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Mushrooms, Mycology and Psychedelics > Advanced Mycology
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Basidiomycete protoplasts, What kind of lysing enzymes???
    #29664130 -

Hi, I’m new here.
I’d like to know if anyone has a solution for replacing two specific lysing enzymes: "Novozym 234" and "Glucanex Lysing Enzymes from Trichoderma harzianum." They appear in many older protocols but are no longer sold. I’m currently using "Novozym Vinotaste Pro," which seems to be the closest match to "Glucanex," but it is an enzyme cocktail intended for the wine industry. I am looking for an enzyme cocktail better suited to digesting the cell walls of basidiomycete fungi. If anyone has a solution—either a complete cocktail and a supplier name, or a list of enzymes (chitinase, 1,3-glucanase, Driselase, and cellulases) along with the dosage for each to create a custom blend—please let me know.
Thanks.


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Re: Basidiomycete protoplasts, What kind of lysing enzymes??? [Re: TonyGrow4All]
    #29664433 -

(A quick web search says Trichoderma harzianum and beta-D-glucanase Driselase can replace Novozym 234 and Glucanex in Trichoderma harzianum, but you probably already knew that). :what:

We will definitely let you know if we come up with a solution.

But in the meantime do you have a question related to the topic of this forum being the cultivating of psychoactive mushrooms?

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Re: Basidiomycete protoplasts, What kind of lysing enzymes??? [Re: veggie]
    #29664798 -

Thanks—to be honest, not right now. But that will come soon enough. For the moment, I'm still just looking around the forum and I'm already finding all the answers I need. The only thing I’m stuck on is non-mechanical dedikaryotization. I work mainly with medicinal mushrooms and wild Psilocybe species, especially the hot woodlover varieties, like Niveotropicalis, Indian Woodlover, Xgriseo... I’ve realized that isolating monokaryons via spores has its limits and that protoplasts offer greater possibilities, but the process is still quite complicated for a novice.:blush:


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Re: Basidiomycete protoplasts, What kind of lysing enzymes??? [Re: TonyGrow4All]
    #29664858 -

You can ferment your own enzymes. Not too hard, all you need is trick basically, there are more advanced ways to do it.

You might consider a chemical cell wall lyzers.

I've also grown some griseos.

While spore monos do have there limitations they also have their values. Not only are ddk monos incredible hard to get, recovering them systematically is where it gets really tricky. And just because a strain is a banger doesn't necessarily mean one of the monos will be.

And it's not necessarily protoplasting. Protoplast regeneration, and chemical/mechanical dedikaryotion have very different functions, outcomes and methods.

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Re: Basidiomycete protoplasts, What kind of lysing enzymes??? [Re: veggie]
    #29664860 -

It's a totally relative question for the cultivation of psychoactive mushrooms. If one plans to breed, mainly inter specific or inter generic, psychoactive mushrooms.

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Re: Basidiomycete protoplasts, What kind of lysing enzymes??? [Re: unapprovedpuppet]
    #29664883 -

unapprovedpuppet said:
It's a totally relative question for the cultivation of psychoactive mushrooms. If one plans to breed, mainly inter specific or inter generic, psychoactive mushrooms.



Actually it isn't relative to normal cultivation at all, it would be better off in the Advanced Mycology thread than in the cultivation thread. Would likely get more responses as well,

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Re: Basidiomycete protoplasts, What kind of lysing enzymes??? [Re: RockinRobot]
    #29664886 -

"normal cultivation" 😆

Yes officer. Take him. To jail. Throw away the key. Hang him by his smallest toes. Ha

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Re: Basidiomycete protoplasts, What kind of lysing enzymes??? [Re: RockinRobot] * 1
    #29665030 -

RockinRobot said:
unapprovedpuppet said:
It's a totally relative question for the cultivation of psychoactive mushrooms. If one plans to breed, mainly inter specific or inter generic, psychoactive mushrooms.



Actually it isn't relative to normal cultivation at all, it would be better off in the Advanced Mycology thread than in the cultivation thread. Would likely get more responses as well,



Would likely be the only actually advanced topic posted in there in years :lol:

I remember a guy on Facebook doing just this. He had some very specific applications for doing the labor needed. He was also a legit mycologist who actually did his thesis on this topic so it was very much in his wheelhouse. However the applications he was using it for had nothing to do with active cult or crossing. I don't even think he ever grew a cube.

Personally if I'm just trying to find good producing genetics or something I would just stick to cloning. Breeding monokaryons is kinda pointless for the average grower.


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Re: Basidiomycete protoplasts, What kind of lysing enzymes??? [Re: Pastywhyte]
    #29665036 -

I'm way out of my depth here, but wouldn't this achieve the same outcome if you're hunting monos

https://www.shroomery.org/forums/showflat.php/Number/28117746#28117746


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Re: Basidiomycete protoplasts, What kind of lysing enzymes??? *DELETED* *DELETED* *DELETED* *DELETED* [Re: PuffM4gicDr4gon]
    #29665075 -

Post deleted by TonyGrow4All

Reason for deletion: I replied to each response individually, and that seems to be bothering users, so I'm correcting my mistake ☺️


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Re: Basidiomycete protoplasts, What kind of lysing enzymes??? [Re: TonyGrow4All]
    #29665096 -

Off topic for Mush Cult. Will be moving over to Advanced.

