Sciencemadness Discussion Board

Tryptophan conversion using Phalaris grass protein extract

Master Triangle - 8-1-2017 at 08:33

I would like to find out whether the mixed protein extract of Phalaris grasses would convert tryptophan at an appreciable rate. These enzymes are active and are known to produce DMT when isolated from the plant cells although I'm not sure how careful the preparation must be to prevent proteolysis for long enough.

Phalaris seedlings would be mulched, the cell membranes broken, and juice filtered off.
The proteins in the juice could be precipitated by ammonium sulfate precipitation or filtered out with dialysis tubing (they are in the dalton range of common and cheap tubes) or both to separate them from the small molecules.
Filtering with different dialysis tubes or doing a proper fractional precipitation would eliminate more of the unwanted proteins. An antioxidant and protease inhibitors might be needed for preservation.

This extract would contain the enzymes of the DMT production pathway along with other enzymes and proteins.
The enzymes must be fed with tryptophan and SAM-e.
The used SAM-e products should be easily removable as they are highly water soluble, even if they get broken down further by remaining SAM-e cycle enzymes.
The only difficult impurities should be the tryptophan to DMT intermediates which are all fine and are hopefully mostly used up anyway.

Here is a paper that has details on the three relevant enzymes in phalaris and their behavior:
My biochemistry knowledge does not extend far enough yet to interpret the activity data but this seems likely to be practical.

One possible problem could be if it were far too slow once the products built up (they compete with the substrate molecules) although if the enzymes are not attacked by anything you could simply add an antibacterial and leave it sitting for a month. From my limited understanding of the rates of reaction this should not take anywhere near that long with a achievable amount of enzyme.
Another problem could be if there are other enzymes that break down either the DMT pathway enzymes or the DMT itself that cannot be removed easily but I hoping that those enzymes need additional substrates since only the tryptophan and SAM-e would be supplied.

I don't know how appropriate posting this here is, but finding people who have experience in biochemistry is not easy. If this is workable it could lead to a much safer and cleaner method than most that are currently used, I don't know of such crude enzyme extracts being used like this before, and in this field of synthesis I have not heard of any enzymatic processes being used at all. INMT enzyme is a pre-prepared substitute for all this but until they make it with recombinants all the rabbit lungs you need for it makes it extremely expensive to buy.

If anybody is familiar with functional protein extractions and using them for reactions I would love to hear about potential pitfalls in the process.

Scalebar - 13-1-2017 at 04:46

I suppose it's not a complicated pathway but you'd definitely need a protease inhibitor, the goods ones we use in the lab tend to be on the slightly iffy side of 'lethal' though - ( like they take out acetyl choline esterase too. )
A crude ammonium sulphate precipitation of total protein after protease inhibition would concentrate everything present and might be sufficient.

Metacelsus - 13-1-2017 at 12:40

I've done work with protein purification and kinetic analysis. This probably isn't a viable route to making DMT, because even under ideal conditions proteins tend to degrade. Also, where would you get the S-adenosylmethionine?

However, it is likely possible to express these three enzymes in yeast or bacteria, and integrate them into the existing tryptophan biosynthesis pathways. Ultimately, this would enable DMT production from glucose and ammonium salts.

[Edited on 1-13-2017 by Metacelsus]

Tsjerk - 14-1-2017 at 04:09

I like this kind of stuff, but don't expect this to give more than 10s of milligrams per liter. But nevertheless academically seen this is interesting stuff.

The first thing to do is to find out how to calculate how concentrated the different compounds should (can) be, also during this calculation you will find out the speed of conversion of the different compounds and thus how fast the production of DMT. And how many liters(?) of reaction you need to get measurable amounts of compound. Keep in mind though, that the Ki probably goes up when the concentration of protein goes up, at some point though, your protein of interest will precipitate out.

You have to minimally learn how to run, stain and de-stain SDS-PAGE gel when working with protein. Don't forget you need a reference protein for these gels to see where your protein of interest is running (you can prepare your own if you are inventive, keratin eg).

You also have to think of a way to isolate and indentify your product (TLC?) while differentiating between the different intermediates.

Don't leave the reaction running for longer then strictly necessary, I would personally stop the reaction at the earliest calculated end and possibly earlier depending on the price of the substrates. This way you avoid bacterial and fungal growth (they will grow, also with antibiotics). Nevertheless, try to find 0.2 micrometer filters so you can filter sterilize the pre-reaction. Bacteria and fungi secrete lots of stuff you won't be able to detect.

I don't have experience with Phalaris species, but I guess Protease inhibitors are necesarry. I can isolate protein from Bacillus subtilis without, but it takes some practice and proper handeling like proper washing with buffer between steps and never ever let the temperature rise above 4 degrees Celcius. I isolate quit specific fractions before using the protein at elevated temperatures though and I'm pretty sure an ammonium sulfate precipiation won't do for this purpose.

