Depending on the type of metabolite (if peptides...) you can use a solid phase extraction with a polymeric reversed phase cartridge. 

Measuring extracellular space (ECS)  in fixed tissue may be a waste of time as it has little physiological meaning. Fixation induces a swelling of the astrocytes who tend to occupy all available spaces. A better way to measure ECS would be fast freezing and electron microscopy of freeze-substituted tissue.

Hi Peter

For beta glucosidase add cheap substrate such as esculin (as the only carbon source) to the minimal medium, in this case you'll separate beta glucosidase producer only, once you have it as a pure culture then you can test other substrates (expensive ones).

Hope this helps 

After following the steps outlined by Becky Aloo, what do you do next? It depends on what you are trying to accomplish. Do you want to purify the enzymes? Do you just want to know which enzymes are there?

If you want to know which enzymes are in the culture supernatant, a modern way would be to use proteomics. You would digest the proteins from the culture supernatant with trypsin and subject the resulting mixture of peptides to mass spectrometry-based proteomics methods to obtain amino acid sequences that could be compared with a database of sequences derived from the genomic sequence of the organism.

If you want to purify the enzymes, you need to start with an idea of what catalytic activity they possess. Then you can subject the culture supernatant to a variety of purification procedures (ammonium sulfate precipitation, various types of chromatography) and use an assay for a specific enzyme activity to test fractions for the presence of the enzyme in order to follow each enzyme through the purification steps.

Dear Joseph Konnie,

An exoenzyme, or extracellular enzyme, is an enzyme that is secreted by a cell and functions outside of that cell. Exoenzymes are produced by both prokaryotic and eukaryotic cells and have been shown to be a crucial component of many biological processes. Most often these enzymes are involved in the breakdown of larger macromolecules. The breakdown of these larger macromolecules is critical for allowing their constituents to pass through the cell membrane and enter into the cell. For humans and other complex organisms, this process is best characterized by the digestive system which breaks down solid food via exoenzymes. The small molecules, generated by the exoenzyme activity, enter into cells and are utilized for various cellular functions. Bacteria and fungi also produce exoenzymes to digest nutrients in their environment, and these organisms can be used to conduct laboratory assays to identify the presence and function of such exoenzymes.Some pathogenic species also use exoenzymes as virulence factors to assist in the spread of these disease causing microorganisms. In addition to the integral roles in biological systems, different classes of microbial exoenzymes have been used by humans since pre-historic times for such diverse purposes as food production, biofuels, textile production and in the paper industry. Another important role that microbial exoenzymes serve is in the natural ecology and bioremediation of terrestrial and marine environments.

Kong F, Singh RP (Jun 2008). "Disintegration of solid foods in human stomach". Journal of Food Science 73 (5): R67–80. doi:10.1111/j.1750-3841.2008.00766.x. PMID 18577009.

 Roberts, K. "Exoenzymes". Prince George's Community College. Retrieved 8 December 2013.

Duben-Engelkirk, Paul G. Engelkirk, Janet (2010). Burton's microbiology for the health sciences (9th ed.). Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins. pp. 173–174. ISBN 9781605476735.

Thiel, ed. by Joachim Reitner, Volker. Encyclopedia of geobiology. Dordrecht: Springer. pp. 355–359. ISBN 9781402092121.

Arnosti C (15 January 2011). "Microbial extracellular enzymes and the marine carbon cycle". Annual Review of Marine Science 3 (1): 401–25. doi:10.1146/annurev-marine-120709-142731. PMID 21329211.

Hoping this will be helpful,

Rafik

Dear Shwethashree Malla,

Please read the following text:

In eukaryotic cells, exoenzymes are manufactured like any other enzyme via protein synthesis, and are transported via the secretory pathway. After moving through the rough endoplasmic reticulum, they are processed through the Golgi apparatus, where they are packaged in vesicles and released out of the cell. In humans, a majority of such exoenzymes can be found in the digestive system and are used for metabolic breakdown of macronutrients via hydrolysis. Breakdown of these nutrients allows for their incorporation into other metabolic pathways.

An endoenzyme, or intracellular enzyme, is an enzyme that functions within the cell in which it was produced. Because the majority of enzymes fall within this category, the term is used primarily to differentiate a specific enzyme from an exoenzyme. It is possible for a single enzyme to have both endoenzymatic and exoenzymatic functions.

Example:Glycolytic enzymes,enzymes of Kreb's Cycle. enzymes are a type of protein that speed up chemical reactions in cells. enzymes are specific to the job they do. only molecules that are the correct shape can fit into the enzyme. this is called the lock and key model. enzymes work outside of the cell (extracellular enzymes) as well as inside the cell (intracellular enzymes) In most cases the term endoenzyme refers to an enzyme that binds to a bond 'within the body' of a large molecule - usually a polymer. For example an endoamylase would break down large amylose molecules into shorter dextrin chains. On the other hand, an exoenzyme removes subunits from the polymer one at a time from one end; in effect it can only act at the end ponts of a polymer. An exoamylase would therefore remove one glucose molecule at a time from the end of an amylose molecule.

intra (meaning --within) cellular enzymes are the common metabolic enzymes that are responsible for catalyzing all those processes we need in our cells---relaese of energy from glucose or carbohydrates, lipids, proteins------- replication of DNA----synthesis of proteins and hundreds of others------(a really cool one is---- acetylcholinesterase--check it out)

extra (meaning outside) cellular enzymes are released by the cells after being packaged by the golgi body or released straight out the membrane to perform a job external to that cell (ie digestive enzymes of our own digestive system--salivary, peptic and pancreatic enzymes to name some general types).

