Mixing mushroom enzymes with tryptamines

[Loveall]

This maybe a silly question, but I have to ask. This likely has been asked before but I can't find the question.

What would happen if one got fresh mushroom and ground them to a soup. Add water and any stuff that keeps enzymes nice and happy (active). Then add a bunch of tryptophan from the vitamin store. Excess amounts. Based on the image below also add ATP to get to (nor) baeocystin. Then one would need S-Adenosyl methionine (SAM) to get to psylocybin. All these added items are available as supplements (SAM is called SAMe I believe).

Would the mushroom benzymes act on the tryptophan with the added supplements and synthesize psylocybin? According to the attached paper all one needs is a few enzymes to be active.

If this works would this be a use for old innactive mushrooms? Could you setup a fermentation bucked with the mushroom enzymes and add the health supplements to get psylocybin?

 

[downwardsfromzero]

would this be a use for old immactive mushrooms?

If the mushrooms have degraded through enzymatic activity, what's to say that you'd not be plagued by the same issue in the enzymatic broth?

And if the actives have degraded through age and poor storage alone, the enzymes probably won't be faring that well by then either.

 

[Loveall]

would this be a use for old immactive mushrooms?

If the mushrooms have degraded through enzymatic activity, what's to say that you'd not be plagued by the same issue in the enzymatic broth?

And if the actives have degraded through age and poor storage alone, the enzymes probably won't be faring that well by then either.

True. We could only have a meaningful positive result. A negative result would be meaningless since as you point out we would not know if it failed because the enzymes are gone or because the simple home in vitro process will not work.

Anyway, what do you think the chances are of grinding fresh mushrooms with a dense solution of tryptophan, ATP, SAMe and having the mushroom enzymes convert tryptophan to psylocybin naturally? Sounds too good to be true / too simple...

 

[Loveall]

Starting with 4-HTP would be a more direct route (3 mushroom enzymes needed instead if 4), but I don't see that in the supplement market.

Also, the paper does not specify temperature or pH for each step, it just says it mixed different combos of stuff and examined the results to deduce the full chain of events. Anyone know what the ideal conditions are likely to be?

One coralary in the paper seems to be that by adding ATP to one may be able to push psilocin to phosphorylate?

 

[benzyme]

This maybe a silly question, but I have to ask. This likely has been asked before but I can't find the question.

What would happen if one got fresh mushroom and ground them to a soup. Add water and any stuff that keeps enzymes nice and happy (active).hen add a bunch of tryptophan from the vitamin store. Excess amounts. Based on the image below also add ATP to get to (nor) baeocystin. Then one would need S-Adenosyl methionine (SAM) to get to psylocybin. All these added items are available as supplements (SAM is called SAMe I believe).

Would the mushroom benzymes act on the tryptophan with the added supplements and synthesize psylocybin? According to the attached paper all one needs is a few enzymes to be active.

If this works would this be a use for old innactive mushrooms? Could you setup a fermentation bucked with the mushroom enzymes and add the health supplements to get psylocybin?

excess amounts will inactivate the pathway, because decarboxylation is the rate-determining step; it is probably a coupled reaction, which requires the most gibbs free energy. otherwise, it would require a buffer solution, probably around pH 5. best to start with mycelial growth, I'd experiment with LC and tryptamine, or even better, NMT. DMT would yield the highest conversion; but avoid excess amounts, or feedback inhibition will occur.

 

[downwardsfromzero]

Having an independent process for the Trp-decarboxylase would be handy, perhaps. Tryptophan is the more readily available starting material and thermal decarboxylation can be a bit messy.

Would the enzymatic decarboxylation require ATP to function?

And of course, this suggestion raises the question of how one isolates the Trp-decarboxylase in the first place.

 

[Loveall]

Ok, a suggestion is to have a LC to which we add vitamin store supplements.

-Tryptophan
-ATP?
-SAMe?

What would the concentration be? pH would be set at 5, right?

Then what would one do? Blend the mycelium, filter the water, extract the water (e.g, with the PIXIE method we are trying to get to work and verify), replenish the mycelium using fresh honey or caro water (4%) spiked with with Tryptophan/ATP/SMAe, wait a couple weeks and repeat?