Protease inhibitors are notoriously toxic, so keep this in mind in case you where thinking about any ''bio-assays''.

Personally I was thinking about performing the bio-chemical stereospecifically transformation of 2-carbomethoxy-3-tropinone to 2-carbomethoxy-3β-tropine, someday. Purely academic, as the co-substrate of MecgoR, NADPH, costs about 1000 euro's per gram.

For an biochemicall factory like what Metacelcus is talking about would require something like the following work-flow:
Prepare cDNA from your grass.
Design primers to PCR out the genes of interest.
Make sure the product or the intermediates are not toxic for the host.
Make sure your host produces the co-substrate.
Make sure your host can secrete the product.
make sure your host can absorp secreted intermediates (as it will probably do if it is secreting the product).
Clone the genes into integration vectors suitable for your host.
Induce the genes with suitable inducer.
Add tryptophan.
See if it works.

So: a lott of work for a little product, if any at all.

[Edited on 14-1-2017 by Tsjerk]

Metacelsus - 14-1-2017 at 06:19

Come to think of it, I've isolated beta-lactamase from E. coli lysate without treatment with protease inhibitors (all work was done at 4 °C). This was because the protease inhibitors also inhibited the lactamase, due to its similar active site serine. However, Phalaris is not E. coli, and this might change things.

For SDS-PAGE, it's probably easier to buy protein MW ladders than it is to try to make your own. It is possible to cast your own gels, but be warned that acrylamide is neurotoxic.

If you plan to purify the proteins, it's very helpful (almost necessary) to have an assay so that you can track the activity of your fractions.

Tsjerk - 14-1-2017 at 06:47

The reason why I suggested making the MW marker yourself is because they are ridiculously expensive, although you might be lucky and get a sample for free if you ask nicely. You can check what proteins are used in commercial markers, IIRC one usual one is a major protein of egg-white, you don't need a whole range of proteins if your protein of interest is near the MW of your isolated reference protein.

@Metacelsus; some proteins are more stable towards proteases than others, I don't know about beta-lactamase though. How well did you isolate it? With a proper isolation before rasing the temperature you should lose most proteases, also many coli strains used to express proteins are deficient in there protease expression (BL21 for example).

[Edited on 14-1-2017 by Tsjerk]

Metacelsus - 14-1-2017 at 10:19

The E. coli was strain J53, and it was transformed with a plasmid from K. pneumoniae that codes for a beta-lactamase. The general procedure to isolate it was:
1) Pellet cells by centrifugation; resuspend pellet in 0.1 M pH 7 phosphate buffer.
2) Lyse cells by freeze-thaw cycles (-78 °C to 4 °C, four times).
3) Centrifuge lysate and collect the supernatant.
4) Add supernatant to dialysis bag, and dialyze against 30 mM pH 7.5 TES buffer overnight at 4 °C.
5) Purify the crude extract by anion exchange chromatography on QAE Sephadex (eluent was 30 mM pH 7.5 TES buffer, with increasing NaCl concentration up to 2 M).

After this, the beta-lactamase retained activity for two weeks when stored at 4 °C (it might have been stable for longer, but I was done with it after two weeks). The beta-lactamase band was the only one visible on SDS-PAGE (stained with Coomassie blue), and the specific activity was within the range expected for the pure protein.

Tsjerk - 14-1-2017 at 12:18

The column purification is quite efficient when used with a slow gradient, but in the case of no column purification you probably need inhibitors.

Scalebar - 16-1-2017 at 01:04

I used to work in food chemistry, there are some precedents for whole cell lysate reaction - a couple of the fruit ester flavours used to ( possibly still are ) by mixing yeast cell lysate with apple pulp and steam distilling after incubation. Don't ask me what the reaction components are or the enzymes 'cos this was thirty years ago.

It wasn't the most efficient approach but it was commercially viable and has a big advantage - you don't have to declare the flavouring as being artificial.

'course it'll depend on relative levels of enzymes...

EDTA might give you sufficient protease inhibition.

There's also the possibility that as plant enzymes tend to be quite highly compartmentalised, you may get problems when you break open the cells and end up with side products that may or may not be easy to remove

Tsjerk - 16-1-2017 at 04:55

This reaction was shown to work so I don't see any problems there, the problem with EDTA is that it also captures ions necessary for the enzymes of interest to work, if they need them (I guess so)

Scalebar - 16-1-2017 at 07:42

Good point. What about soybean trypsin inhibitor?
the isolation looks well within kitchen chemistry range..

Tsjerk - 16-1-2017 at 10:04

The soybean inhibitor looks good, usually a combination of inhibitors against trypsine proteases and serine recognizing proteases is used. The usual serine protease inhibitor is pmsf, which is really toxic. I don't know a alternative by heart

Edit you just need a dialysis bag for the Science method

[Edited on 16-1-2017 by Tsjerk]