# Enzymatic cleaner question



## Wladziu (Mar 4, 2010)

I'm gonna try this forum thing once more. 
Be nice, please. 
I'm not so great at trusting people. Pretend I'm an abused stray or something.



My interest in organic chemistry leads me to ask the question:
*Will warming enzymatic cleaner increase its effectiveness? *And, if so... to what extent and at what temperature? 


I don't have a bottle in front of me, so I can't read the label. 
I'm assuming that brands available to the average consumer have true enzymatic function.
If that's so, then there's an optimal temperature range at which they operate. Also, there may be a key temperature that accelerates their function without denaturing the effective compounds. 



Please pardon me if this seems odd. I'm weird. 
Lately, I've been wondering exactly how the polyvinyllic compounds in carpet accept stains. 
I understand how phenols (like in grape juice) add across the double bonds, and that they're present in poo. Haven't yet looked into urea or others. 
But, I'm interested in the science of it. 
Does alkene reduction lead to stain rejection? If so, won't enthalpy have an effect... and certainly in the biological extent with enzymatic function? 

Any other chemistry nuts out there? 
Or anyone with a stained carpet, a stove, and some time on their hands?


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## clickntreat (Feb 19, 2010)

Wow well I don't have an answer for you, but if anybody actually knows the answer to that question I'd love to hear it!


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## Wladziu (Mar 4, 2010)

Well, I picked up a bottle of Nature's Finest (or whatever it's called).

It looks to have actual enzymatic function, not just pH balance restoration or something. Unless the 30 minute (maybe 1 hr, can't remember) wait period is simply consumer misdirection. 
I never wanna trust the labels that those wacky consumer scientists dream up. 

Haven't yet had a chance to test it. 
Millie's not taking food yet, after her spaying. Only small amounts of water so as not to induce peristalsis and thermal shock which both lead to post-care vomiting. Just my opinion, but it works.


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## Wladziu (Mar 4, 2010)

Sorry for the double post!

Just remembered: This brand contains isopropyl. Unfortunately, unless it's a pretty rugged protein that I don't yet understand... such a non-polar, protic solvent would break down the beta structure. 

Simply put: enzymes are proteins, and alcohol breaks down proteins. Especially in such high concentration. 


I'm wondering how it removes stains, though...
The alcohol could simply be acting as a solvent with the organics on the carpet. Urea protein hydrolysis. Would also take about half an hour. 
It'd certainly be easier to ship in non-refrigerated containers. And, it'd explain the lack of expiration date.





Bottom line, though:
Unless I'm mistaken, this "enzymatic cleaner" could be simply a $12 bottle of isopropyl alcohol with a fancy label and squirt trigger. 
I'll try to find a biological "litmus test" today. Gonna be fun. I haven't done one of those before!

Just need a test for nitrogen.
If it tests positive, that'll be a good enough indicator of either enzyme presence or their constituent amino acids.


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## BaileysMama (Mar 5, 2010)

Wow..thats seriously deep thought, I just buy the pet stain and odor formula by bissell =) 

You lost me at enzyme lol. Interesting read GL with your results ill be watching this one to see what you come up with.

Lol my bissell formula is over 20 bux a bottle.


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## Spitzy (Oct 13, 2007)

Indeed, optimal temperature would likely depend on the particular enzyme and its organism of origin. It seems possible that archaea, including thermophiles, would be looked to as a source of enzymes as their proteins' structures are often more rigid/stable due to their environments. They are certainly taken advantage of for other commercial industrial and biotechnological uses.

However, although a higher temperature may be optimal for the enzyme, it may also destroy whatever sequestering/protective system may be in place to preserve the protein in its native state.

I remain skeptical of the enzyme(s) activity in a cleaner such as Nature's Miracle, though there certainly seems to be a broad industry of "enzyme cleaners" of various sorts and surely there've been studies done in the R&D departments of companies to optimize effectiveness. The work doesn't seem to be particularly accessible, which is unsurprising since it would be in the companies interest to keep their results as trade secrets and/or patent them.

Section 7 and 8 of this patent was about the best review of the history of enzymatic cleaners that I pulled up in a quick search. It speaks of enzyme stability issues, and mentions tactics that prior art has used to address the problem.

