#32 from R&D Innovator Volume 2, Number 4          April 1993

Enzyme Catalysis Without Water
by Alexander M. Klibanov, Ph.D.

Dr. Klibanov is professor of chemistry and a member of the Biotechnology Process Engineering Center at the Massachusetts Institute of Technology.  He has received many awards, including the 1991 International Enzyme Engineering Prize.  He is a member of the National Academy of Engineering.

One of the fastest growing areas of industrial biotechnology is the use of enzymes in non-aqueous media.  Water has been the conventional reaction medium because it is an enzyme's natural habitat.  I can still remember people telling me at scientific meetings, after our first paper on enzymatic catalysis in anhydrous solvents was published in Science  in1984, "Professor Klibanov, you are doing interesting work but, frankly, I don't believe enzymes can function in organic solvents!"

But why bother using enzymes outside their natural habitat?  Because using an enzyme in organic solvents eliminates several obstacles that limit its usefulness in water.  For example, most compounds that interest organic chemists and chemical engineers are insoluble in water, and water often promotes unwanted side reactions.  Furthermore, in many aqueous reactions, only a small amount of product is formed.  Also, because of its high boiling point, water is far from the ideal milieu for product recovery.

Consequently, once it was established that enzymes can work in organic solvents with little or no water, R&D in the area surged.  Numerous applications are being commercialized or developed in the pharmaceutical, food, and specialty chemical industries, and further development is on the horizon. 

Where did our concept originate?  What is the anatomy of its invention?  Despite a few scattered reports (which were either ignored or considered unique cases), the idea of using enzymes in pure organic solvents so flouted the conventional wisdom as to seem absurd.  Therefore several laboratories, including mine, were doing the next best thing—using a solvent with water in biphasic mixtures.  In such mixtures, the water and solvent (such as benzene or ether) remain separate.  The enzymes stay dissolved in the water, since they are insoluble in the solvent.  Substrates added to the organic solvent diffuse into the aqueous phase, undergo enzymatic conversion, and the products diffuse back into the organic phase for recovery. 

Such biphasic systems were useful, but they told us little about how effective a monophasic organic solvent would be by itself.  In 1983, I decided to investigate the relationship of enzymatic activity to water content in biphasic systems.  I expected a threshold phenomenon, and thought the location of the threshold might indicate the minimum amount of water needed by the enzyme.

Suspicious Results

I suggested that my graduate student, Alex Zaks, carry out an enzyme reaction in a biphasic system, starting with a 95:5 mixture of benzene and water, and then reducing the water content.  At what point would the reaction cease?

A few days later, Alex came to my office and said that he'd finally removed all the water, and the enzyme was still catalyzing the reaction!  I was surprised and skeptical and asked him about various controls—he'd done them all and nothing seemed to be skewing the results.  I asked him to repeat the experiment; he grudgingly agreed—and obtained the same result.

Still suspicious, I went to the lab and performed the no-water experiment myself (without telling Alex).  I found the same result—the enzyme worked in an anhydrous organic solvent! 

We were finally convinced that the effect was real, and Alex found the phenomenon working with different solvents, enzymes and substrates.  Not once did he see the threshold of water concentration I anticipated.  I put several other members of my research group on the project, and over the years we've learned to activate other enzymes in organic solvents and have discovered that enzymes exhibit remarkable properties in these solvents.  For instance, we've seen enhanced thermostability, altered selectivity, and the ability to catalyze new reactions.

Furthermore, many of these properties can be profoundly altered simply by switching the organic solvent.  Hence the new "enzyme-solvent engineering" represents an alternative to protein engineering, where enzymes are changed rather than the reaction medium. 

Today, dozens of laboratories throughout the world are working in this area, and hundreds of research papers and dozens of patents have appeared.  No longer does anyone doubt that enzymes can work in organic media.

This Discovery Could Have Been Made Decades Ago

But why did the discovery wait until the mid-1980s?  After all, the need to carry out enzymatic reactions in non-aqueous media has long been recognized.  My reading of the literature reveals a problem with general significance for innovation (or the process of stifling innovation).  Although it is admittedly difficult to reconstruct the reasoning of scientists from years past, the following thought process seems to have dominated in the field of enzyme research:

"It would be nice to use enzymes in pure organic solvents.  But everybody knows enzymes are destroyed in that environment, so let's not waste time doing something ridiculous.  Instead, we'll start by gradually adding a water-miscible solvent, say, ethanol or acetone, to an aqueous solution of an enzyme and see what happens." 

As more organic solvent is added, the enzymatic activity is gradually destroyed; with 50 to 60 percent solvent, virtually no activity is left.  There is clearly no incentive to proceed to 100 percent organic solvent.

This last conclusion sounds plausible, but it's wrong.  The key is this:  Although enzymes work in water (or in water with a very small percent of solvent), they also work in organic solvents with little or no water.  They just don't seem to work in intermediate mixtures. 

This counterintuitive behavior is explained by the fact that enzymes are in their most stable state in pure water.  In pure organic solvent, they "want" to lose their 3-dimensional structure, which is essential for activity.  But they can't because they become too rigid (water, which acts as a molecular lubricant, is absent).  However, in aqueous-organic mixtures, enzymes both want to and can lose their structure—and that produces the inactivation.  So much for logical extrapolation!

As a result, I've started telling students that if it's easy to test a striking but improbable hypothesis, then test it.  If it works, they will be heroes.  If not, they can keep mum and avoid embarrassment.  How many inventions have been missed simply because intelligent, knowledgeable researchers decided to forego "ridiculous" experiments?

To finish the story, I've noticed there's no stopping innovation once the critical barriers are down.  After proving that enzymes work in organic solvents, we discovered that antibodies can do likewise.  This work stimulated us and others to determine other "strange"—but potentially valuable—milieus for enzyme activity.  These include supercritical fluids, whose fundamental characteristics can be markedly altered by pressure, thus affording pressure control of enzyme performance as well. 

Some enzymes need no liquid phase at all—aqueous or otherwise—and catalyze gas-phase reactions.  For example, solid alcohol oxidase can react with, and thus detect, ethanol in the breath or the carcinogen formaldehyde in the air.  Several research groups are now pursuing reactions by whole microbes in organic solvents.  Thus, complex multi-enzyme pathways (naturally occurring in the microbial cell) may produce valuable products like pharmaceuticals.

And all of this stems from an important, albeit humble, experiment that was done for the wrong reason.  We were merely interested in watching an enzyme lose activity and had no inkling that the condition we thought would totally kill the enzyme would actually sustain it. The conventional wisdom is what we already know.  But our job is to find something new.  Perhaps the only thing we deserve credit for is recognizing the significance of an unexpected result and exploring, rather than dismissing, it.