Downwardsfromzero, as I understand the diagram/paper, starting with two molecules of tryptophan in the presence of mushroom enzymes, this is the global reaction:

2 Tryptophan + O2 + 2 ATP + 4 SAMe -> 2 Psilocybin + 2 CO2 + 2 ADP + 4 SAH

Also, there is this reaction to consider (again mushroom enzymes must be present for catalys):

Psilocin + ATP -> Psilocybin + ADP

I don't understand the rate limiting reaction comment. What execss would hurt (tryptamine, ATP, or SAMe?).

We'll also need to understand if PIXIE would extract tryptophan (another zwitterion with pI ~ 5.9), this can be tested after a trip to the vitamin shop, but Mindlusion doesn't think it would based on Xylene solubility. Interestingly, Tryptophan would have the same MS peak as psilocin, right?

 

[benzyme]

I don't understand the rate limiting reaction comment. What execss would hurt (tryptamine, ATP, or SAMe?).

potentially any of those. rate-determining basically means it is the step in the reaction which occurs the slowest, and is reversible, because it requires the most energy to proceed. a cofactor, in this case it would be ATP, is required to move the reaction forward...but too much of the precursor (tryptophan), or cofactor, would potentially reverse the reaction...it would not proceed forward, just result in stalled growth. I've mentioned before, mushrooms derive their own tryptophan from the decomposition of proteins in substrates, i.e. grains, dung, etc. It's a shot in the dark to determine, quantitatively, what concentrations would be necessary for an effective conversion. that can only be determined experimentally. I've never seen any data/experiments to suggest conversion would occur with supplemented tryptophan. Gartz has shown that supplemented tryptamine, NMT, and/or DMT will increase yields.

Interestingly, Tryptophan would have the same MS peak as psilocin, right?

the parent ion, yes...the fragments would be different.

 

[Loveall]

Many thanks for the explanation benzyme. The good news is that (nor)baeoe appears very early in the reaction chain, we believe that is active, based on my bioassay and your analysis where small (nor)baeo peaks were seen.

Also, I think we can now lay out the path for the activity boost with tryptamine, NMT, and/or DMT.

Triptamine: already in the chain in the OP (labeled as 7). Is a more direct route than Tryptophan since the following step is not longer needed:

Tryptophan -> Tryptamine +CO2, catalized by psiD​

DMT looks like a big shortcut (methilation steps are avoided). Proposed reaction would be

DMT + O2 -> 4OH-DMT (psiH catalisis), 4OH-DMT + ATP -> Psilocybin (psiK catalisis)​

NMT, needs one more step than DMT (along with one more enzyme)

NMT + SMAe -> DMT + SAH (psiM catalist)​

So by reviewing the reaction chain, rankimg most direct paths,

DMT (2 steps, needs ATP, psiH/K) > NMT (3 steps, needs SAM, ATP, psiH/K/M) > Triptamine (4 steps, needs 2xSAM, ATP, psiH/K/M) > Tryptophan (5 steps, needs ATP, 2xSAMe, psiD/H/K/M)

Also, the authors mention that if only certain enzymes are allowed to act, one could get DMT from triptamine:

Based on the proposed order of
1 biosynthesis events,[6] PsiM is expected to catalyze iterative
methyl transfer to the amino group of 7 to yield
N,N-dimethyltryptamine (10).

Another interesting possible reaction is putting together psiD, psiM, SAMe and tryptophan, that should yield DMT :want:.

So.... If one could separate the mushroom enzymes psiD/M from mushrooms, and keep them active in vitro, in theory it may be possible to convert over the counter supplements (tryptophan + SAM) to DMT.

Here is the interestimg part. Different enzymes precipitate out of an ammonium sulfate solution at different concentrations. This is a pretty standard way to separate protein/enzymes. I've seen this happen at home in mushroom extracts (moar stuff precipitates as ammonium sulfate is added). So there may be a way to separate the psiD/H/K/M enzymes at home.

Or am I talking nonsense?

 

[benzyme]

no, that's common practice. it would help to run some SDS-PAGE gels to identify the protein of interest.