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Re: Basidiomycete protoplasts, What kind of lysing enzymes??? (moved) [Re: TonyGrow4All]
    #29665102 -

This thread was moved from Mushroom Cultivation.

Reason:
Off topic for Mush Cult. Moved to Advanced.

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Re: Basidiomycete protoplasts, What kind of lysing enzymes??? [Re: TonyGrow4All]
    #29665117 -

Please do not double or triple post on the boards. Use the quote function to make a single post while replying to multiple people.

If you want to do it for the sake of doing it then fill your boots. But as far as things like maximizing yeilds, potency, etc, I personally feel you can find the results you want faster and with more to show for it with more conventional methods. It would be like putting a leveling kit and mudders on a Ferrari cause you want an off road vehicle. Can it be done? Sure but most people will just go get a SUV.


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:headbang3: Pastywhyte's Easy Writeups :headbang3: My Favorite Grow Logs :headbang3:
:headbang3: Avoiding the dreaded facepalm in cultivation :headbang3: The worst threads all have the same subjects :headbang3:
:headbang3: Q & A.M.U. :headbang3:

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Re: Basidiomycete protoplasts, What kind of lysing enzymes??? [Re: unapprovedpuppet]
    #29665129 -

[Re: unapprovedpuppet]
It is true that protoplasts are more complicated to work with than monokaryons, but they offer the advantage of starting from a specimen with specific, established traits—unlike monokaryons, monokaryon may carry a variety of characteristics. They are also very practical in cases of non-spore-producing mutations (such as Enigma). With a suitable osmotic buffer, the yield of viable cells is generally satisfactory. In my view, chemical digestion of cell walls is more complex. As for the mechanical method, I do not own an autoclavable blender, as they are too expensive. As for enzymatic fermentation, it is possible, but it seems relatively complicated to me; furthermore, it would likely make it much harder to determine the dosage needed to dissolve the membrane without damaging the cell itself. However, I’ll look on Academia or Scribd. There are a huge number of microbiology and breeding protocols on those platforms.

[Re: Pastywhyte]
To be honest, this isn't my profession. I’m nothing more than a mechanic 😆. But I’m passionate about mushroom cultivation and plant propagation. So, sooner or later, working with protoplasts was a must for me. Online forums and the advent of consumer-facing AI did the rest.

[Re: PuffM4gicDr4gon]
Monokaryons are certainly interesting. However, protoplasts allow you to obtain monokaryons from specific specimens or to work with sterile genetics like blobs. I would say that isolating monokaryons using the "grab and drag" method for swabs and serial dilution for spores prints is perfect for any grower looking to breed strains while "simply" having a flow hood, a microscope, and two micropipettes. Protoplast techniques are used far more by lab technicians than by casual growers. In my case, it is more out of educational interest and fun than necessity.


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Re: Basidiomycete protoplasts, What kind of lysing enzymes??? [Re: TonyGrow4All]
    #29668261 -

Protoplast fusion is cool if you can pull it off.  I go a different route I’ll share one day. 

Di mon mating is also heavily underused when it comes to pairings, goin that route may nail down what you are attempting, if it is breeding without traditional meiosis.

You can check out another thread in this section and a paper on cytoplasmic mixing will crop up, I think  going the route you are attempting will have to consider the self/ non self  phenomenon.

From what I gather, even if you can get proficient at lysing cell walls , producing a stable crop of mushrooms worth growing  from classic protoplast fusion techniques may require a heavy test load..

Instead of hunting down those lysing enzymes, it may be easier to find and research rattle  snake venom for somatic hybridization.

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Re: Basidiomycete protoplasts, What kind of lysing enzymes??? [Re: trippleblack]
    #29668320 -
Log in to view attachment

Yes, I’ve seen protocol sheets where venom is used to force "interspecies" crosses—keeping it relatively close, of course—but that’s tricky for me since I live in France; given the regulations, I don't think I could acquire that kind of venom. At a stretch, maybe at a reptile shop, but even then... On the other hand, by using a blend of enzymes like Vino Tast Pro and Triton X-100, you can achieve pretty much the same result. And it’s much more accessible—you can get them on AliExpress or from any shop selling winemaking enzymes. I dug up this short document that might interest you. I used Google Translate because my English is terrible... 🫣


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Edited by TonyGrow4All (09/14/26 01:47 PM)

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Re: Basidiomycete protoplasts, What kind of lysing enzymes??? [Re: TonyGrow4All]
    #29669626 -

Understood.  I know what you are trying to do.  I did the parasexual thing when i created a cube strain going around called tmac3.  I have a hunch that this is a way to reset the clock on older genetics; by breeding away from the arrangements set in stone by constant spore >meiosis spore > meiosis. 

If you can find a protocol to do it with those enzymes, and get it to work, that would be great work. I’m fascinated by others adopting these theories that would lead to this type of breeding style.

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