Anyway, good luck on your home experiments!


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## Wladziu (Mar 4, 2010)

*BaileysMama, *
Enzymes are like...wrenches. They pull apart chemicals in certain places, but the wrench has to fit the bolt. Certain sizes for certain bolts / certain enzymes for certain chemicals. 
If you dissolve the wrench in acid, the metal is still there... but it can't unscrew the bolt anymore. Alcohol is like acid to enzymes. Changes their shape, and they're useless. The damage is irreversible. When the alcohol evaporates, the enzymes are still "dead". 
Nature's Miracle is filled to the brim with simple rubbing alcohol. 


The reason your dog can't smell the pee anymore: urea (the thing they smell in pee) is broken apart by alcohol, too. 
These magical "proteins" that dogs smell in pee... also broken down by alcohol.

Nature's Miracle is charging you $12 for a $2 bottle of simple rubbing alcohol that you can buy at the drug store in much greater quantities. 
It just won't be lemon scented.

I've got access to a lab that can prove it, with a single test. 




Spitzy, 
I thought archea, also, originally. Thermophile derived nitrases and such as used for DNA extraction.
I used them in my fellowship last year, but they always had expiration dates and needed constant refridgeration. 
They were also extremely expensive. 
Very hard to get. Remember, they only come from deep sea thermal vents. The lab bred stuff is just too difficult and financially inefficient to mass produce. 
I'm not sure if hydroxiphiles even exist. Only salino- and various thermophiles (on either end of the mercury). Oh, and the sulfurs in that lake in Yellowstone. 

Hell, urea itself will denature these magical "proteins" they keep harping on.


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## BaileysMama (Mar 5, 2010)

So I can basically go buy rubbing alcohol, at teh dollar store and pour it onto pee spots and the will kill the stuff the dog can smell? That is pretty pretty cool I just went and bought that Natures balance stuff whatever its called.. Lol waste of money. 

Unless ive misunderstood something.


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## Wladziu (Mar 4, 2010)

The other post on this stuff mentions it's ability to wash away ammonia smell.

Yeah, alcohol tends to do that. 

Ammonia: NH3
"Business end" of alcohol: -OH

The -OH binds to one of the H's on ammonia, makes water and NH2-. Much more stable. Washes right away. No more smell or white crusty stuff. Magic. 

Simple acid-base reaction, taught in high schools across the country.


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## Wladziu (Mar 4, 2010)

BaileysMama said:


> So I can basically go buy rubbing alcohol, at teh dollar store and pour it onto pee spots and the will kill the stuff the dog can smell? That is pretty pretty cool I just went and bought that Natures balance stuff whatever its called.. Lol waste of money.
> 
> Unless ive misunderstood something.



You understand perfectly.

It just won't smell like lemon.


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## Jax (Feb 14, 2010)

Is there something that is keeping it from eating the surfaces or fading color though?

Rubbing alcohol is notorious for taking out color (people use it for dye stains) as well as the finish. Rubbing alcohol poured straight on my hardwood floors = not so good for the finish. This stuff doesn't seem to bother them, or hurt the rugs (which I'm afraid to pour rubbing alcohol on it to try it out, lol).

Science isn't my thing, so I don't know. I also really dislike the smell of straight rubbing alcohol, myself. It infiltrates everything and seems to even get in my taste buds. yuck.


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## Wladziu (Mar 4, 2010)

Contents:
*(Verbatim from the bottle)*
Water
Nature's Enzymes
Isopropyl Alcohol
Natural Citrus Scent


The alcohol's watered down. Keeps it from damaging floors.

Still works for proteins, just not enough of it to damage the cellulose in wood or the vinyll in carpet.
Cellulose and plastics are much more stable than proteins. 
Watered down, it's too weak harm something so relatively stable.



But, you're exactly right. It's a solvent: breaks down biological components like gasoline breaks down styrofoam. 
Water itself is a biological solvent, in fact.
Ask a wood floor owner. 
Or, keep a piece of rawhide in a glass for a while. 
Same process. Rawhide's also a protein, just like those "enzymes". 
How long have these bottles been in storage/transit? 


This stuff is junk in my opinion. And, I'm gonna prove it, just as soon as I get access to the NMR scanner.
A few cap-fulls of rubbing alcohol in some water with a few drops of lemon juice in a spray bottle...
I can make that in my kitchen.