 

[Loveall]

One word of caution: Adding 5-OH substituted tryptamines to the mushroom substrate (e.g. bufotenine or 5-HTP) could produce the 4,5-dihydroxy-DMT which may have some toxicity (but I'm not sure). More details here Safety warning if adding 5-substituted tryptamines to the mushroom substrate - Cultivation - Welcome to the DMT-Nexus.


I would proceed with a lot of caution around this. I was not aware of this possibility when starting the thread.

 

[Loveall]

Ok, so I've learned a little more about this subject.

Essentially, the easiest thing to do is a biotransformation (mentioned previously in the Nexus several times).

For anyone unfamiliar with the subject wondering "What is a biotransformation?" Essentially is adding a substrate to a live cell line so it is transformed into the compound of interest (such as a drug). Humans have been doing this for thousands of years when brewing alcohol. The yeast would be the live cells, the sugar the substrate, and the alcohol the desired drug. Making vinegar is another ancient example.

Isolating specific enzymes is tricky. However, doing a liquid brew (or a mushroom grow) to get a biotransformation going should be accessible to the kitchen alchemist.

To make a brew, the key is to start with the right cell line and a dissolved substrate.

We would be using cubensis mycelium as the cell line. For brewing purposes we would use a liquid culture (e.g. 5% honey).

Promising substrates are mexamine and tryptamine. There may be kitchen safe methods to synthesize these from melatonin and tryptophan (which themselves could be interesting substrates). Work is ongoing on the kitchen safe synthesis and workup (extraction) for these.

In general the steps for a fugal liquid biotransformation brew are:

1) Grow a liquid culture (LC)
2) Feed a dissolved substrate to the LC. Not sure if the salt or frebase form matters or not (?). Salt would be easy to dissolve. HPBCD is mentioned as a cosolvent to help dissolve substrates (see first attachment).
3) Incubate
4) Extract the goods. Mushrooms will tend to brew zwitterions because they like to add the -OH group, so this may be tricky, but we are working on kitchen methods for that too using high ionic strength at the right pH and the right solvent.

Attached are a few documents that go into some details (first two use liquid cultures, second one being a cubensis example, and the 3rd document does a mushroom grow which benzyme has reproduced).

One word of caution, is to not ingest any of the stuff we may make until we understand what we did and have an analysis done. Even if we ever get to that point start very slow.

Anyone interested in this or have more info? Feedback and collaboration welcome

 

[Loveall]

Attached is another article of interest on this subject.

Also, can anyone find this article:

Google Scholar

scholar.google.com

I can't get it.

Thank you.

 

[KloudQ7]

My neighbor likes to take my spent bark and use it for his mushroom projects. We have noticed that the fruits grown this way may feel stronger, though it's hard to tell really. I'd love to be able to to test the difference. If anyone ever figures out a good extraction I'll be sure to try it. Anything promising so far?

 

[Loveall]

Yes, we have promising results using high ionic strength liquid-liquid extractions near the isolectric pH. Still need to check yields and make sure it is practical and reproducible. We should know more as time goes on and more experiments are done.

 

[antares]

My neighbor likes to take my spent bark and use it for his mushroom projects. We have noticed that the fruits grown this way may feel stronger, though it's hard to tell really. I'd love to be able to to test the difference. If anyone ever figures out a good extraction I'll be sure to try it. Anything promising so far?

That is an interesting idea. Does he acidify it to bring it back to neutral PH? I would have thought that the high alkalinity wouldn't be very conducive for mycelial growth.

 

[KloudQ7]

The bark was just boiled with some vinegar. Loveall, how pure of an extract have you made? I'd love to do some experiments myself, any tips?

 

[Loveall]

Oh yes, it would be great if you can help with the saga of OTC mushroom extraction. First off: be ready to lose some mushrooms. Not everything is working yet.

You'll need the following OTC chemicals: ammonium sulfate, Xylene, Ca(OH)2, vinegar, fumaric acid, acetone, magnesium sulfate (last 3 chemicals are to make FASA).

Process:

-Start with mushrooms (fresh or dry). Make a thick mushroom water paste saturated with Ca(OH)2 in a grinder. Want no color change which can indicate degradation, for some reason this does not happen with Ca(OH)2. Starting with fresh mushrooms a rough ratio of 1:1:0.5 for mushroom:water:Ca(OH)2 mass seems roughly adecuate. If not enough Ca(OH)2 is added mushroom will change color, you want a white/lite brown color, not gray or blue.

