querjek
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Use of lipase enzyme for ester hydrolysis?
(sorry if this is in the wrong forum; I wasn't entirely sure where to put it)
A hobby chemical supplier who rarely/never responds to email (but who does reliably send out orders) lists "lipase" as a product. According to
Wikipedia, the various lipases are used to hydrolyze esters.
I'm interested in attempting an ester hydrolysis with this, but have no idea where to start so far as calculating amounts necessary are concerned.
Even a hint in the right direction would be greatly appreciated--since "lipase" is a relatively broad term, Google hasn't gotten me anywhere as far as
use in the lab is concerned.
Thanks!
it's all about chemistry.
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solo
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this may be of interest..........solo
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Enzyme catalyzed hydrolysis of esters using reversibly soluble polymer conjugated lipases
CHARUSHEELA Amritkar ; ARVIND Lali
Enzyme and microbial technology 2002, vol. 30, no1, pp. 19-25
Abstract
A novel form of lipase covalently 'immobilized' on reversibly soluble polymers is proposed as a reusable form of enzyme that is free from steric and
diffusion limitations associated with enzymes immobilized on porous solid supports. Lipase (from Novo Nordisk's Lipolase 100T) was conjugated onto two
water soluble polymers viz. Eudragit S100, a copolymer of methylmethacrylate and methacrylic acid from Rohm GmbH; and carboxymethyl cellulose (CMC), a
readily available semi-synthetic polymer. Lipase 'immobilized' on Eudragit (E-lipase) exhibited reversible solubility in water, and was insoluble
below pH 5.0 and soluble above pH 5.5. CMC-lipase could be reversibly precipitated from aqueous solutions using combination of 7% w/w polyethylene
glycol-4000 and 50 mM Ca2+. The prepared E-lipase could completely hydrolyze 20% w/v olive oil in isooctane at ∼35°C, at an enzyme
concentration of 12.5 U/ml, and an organic:aqueous phase ratio of one. The enzyme preparation was stable under stirred conditions, and could be reused
multiple times without loss of enzyme activity. It was found that the nature of the polymer used for enzyme conjugation had a significant effect on
the activity of the lipase used. E-lipase showed increased specificity for water insoluble substrates, while CMC-lipase showed increased specificity
for soluble substrates. Thus, E-lipase was found more than four times active than free lipase for hydrolysis of olive oil. CMC-lipase on the other
hand, catalyzed hydrolysis of water soluble p-nitrophenyl acetate at a rate more than four times over free lipase.
[Edited on 17-9-2008 by solo]
Attachment: Enzyme catalyzed hydrolysis of esters using reversibly soluble polymer conjugated lipases.pdf (89kB)
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It's better to die on your feet, than live on your knees....Emiliano Zapata.
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chemoleo
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Ok, lipases are like most enzymes proteins, and most proteins are only happy under physiological conditions. This means, your lipase solution will
have to be in something like 50 mM potassium phosphate, 100 mM NaCl, pH 7.5. I'm not sure how well it would work with small esters such as
ethylacetate - as lipases have evolved to attack the glyceride variety (of course there are many other ones, but this is the main one) - and
glycerides are >C6 fatty acids esterified to glycerol, as a mono, di or tri ester. These are of course oils - olive oil as mentioned in the ref
above. So mixing is the next factor - you need to form a good emulsion, or best, add some solvent that stabilises triglycerides as micelles, sheets,
and so on but leaves the activity of the lipase intact. I'd use an emulsifier to start of with (such as lecithin, available in food stores).
Also, regarding enzyme amount- generally the reaction rate is a direct function of the enzyme concentration, as long as you remove the product formed.
So the more the better, i.e. 10 micromolar enzyme concentration would be a decent amount of enzyme (you can calculate molarity if you assume a
molecular mass of i.e. 30000 Dalton). I.e. 1 g/ litre should be more than enough for any enzyme.
Doing the reaction at 30 something deg C is probably also a good idea.
Most likely the lipase you bought will be engineered for high stability, solubility and substrate specificity, so I wouldn't worry too much.
Do report back with your findings!
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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starman
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A search of US patent office is good place to start.There has been a lot of recent work with these compounds,particularly with triglycerides as
mentioned earlier in the thread.
It also a good idea to know the origin(fungal,bacterial,pancreatic etc) and activity,normally given as lipase units (l/u).Some compounds are more
suited to specific reactions than others.Note also that some water is always required(usually added as phosphate buffer as already mentioned) for the
lipase to effect hydolysis.
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