Lodish, Harvey (2008). Molecular cell biology (6th ed., [2nd print.]. ed.). New York Freeman. ISBN 0716776014.

Andrews, Lary. "Supplemental Enzymes for Digestion". Health and Healing Research. Retrieved 9 December 2013.

Hoping this will be helpful,

Rafik

Storage at 4° should be OK, better than freezing anyway, which will destabilize the solutions. And as pectin takes 24h to properly dissolve, I would not advise "freshly prepared". Just remember to add some merthiolate or sodium azide (100 mg/L) for microbial stability.

Hello Tishakorn Singto, while agreeing with all comments above I like to make some additional remarks. To my knowledge, having worked also with defined serum-free media in the past, commonly cells need for the initial attachment to the plate adhesive molecules like fibronectin or vitronectin (being serum components) or other matrix/ ECM proteins, alternatively polylysine coating of the dish. The latter provides very strong adhesion though this is not physiological at al.  Just a few reasons for an excessive release into culture media are lack of or aberrant cell polarity in 2D-culture, free difusion of the secreted ECM molecules, compromised assembly for that reason or lack of bridging and adapter molecules. In vivo higher ECM concentrations at the cell surface ('molecular crowding', Michael Raghunat et al.) will favor ECM assembly. This can be also achieved by more complex 3D-culture conditions.

Dear Maliha,

An easy way to separate intra from extracellular protein is to simply spin down the cells.  Indeed, gel filtration chromatography can be used to fractionate proteins but I would only use this if you do not know anything about the isoelectric point of the proteins that you want to separate.  Ion exchange chromatography is a much more powerful purification tool than GFC.

I assume that you know what enzymes that you are trying to separate, than you should be able to find or calculate their isoelectric point.  I still feel that mass spectrometry may be the way to go to assay many enzymes at the same time.  As I mentioned before you must know if any substrate or product will interfere with any of the reactions you want to study.

Marc

You can find some information in attached publication about phenol oxidase and peroxidase. Also, you can find more in References.

 

In sucrose, both hexoses are liked by their reducing carbon (C1 for glucose, and C2 for fructose). The glucosyl residue is alpha, not beta, hence beta-glucosidases won't  work. The fructosyl moiety is beta, but that's irrelevant.

EMS and MMS are popular along with EBr. You may refer to the attached paper for further understanding

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

apart from the above two points, presuming the fermenter conditions are good, may be enzyme you are interested in oxygen or aeration sensitive. Obviously, the mass transfer of oxygen is much higher in fermenter as compared to shake flasks. in addition, do you see cell lysis in the fermenter due to sheer forces caused by the high tip speed velocity. if the cells are lysed, they might release proteases which would degrade your desired enzyme. As David mentioned, your SDS-PAGE profile would show some light on this possibility.

Estimation of total protein give you the overall concentration of proteins which have been produced by living organisms  and the total enzyme activity determine the concentration of particular enzyme produced which is protein in nature. So the specific activity of particular enzyme give information about  the conc. of particular enzyme in per mg of protein. It mean that how much enzyme units are present in 1 mg of total protein.  

Dear Agnieszka, I have copy and paste the method from my PhD thesis, I had worked with this method for years with no problem. Please compare with what you are doing and let me know for any problem, good luck

Lipase activity assay

Determination of liberated free fatty acid was measured by colorimetry method (Kwon and Rhee, 1986) using olive oil as substrate. The reaction mixture, consisting of 1 ml crude enzyme (culture filtrate), 2.5 ml olive oil emulsion (properly mixed of an equal volume olive oil with 50 mM sodium phosphate buffer, pH 7.0) and 0.02 ml of 20 mM CaCl2, was incubated in a water bath shaker for 30 min at 50°C under 200 rpm agitation. The enzyme reaction in the emulsion system was stopped by adding 6 M HCl (1 ml) and isooctane (5 ml), followed by mixing for 1 min. The upper isooctane layer (4 ml) containing the free fatty acid was transferred to a test tube and properly mixed with 1 ml copper reagent.

The reagent was prepared by adjusting the solution of 5% (w/v) copper (II) acetate-1-hydrate to pH 6.1 with pyridine. The free fatty acid dissolved in isooctane was determined by measuring the absorbance of the upper layer at 715 nm after mixture settlement. Lipase activity was determined by measuring the amount of free fatty acid released based on the standard curve of oleic acid (0-50.0 µmole) in isooctane. One unit of lipase activity was defined as 1.0 μmol of free fatty acid liberated min−1 and reported as Uml−1.

 

The access of extracellular proteases may be controlled by certain factors; one of them is permeability, as mentioned above. These proteases may exist in their apoenzyme (devoid of their coenzymes required for catalytic activity) state or holoenzyme (with coenzymes, and fully fuctional); this would influence their possible effects on the bacterium (B subtilis). The same may be said of the enzymes within the cell. Proteases may have a reduced effect on cell apoenzymes. Another factor is how different proteins and enzymes within the cell organelles are tightly held (sequestration). This might keep them from proteolytic attack.

Enzymes such as xylanase, cellulase, phytase

Holger's suggestions are good.

BB-94 (Batimastat) sold by Tocris, SantaCruz Biotech, others is a hydroxamate-type with potent broad-spectrum activity against several MMPs.

For something more generic, generally o-phenathroline (1:10-phenthroline) at 50 uM and EDTA at 1 mM can also be used in the buffers - wash couple of times and retain in buffer in further steps if it does not interfere downstream.