-Spread the paste in a shallow pan and dry it in the oven at low heat (~170C).

-Collect the paste and put it in a mason jar. An optional Xylene clean can be done at this point.

-Add water and rehydrate. You want to have excess water for the actives to dissolve into. At this point I guess you could filter to make the next steps easier, but I have not done that here.

-Add 35% ammonium sulfate. You'll smell ammonia as the Ca(OH)2 is neutralized. The pH will drop, shoot for ~pH9.

-Pull with Xylene, then perform FASA. This powder has been shown to have psilocin fumarate.

-Drop pH to 4 using vinegar that also has 35% ammonium sulphate. Pull again with Xylene, then perform FASA. This powder has been shown to have Psilocybin fumarate but also had a lot of excess fumaric/fumarate (I think I had an issue with my FASA process, not sure).

Some notes:
1)Limonene+FASI seems to also work instead of Xylene+FASA.
2)Test your FASA on fresh Xylene, make sure nothing crashes out. If it does, maybe your FASA is too concentrated (I'm looking into this more, not sure - officially/historically fresh Xylene and FASA does not crash out Fumaric acid based on previous work, but maybe not all Xylene and all FASA is created equal).
3)You can also test FASA on the optional Xylene wash on the dry powder. Nothing should crash out.
4)This process is not optimized. It could be that higher Ammnium sulfate concentration helps, especially for the psilocybin extraction at pH 4.
5)Work in low lighting conditions to protect the actives.
6)It may be possible to filter at pH4, then increase ammonium sulphate to 55%. The resulting precipitate may be active if Psilocybin crashes out at such high ionic strength. Or the new precipitate may just be more enzymes/proteins. This is currently an open question.


Theory:
Mushroom enzymes are deactivated by Ca(OH)2 and heat. Seems like Ca(OH)2 is safe for the actives based on lack color changes, but this is not well understood as basic conditions are expected to degrade actives (for example, if you use ammonia to basify, the mushrooms turn black in a couple minutes). It could well be that Ca(OH)2 hurts the mushroom actives, it's just that we don't have clear evidence of that at this point. Deactivated enzymes are less likely to form emulsions and convert actives. At the high pH of Ca(OH)2 actives are water soluble, but at the isolectric point and high ionic strength they prefer to move to the solvent instead of staying in the water (and proteins and enzymes that want to bond and trap the actives are also made inactive by a high ionic strength "shield" ).

A lot of open questions and work needed. Good luck! Looking forward to any results.

 

[Loveall]

Loveall, how pure of an extract have you made? I'd love to do some experiments myself, any tips?

Update: incase you are still looking, there could be a more straight forward path to OTC mushroom extraction, steps described here.

The method in the previous post of this thread (post #18 ) did give a psiclocin signal (benzyme's work), but the extract process is cumbersome and the yields are questionable. I'm abandoning that path for now in favor of the one linked above.

 

[Loveall]

Promising substrates are mexamine and tryptamine. There may be kitchen safe methods to synthesize these from melatonin and tryptophan (which themselves could be interesting substrates). Work is ongoing on the kitchen safe synthesis and workup (extraction) for these.

The wise good people in the nexus chat suggested to simply add melatonin to the substrate to see what happens. That was done (10mM melatonin added to the water in a monotub grow) and we are trying to sort out the results. Early contributions from dreamer042 (TLC) and benzyme (MS) suggest that a bio-transformation could have occurred. More work is needed to verify (could simply be a sample contamination instead).

If a bio-transformation did happen, what could it give? Melatonin is interesting because the nitrogen that attaches to the acetyl group seems prime for methylation, similar to how tryptamine gets its second methylation when transforming to psilocin. One bio transformation possibility is shown below consisting of two simple steps (see first image where melatonin is in the first row, and the ~equivalent psilocin steps are in the second row).

Mass-spec would show a peak in the 262-264 range (see second image). It could be named 4-OH-5-MEO-MACT (where MACT = MethylACetylTryptamine). A close relative to it could be 4-HO-5-MeO-DMT.

To be clear, this is not a claim of a result, only discussing one of the possibilities there could be.