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OfflineFeelers
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Where to get cheap Lytic Enzyme used to produce Protoplasts?
    #22001124 - 07/26/15 06:13 PM (8 years, 7 months ago)

The "lytic enzyme" used in these processes is typically a cocktail of Protease, cellulase, chitinase, and others. I want to create protoplasts, but so far have only found a good supply of cellulase.

I have been searching on Alibaba, but don't really know how to search for this type of product. The perfect one is here, but from SigmaAldrich, so it's super expensive.

www.sigmaaldrich.com/catalog/product/sigma/l1412

Anyone have any ideas?

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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: Feelers]
    #22001512 - 07/26/15 07:37 PM (8 years, 7 months ago)

It wont be cheap, its a speciality product.
160 for 10g is very reasonable but good luck ordering anything from sigma aldrich :lol:

If you can culture trichoderma harzianum and isolate them yourself it might be cheaper but the initial investment and logistics are almost impossible.

Have you considered other lysins? Market price may be cheaper for slug gut lysins.


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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: Toadstool5]
    #22002042 - 07/26/15 09:24 PM (8 years, 7 months ago)

I actually have my own protein fractionator but it seems like more hassle than it's worth.

There are some products like this one...
http://www.alibaba.com/product-detail/SUKACell-trichoderma-reesei-cellulase-enzyme_60094860046.html?spm=a2700.7724838.35.1.Iw9lZ5

...that appear to be crude extracts from Trichoderma, I'll flick them an email to find out.

Edited by Feelers (07/26/15 09:36 PM)

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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: Feelers]
    #22002136 - 07/26/15 09:48 PM (8 years, 7 months ago)

Quote:

more hassle than it's worth




Exactly, its better to simply pay the 100 to 200 for 10g than making it yourself.

Crude extracts may be less expensive but how effective are they on a gram per gram basis? It might be better to go with the less dilute stuff. Normally economy brand trich products are <5% and 95%+ filler.

Either way i hope you find something. Maybe call a bioengineering company like aloha medicinals and ask for a quote?


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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: Feelers]
    #22002897 - 07/27/15 05:04 AM (8 years, 7 months ago)

Toadstool, you seem to know a bit about this stuff - you you have a technical background?

I would love to have someone to go through things with on projects.

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InvisibleToadstool5
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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: Feelers]
    #22003441 - 07/27/15 08:51 AM (8 years, 7 months ago)

I used to dabble in organic chemistry (DOD) when i was a teenager. Im not nearly as educated as a post-grad of the sciences (esp. Biology) but i like to help when i can lol

At a certain point you basically just give up trying to learn the harder stuff. I guess i just never fully developed my knowledge because i felt i could never become as intelligent as a double or triple doctorate. Very discouraging to work with brilliant minds you can not compete with. :shrug:

So now i engulf myself with my studies and learn at my own pace, learn what i want, and try to stay out of the spotlight so i dont end up in prison. Its a sad time we live in where a man cant start a project like radioactive tracing of a biosynthesis pathway or make a firecracker without getting in trouble :facepalm:



If you ever need a second opinion from someone semi-educated :lol: PM me!


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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: Toadstool5]
    #22004435 - 07/27/15 01:02 PM (8 years, 7 months ago)

I have made protoplasts more times than I can count (academic work in fungal genetics). Our lab used to use an enzyme mix called Novozyme 234. After it became unavailable we moved to something called Glucanex (The same stuff as you have linked from Sigma up there with a different brand name on the jar). Lately we have been using something called Vinotaste, which was designed for use by the wine making industry to clarify and artificially age wines. There is also something called Zymolase that i've seen mentioned but never had the opportunity to use myself.

Vinotaste is relatively cheap ( http://www.dwinesupplies.com/dws2014/itemDetails.asp?catID=15&child=15&pid=3501 ) but requires a different procedure than most. Its a bit... gentler than other lytic enzymes, and can be touchy about its working conditions. Unfortunately I don't have the updated lab protocol we used for the stuff (the tech in charge of that project is out of town for summer) but I can see what I can dig up if youd like.