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## Wladziu (Mar 4, 2010)

I keep double posting! So sorry! 
Hate to annoy anyone actually reading this.



Look, you don't have to listen to me.
Try it yourself.

Pour a little of it in a cup with a raw egg white. Egg white's mainly casein protein. 
Then pour some watered-down alcohol into another cup with another egg white. 
If they both turn white and get kinda thick... like when the stove isn't hot enough... there's your answer. 


I do welcome any and all contrary arguments, of course. 
I highly respect the peer review process. 
Anyone with internet access is welcome to prove me wrong.




Oh, one thing I forgot:
By law, the components of any consumer product must be listed in the order of their component percentage. 
Water's first, showing how dilute the stuff is.
Enzymes are next, but it could be a few CCs of spit for all we know. (I intend to find out, next week.)
Alcohol's next. 
Then, a few drops of lemon juice.


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## Spitzy (Oct 13, 2007)

Wladziu said:


> The lab bred stuff is just too difficult and financially inefficient to mass produce.


Pft - I've had multiple co-workers grow up large batches of Taq polymerase. It is perhaps not cheap to buy, but it seems to me that's more a business/patent issue.




Wladziu said:


> The -OH binds to one of the H's on ammonia, makes water and NH2-. Much more stable.


Uh... NH2- more stable?

You say you're into organic chem... ok. You don't seem to have a very deep understanding of biochemistry. And now I'm wondering about the extent of your org chem knowledge based on the above. It seems as if you've perhaps taken undergraduate courses in these subjects?

You are making claims and assertions that it does not seem like you have the knowledge to make. I think you are doing a disservice to this forum to strongly assert Nature's Miracle and the like are equivalent to:


Wladziu said:


> A few cap-fulls of rubbing alcohol in some water with a few drops of lemon juice in a spray bottle...


I'm not sure what you think you're going to accomplish using NMR. If there are any significantly complex organic molecules, much less large polypeptides, I really question if you have the experience to deconvolute the spectra and assign structure. I can only suppose you think you might find nothing but water and simple alcohol molecules. 

It is cool that you are questioning and experimenting on your own. Certainly, particularly as a scientist, inquisitiveness and skepticism are very important.

I sympathize that it is difficult to communicate science to a broader audience. In general, the public is _not_ good at respecting the qualifiers and skepticism that is so necessary to do good science. In general, it seems people seem to respond to whatever is stated the loudest and with most conviction, regardless of the (lack of) quality of the work and knowledge behind the statements.

But I think here you are falling to far towards the 'loud & w/ conviction', when you just don't know enough to make the statements you are making. I'd like to think you actually know you don't know. Knowing you don't know is very important. (disclaimer: the author of the article is rather free with curse words, but I think the underlying message is very valid.) But right now, you're coming off like you are trying to present yourself as an authority on these subjects, and I think it is misleading and irresponsible to do so.


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## Wladziu (Mar 4, 2010)

That's me!

Irresponsible and misleading. Disservice to the community. 
Awesome.
Damn, I love people. Try to help, and they call you an idiot. 


Reversed a conjugate base, oops.
Back to digging ditches. 
Alcohol's not a solvent, because Nature's Miracle says so. 
And, some guy on the internet will nerd-fight with you instead of helping.


Any other key points you wanna toss in, while you're not helping? 
-I'm too dumb to analyze an NMR (Yeah... you just don't do that. That's like pissing on somebody's 
birthday cake.)
-My chief geneticist's advise on polymerase is wrong, despite her NIH/USDA grant using the stuff 
(Codon marker identification of propensity toward California mastitis in goat/bovine mammaries)
-The public is too stupid to care about the science
-I'm dumb, but I don't know it 
-I'm too arrogant to admit it, and want everyone to believe otherwise
-Oh, and my education is sub-par


Congrats!
You win the nerd fight. 


I've questioned business ethics and tried to find a better/cheaper solution......
Damn right I'm loud and have conviction. It makes me angry. 
At least I'm doing something.
What are you doing, Mr. High and Mighty?

Tell me how an enzyme can exist in isoproponal in a 120 degree shipping container, and you can tell me who's doing a disservice to the community.


