one hand, the enzyme depends on water for the majority of the noncovalent interactions – salt bridges and hydrogen bonding – that help to maintain its catalytically
active conformation [15] but water also participates in most of the reactions which
lead to denaturation. As a consequence, it may be anticipated that replacing some (but
not all) of the water with an organic solvent would retain enzymatic activity. Hence,
it is conceivable that completely anhydrous solvents are incapable of supporting
enzymatic activity because some water is always necessary for catalysis. The crucial
answer to the question concerning how much water is required to retain catalytic
activity is enzyme-dependent [16]. For example, α-chymotrypsin needs only 50 molecules of water per enzyme molecule to remain catalytically active [17], which is
much less than is needed to form a monolayer of water around the enzyme. Other
enzymes, like subtilisin and various lipases are similar in their need for trace
quantities of water [18]. In other cases, however, much more water is required.
Polyphenol oxidase, for instance, prefers a rather ‘wet’ environment and requires
the presence of about 3.5 Â 10
7 molecules of water [19].
The water present in a biological system can be separated into two physically
distinct categories [20–22]. Whereas the majority of the water (>98%) serves as a
true solvent (‘bulk water’), a small fraction of it is tightly bound to the enzyme’s
surface (‘bound water’). The physical state of bound water – as monitored
by differences in melting point, heat capacity, EPR- and IR-spectroscopical properties – is clearly distinct from the bulk water and it should be regarded as a crucial
integral part of the enzyme’s structure rather than as adventitious residual solvent.
Thus, bound water is also often referred to as ‘structural water’. For a picture
displaying the crystal structure of Candida antarctica lipase B with (and without)
structural water molecules see Sect. 1.4.1, Fig. 1.1. If one extends this concept to
enzymatic catalysis in organic media, it should be possible to replace the bulk water
by an organic solvent without significant alteration of the enzyme’s environment, as
long as the structural water remains unaffected.
Biocatalytic transformations performed in organic media offer the following
advantages:
• The overall yields of processes performed in organic media are usually better
due to the omission of an extractive step during work-up. Thus, the loss-causing
formation of emulsions can be avoided and the recovery of product(s) is facilitated by the use of low-boiling organic solvents.
• Nonpolar substrates are transformed at better rates due to their increased
solubility [23].
• Since an organic medium is a hostile environment for living cells, microbial
contamination is negligible. This is particularly important for reactions on an
industrial scale, where maintaining sterility may be a serious problem.
• Deactivation and/or inhibition of the enzyme caused by lipophilic substrates
and/or products is minimized since their enhanced solubility in the organic
medium leads to a reduced local concentration at the enzyme’s surface.
• Many side-reactions such as hydrolysis of labile groups (e.g., epoxides, acid
anhydrides [24]), polymerization of quinones [19], racemization of
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3 Special Techniques
active conformation [15] but water also participates in most of the reactions which
lead to denaturation. As a consequence, it may be anticipated that replacing some (but
not all) of the water with an organic solvent would retain enzymatic activity. Hence,
it is conceivable that completely anhydrous solvents are incapable of supporting
enzymatic activity because some water is always necessary for catalysis. The crucial
answer to the question concerning how much water is required to retain catalytic
activity is enzyme-dependent [16]. For example, α-chymotrypsin needs only 50 molecules of water per enzyme molecule to remain catalytically active [17], which is
much less than is needed to form a monolayer of water around the enzyme. Other
enzymes, like subtilisin and various lipases are similar in their need for trace
quantities of water [18]. In other cases, however, much more water is required.
Polyphenol oxidase, for instance, prefers a rather ‘wet’ environment and requires
the presence of about 3.5 Â 10
7 molecules of water [19].
The water present in a biological system can be separated into two physically
distinct categories [20–22]. Whereas the majority of the water (>98%) serves as a
true solvent (‘bulk water’), a small fraction of it is tightly bound to the enzyme’s
surface (‘bound water’). The physical state of bound water – as monitored
by differences in melting point, heat capacity, EPR- and IR-spectroscopical properties – is clearly distinct from the bulk water and it should be regarded as a crucial
integral part of the enzyme’s structure rather than as adventitious residual solvent.
Thus, bound water is also often referred to as ‘structural water’. For a picture
displaying the crystal structure of Candida antarctica lipase B with (and without)
structural water molecules see Sect. 1.4.1, Fig. 1.1. If one extends this concept to
enzymatic catalysis in organic media, it should be possible to replace the bulk water
by an organic solvent without significant alteration of the enzyme’s environment, as
long as the structural water remains unaffected.
Biocatalytic transformations performed in organic media offer the following
advantages:
• The overall yields of processes performed in organic media are usually better
due to the omission of an extractive step during work-up. Thus, the loss-causing
formation of emulsions can be avoided and the recovery of product(s) is facilitated by the use of low-boiling organic solvents.
• Nonpolar substrates are transformed at better rates due to their increased
solubility [23].
• Since an organic medium is a hostile environment for living cells, microbial
contamination is negligible. This is particularly important for reactions on an
industrial scale, where maintaining sterility may be a serious problem.
• Deactivation and/or inhibition of the enzyme caused by lipophilic substrates
and/or products is minimized since their enhanced solubility in the organic
medium leads to a reduced local concentration at the enzyme’s surface.
• Many side-reactions such as hydrolysis of labile groups (e.g., epoxides, acid
anhydrides [24]), polymerization of quinones [19], racemization of
316
3 Special Techniques