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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: matsc]
    #22007623 - 07/28/15 12:24 AM (8 years, 7 months ago)

Awesome - yeah that Novozyme 234 stuff is what I ran into at every turn. It seemed crazy that they stopped making it because all I could find were people asking where to get it.

Called up about the Zymolase - it's only available in minimum order of 1000 units(although a unit is quite small) and is about 150USD. I think I'm probably going to bite the bullet and go Sigma, although I am interested to hear how difficult the wine stuff is to use.

I'll put a query up on Alibaba and see in anyone has a decent product.

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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: Feelers]
    #22007635 - 07/28/15 12:29 AM (8 years, 7 months ago)

150 just seems to be the magic number  :wizard:


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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: Toadstool5]
    #22007719 - 07/28/15 01:07 AM (8 years, 7 months ago)

Most folks using Vinotaste are working with Aspergillus, but there are a few mentions of it being used by others. As such most protocols are designed around Aspergillus, so you would have to experiment a bit to figure out optimal buffer conditions and timing.

That said, if you dont care about max efficiency, it appears to produce at least SOME protoplasts regardless of what you throw at it. You may get lousy yield, or a ton of bursting, but any reasonable conditions and time seems to be fair game.


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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: matsc]
    #22008035 - 07/28/15 05:05 AM (8 years, 7 months ago)

Buffers and osmotic stabilizers are something I've never had to work out, normally you just follow directions. Are you able to give me a basic idea about the interplay among the generally used reagents?

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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: Feelers]
    #22008504 - 07/28/15 09:00 AM (8 years, 7 months ago)

https://books.google.com/books?id=IlO7BQAAQBAJ&pg=PA142&lpg=PA142&dq=vinotaste+protoplast&source=bl&ots=7GXw5QPZNA&sig=AM1CfkNZEfK81vdjZ9U1cCLYaIM&hl=en&sa=X&ved=0CEsQ6AEwBmoVChMI2vXyg4n-xgIVC5ANCh1eHgww#v=onepage&q=vinotaste%20protoplast&f=false
This paper uses 100mg/mL of enzyme in MgSO4 and phosphate buffer.

http://www.buuconference.buu.ac.th/buureg/getfile.php?file=file/BUU2015/paper63.pdf
This paper uses 25-50mg/mL in 1M NaCl

http://www.amb-express.com/content/pdf/s13568-014-0054-7.pdf
Mag sulfate and phosphate buffer again, but this time at 60mg/mL

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3703436/
Uses a final conc. of 65mg/mL in KCl and citrate buffer.

There are other protocols but those were the first few to pop up in a search.


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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: matsc]
    #22009079 - 07/28/15 11:32 AM (8 years, 7 months ago)

http://what-when-how.com/molecular-biology/protoplasts-molecular-biology/

Quote:

Protoplasts are osmotically sensitive, as in a suspension medium hypotonic to that of its protoplasm, water enters the cells by osmosis. This inflow is prevented in walled cells by the rigidity of the cell wall. It has been calculated (1) that the hydrostatic pressure of the cytoplasm of bacterial cells suspended in a dilute aqueous medium may be as much as 30 atm. In intact cells, this is contained by the inward pressure of the wall. In contrast, protoplasts transferred to a hypotonic medium swell and burst. It is consequently necessary for them to be suspended in an iso-osmotic or hypertonic medium for the protoplasts to survive. In iso-osmotic conditions, protoplasts assume a spherical shape. It is often difficult to be certain that a cell wall has been totally removed because cells become osmotically sensitive and adopt a spherical shape before the cell wall is completely removed, so neither of these can be regarded as an adequate indicator of protoplast formation. Cells from which the cell wall has been incompletely removed are referred to as spheroplasts. When it is not certain whether protoplasts or spheroplasts are being used, these are often referred to as osmotically fragile cells.




With osmotic stabilizers there must be a way to estimate how much potential is needed to create protoplasts without trial and error but it is beyond my comprehension. You could simply try different concentrations and observe under microscope if the cells are bursting but i would start very hypertonic and slowly scale back until you see them start to burst. Basically titrate the cultures if possible or find out a way to calculate the osmotic potential of the cytoplasm then match that or make it slightly more concentrated in the media you are using.

