Wednesday, July 11, 2012

Process Wednesday: Filtering out an enzyme? Whoa.

Kilomentor has an interesting post on the practicalities of running an enzymatic process (based on a process chemistry conference talk by Dr. Jacob Janey of BMS) -- specifically, now that you have an aqueous/organic slurry of product and enzyme, how the heck do you get the product out?:
The method applies to an enzymatic transformation that has been conducted in water with 2-50% of an organic cosolvent such as methanol, DMSO or isopropyl alcohol (IPA). First, the pH is adjusted (if necessary) so that the substrate will be extractable into an essentially organic layer when it is introduced. Then, 0.5 relative volumes of alcohol (IPA or MeOH) is added. Then 0.5 relative volumes of isopropyl acetate or methyl t-butyl ether is added. The order of addition of these liquids is noted to be crucial. Then moderate agitation is employed to cause phase mixing to even out the concentration of liquids in between the  physical layers and then allow phase separation. The goal here is to achieve a 1:1:1 v/v/v mixture of water, alcohol and aprotic organic before the two layers separate. The bottom, more essentially organic,*  layer is then backwashed with 0.5 relative volumes of 30:70 v/v  alcohol/aprotic organic. The upper, more essentially organic wash is combined with the first upper, essentially organic layer and after being combined together they are in turn washed with water. 
The product is to be found in this wet combined alcohol/aprotic organic layer. The advantages of this method claimed are that it is scaleable, gives reliable easily visible quick separating cuts, and is reliable and adjustable to the particular process by adding more or less alcohol as needed to give best separation of the layers. I would judge this makes it the most general and scalable isolation procedure available. The admitted disadvantages are that significant product losses arise for relatively more polar substrate molecules. These are held more strongly in the water rich layer and so are lost to a greater degree. The method is of necessity more volume intensive and can be more lengthy since more additions are made to achieve superior layer separation.
I suspect the most difficult part of this procedure would be adjusting the relative ratios of solvent -- too much alcohol would probably not be a good thing. "Reliable easily visible quick separating cuts" are easy to come by in the lab, but when you're staring at a 3 inch sight glass in the middle of the night, you want your separations to be as foolproof as possible. 

*Less organic? See comment from Anon071120120753a. 


  1. Shouldn't that be the bottom, more essentially aqueous layer? They can't both be organic!

    1. Yeah, I think you're right. All the solvents mentioned (w/exception of DMSO) have densities less than 1.

  2. One thing that is lovely on scale are crystallized and crosslinked enzyme preparations - the highly purified enzyme is crystallized into macroscopic crystals that are then crosslinked with stuff like glutaraldehyde, which makes the crystals water-insoluble but keeps the enzymatic activity and makes the crosslinked enzyme more resistant to denaturation (protein crystals have ridiculous quantities of water, something like 60-70% of water by weight and are flexible enough that small molecules can diffuse through them). I think there are companies selling these crosslinked enzyme crstals on industrial scale