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## lalaperson (Mar 3, 2010)

people were mean to you on a forum? that sucks!  i did not know anything about chem. i fail it twice! once in high school and again last year.  i still have to take three more!


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## Spitzy (Oct 13, 2007)

Wladziu said:


> Tell me how an enzyme can exist in isoproponal in a 120 degree shipping container




Renu Tyagi and Munishwar N. Gupta. Stability of enzymes in water/organic co-solvent mixtures. (1998) _Biotechnology Techniques_ *12*(7), 569–570 [Link]


> The data given in Table 2 shows that in general enzymes can withstand exposure to significant level of organic cosolvents provided the co-solvents have polarity index of 5.8 and above or are alcohols.


Solvents included isopropanol (aka isopropyl alcohol)


Donn N Rubingh. Protein engineering from a bioindustrial point of view. (1997) _Current Opinion in Biotechnology_ *8*, 417-422 [Link]


> An understanding of the relationship between basic properties and structure is far from complete. *Nevertheless, improving protein thermostability is becoming relatively routine.* This is because earlier investigations have identified a number of structural features influencing thermostability, so that mutations with a high probability of success are easy to identify and to test. Improving enzyme activity is more difficult, since the structural parameters affecting activity are not as clearly defined. This is particularly true where substrates are macromolecular and insoluble, as they are for many of the important industrial enzymes.


(empahsis mine)


John W. Shield, Holly D. Ferguson, Andreas S. Bommarius, and T. Alan Hatton. Enzymes in Reversed Micelles as Catalysts for Organic-Phase
Synthesis Reactions. (1986) _Ind. Eng. Chem. Fundam._ *25*, 603-612 [Link]


> Specifically, we are concerned with reactions in an organic medium where the enzyme is in solution, protected from the apolar medium by being sequestered within the polar cores of reversed micelles. We begin by discussing the more general question of why organic solvents may be advantageous for the reactions of interest and follow with a description of reversed micelle systems and a historical perspective on reversed micellar catalysis.
> [...]
> Additionally, Klibanov reported that the thermostability of enzymes may be substantially improved when the enzyme is not fully hydrated, as is often the case in organic solvents (113).
> [...]
> Some enzymes might demonstrate increased stability when solubilized within reversed micelles.



These were just the first articles that came up when I searched the first three things that came to my mind that you were failing to take into account: suitability of alcohol solvents, protein engineering, and micelle engineering. You will note that all of these papers are over a decade old - in other words, expect that there have been significant advances since they were published. And _even then_, thermostability was an issue pretty well under control.

As I alluded to earlier, recent findings and data such as the composition of "nature's enzymes" is likely to be *proprietary *information thus it would be very difficult to specifically address why this particular product is capable of performing as it claims. However, foundation research such as that cited above alludes to how such properties may have been engineered.

Industrial application of biochemistry is a huge field. (And one in which an ag scientist studying mastitis, who like thousands of others implements the very common PCR experimental technique, is unlikely to be an expert.) Protein engineering in particular is a hot field and there's been a lot of progress in the past decade or so. Your replies so far haven't really shown any appreciation or awareness of this. 


I am not attempting to be "mean". If you choose to feel angry and insulted, then there's not really anything I can do about that. I do not know you and I do not know the full extent of your experience and knowledge - I believe I have accounted for that in my responses. Note my liberal use qualifiers such as "seem(s)".

I would hope that instead of reacting negatively, you would take the opportunity to elaborate on and clarify your previous statements in the case that you do have deeper knowledge than was first apparent, or if you do not have that knowledge to remain open minded and attempt to learn something new. (Did you even look at the text of the patent which I previously linked, speaking to the experimental history of enzyme cleaners?) I can only respond to what has been written - and in my opinion what you have written has edged towards misinformation.


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## Wladziu (Mar 4, 2010)

I see.
Very nice. Didn't see the co-solvent involvement with quarternary polarity sheilding at all.
I yield the floor.


I'm no protein cowboy, just a dabbler with honest intent. 
I remain distrustful of mysterious "proprietary" contents that could enable price hiking, but recognize room for growth from my end, as well.
PCR is indeed only as valuable as a calculator. I'm biting at the bit for more interesting work.


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