Quote:

Protoplast production was generally greater
when 0.5 M MgSO, was used as the osmotic stabilizer, although the differences between that
stabilizer and 0.5 M mannitol were generally
small (Fig. 2). Both stabilizers are used for re-
leasing basidiomycete protoplasts; MgSO, per-
haps more commonly (Gold et al. 1983; Tro-
janowski et al. 1985). Increasing the
concentration of MgSO, from 0.3 M to 0.5 M
resulted in increased release of protoplasts with
P. chrysosporium, G. trabeum, and T. versi-
color, but release declined at 0.6 and 0.7 M
(Fig. 1B). P. placenta appeared least affected
by pH, but only low levels of protoplasts of
this species were released at any pH level test-
ed. Protoplast production declined in all test
fungi when 0.7 M MgSO, was used as the os-
motic stabilizer. The pH of the maleic-NaOH
buffer also appeared to influence protoplast
generation with G. trabeum. Optimum num-
bers of protoplasts released appeared at pH
5.5-6 and declined on either side of that range
(Fig. 1C).




Quote:

PRODUCTION OF FUNGAL PROTOPLASTS FROM SELECTED WOOD-DEGRADING FUNGI by Chen Rui and Jeffrey Morrell




Seems like you should buffer to the most appropriate pH for the bacteria, yeast, fungi that you are working with. Chances are you will want to buffer at 5.5 and magsulfate or mannitol are your best bet because they buffer very well, are cheap, and easily digestable by most microbes.

Quote:

or a ton of bursting,




Like matsc said, you can use almost anything but will suffer conversion yields and bursting of the converted. Thats why i wouldnt suggest low-quality lysins, i have seen a few papers mention impurities in commercial batches leading to bursting or even no reaction at all.

If you want to innovate you have to research as much as you can and then experiment. Without a procedure to follow you basically have to just figure it out and try to not burn through all your resources at the same time. :shrug:

I dont know if you know this but forming protoplasts is beyond most peoples' skills :lol: its going to be a challenge until you have your own procedure and experience. Seriously, matsc is probably the only guy on the shroomery that knows that much about biology :thumbup:

On a side note,

Remember that anything that dissociates in the media will also contribute to the overall osmotic stability. That may account for changes in which buffer is used, how much stabilizer is used, and how nutritious the media is.

A modified mannitol salt agar might work. Use 1M for salinity, add extra mag sulfate if you want to, and your enzyme mixture.



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Edited by Toadstool5 (07/28/15 11:41 AM)

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OfflineMrcloudy
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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: Toadstool5]
    #22010250 - 07/28/15 03:12 PM (8 years, 7 months ago)

Chitinase can be found in Puffball mushrooms, specifically Lycoperdon sp. I am not sure to what degree.


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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: Feelers]
    #22012568 - 07/28/15 10:46 PM (8 years, 7 months ago)

That's good about the basic reagents. Cheap is good! :cool:

I rang Sigma, they said they only sell to institutions and to businesses, but said that I can ask their customer service team specifically for an exception via email. Pain in the ass though.

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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: Feelers]
    #22012788 - 07/29/15 12:10 AM (8 years, 7 months ago)

Quote:

Pain in the ass




Synonymous with sigma-ald, they are a nightmare on a good day. Seriously the worse customer service on the planet and red tape, red tape, photo ID faxes, finger prints, signature after signature, just for something like potassium hydroxide :facepalm:


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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: Toadstool5]
    #22012790 - 07/29/15 12:12 AM (8 years, 7 months ago)

They arent much nicer from an academic stand point. I did a price quote of one thing for my lab, and the very next day I was getting phone calls from sales people. They have people who literally come and visit you in lab trying to upsell you on stuff. Argh leave me alone I just wanted to see how much primers cost!


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Re: Where to get cheap Lytic Enzyme used to produce Protoplasts? [Re: matsc]
    #22013104 - 07/29/15 02:25 AM (8 years, 7 months ago)

I have found a small suppler in Australia that can make purchases from Sigma Aldrich, which is handy. I've been fishing on Alibaba and it seems like no one makes this specific type of enzyme, although they have very similar mixes.

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