Enzyme

Functions
In Organic Solvents
 ● Shabat Fauzi Abdullah (202051142)

Kel 12 ● Shella Amanda Putri (202051144)

● Septia Ningsih (202051141)
● Sekar Ismawati (202051139)
 Enzyme catalysis in organic solvents is increasingly being used for a wide
variety of applications. Of particular interest are cases in which the media is largely
non-aqueous and contains little water. The display of enzyme activity, even in
anhydrous solvents (water <0.02% vol.), May reflect that the minimum requirement
for water is to form bonds with polar amino acids on the enzyme surface.

The rigidity of the enzyme structure at low water content results in new
substrate specificity, pH memory and possible techniques such as molecular
printing. Limited data suggest that, while improved thermal stability always results,
the optimal temperature for catalysis can occur unchanged. If true in general, this
increased thermostability would have very limited benefits.
 For classical biochemists, it is difficult to visualize the enzymes catalyzing reactions in
the absence of water, that is, in a non-aqueous medium. The addition of organic solvents
is done either to precipitate enzymes or to study denaturation. Thus, the study of the
action of enzymes in organic solvents is a relatively new aspect of enzymes. Singer
(1962) reviews the status of this region and calls it the 'rapidly developing field of
biophysical chemistry'. Whereas we have made limited progress since then in our
understanding of the biophysical chemistry of non-aqueous enzymes, their
biotechnological implications have resulted in the explosive growth of literature
(Linhardt, 1986; Dordick, 1988).
 Low Water Systems
Mixing organic solvents with water causes many interesting variations and possibilities depending
on the solubility of solvents with water and their relative proportions, this review will take a
broader view and look at the total scenario when water content is low. As will be discussed in this
section, even if the amount of water is kept low, it is assumed that in the short term the exact
amount is still critical. Therefore, we need an appropriate method to measure the amount of water
in the system. The Fischer titrimetric method (Zaks and Klibanov, 1988) and gas chromatography
(Reslow et al., 1988) are two easy methods. Recently, membrane inlet mass spectrometry has been
used to measure water activity in organic solvents (Lundstrom et al., 1990). Apart from being able
to adapt to continuous measurements it is claimed to be at least 50 times more sensitive than the
Fischer method.
 Advantages of using enzymes in low water systems

• When the substrate has greater solubility in organic solvents

• Shifting the equilibrium of the reaction in the desired direction such as
the use of hydrolases for synthetic reactions
• Reduces the risk of microbial growth
• Improved thermoslibility
• Enzyme recovery and reuse even without immobikation
• Downstream processing is more energy efficient when the solvent used is
volatile
• Convenient to use 'moisture sensitive' substrates / reagents such as acid
anhydrides
• Possible control for substrate specificity, re-specificity
 Conformation rigidity, 'memory' and 'protein molding'.

Dry enzymes have roughly the same gross conformation as fully hydrated enzymes. Fully hydrated molecules
(based on heat capacity data) have 0.38 g water / g protein (300 moles water / mol lysozyme). This water is barely
sufficient for the formation of a single layer and about half as much as is present in the protein crystals. 
The onset of activity begins as protein molecules approach total mobility. This is in accordance with Karplus
and McCammon (1983) who showed this mobility to be important for enzyme activity. Perhaps in organic solvents,
activity is observed immediately because the hydration corresponding to the first hydration event, i.e. hydration of
the surface charged groups, is complete. It has been shown (Zaks and Klibanov, 1988a) that the main role of water is
to form bonds with functional groups that are present in proteins.
Perhaps the most critical partners are billed groups which, even in the absence of this minimum water, interact
with each other and produce a 'locked' inactive conformation. The added water makes the protein more flexible, as
has been reported during the hydration process. So, at a water level corresponds to about 50 subtylisin or
chymotrypsin molecules / molecules, while active in organic solvents, these enzyme molecules are still very
rigid. Undoubtedly, it is this stiffness that causes some of the interesting consequences observed in anhydrous organic
solvents (water <0.02% vol.).
 Improved thermostability

Another consequence of the stiffness in low water medium is an increase in protein /

enzyme thermostability (Gupta, 1991). This has been observed with various enzymes:
porcine pancreatic lipase (Zaks and Klibanov, 1984), terpene cyclase (Wheeler and
Crotean, 1986), lysozyme (Klibanov and Ahern, 1987), chymotrypsin (Zaks and Klibanov,
1988), mitochondrial cytochrome oxidase. (Ayala et al., 1986) and FATPase (Garza-Ramos
et al., 1989).
The thermostabilization achieved is quite impressive. Lipases remain stable at 100 °
C for hours, so do lysozymes. Such dramatic thermostabilization is rarely possible using
other approaches such as chemical crosslinking (Kamra and Gupta, 1988; Khare and
Gupta, 1990; Rajput and Gupta, 1988), immobilization (Khare and Gupta, 1988) or even
protein engineering ( Nosoh and Sekiguchi, 1988).
Apart from stiffness, another reason for enhanced thermostability is a number of
covalent processes involved in irreversible inactivation such as deamidation, peptide
hydrolysis and cystine decomposition requiring water. This irreversible process is also
very slow in low velocity aqueous media (Gupta, 1991).
 Intermediate Engineering

There appears to be general agreement that enzymes in largely non-aqueous or low-

water environments can function provided there is an essential water layer around
them. they are not stripped (Klibanov, 1986; Laane, 1987; Aldercreutz and Mattiasson,
1987; Khmelnitsky et al., 1988). Thus the golden rule of non-aqueous enzymes (Zaks and
Klibanov, 1988), that non-polar solvents are better than polar solvents, can be rationalized
by showing that polar solvents, because they dissolve in water, release the essential water of
the protein layer. the influence of organic solvents on an enzyme occurs due to interactions
with the water shell around the enzyme and not with the enzyme itself. They also found that
enzyme activity increased rapidly after increasing the water content of the medium. 
It must be emphasized that this generalization is in the context of anhydrous media
when the water content is below the limit of solubility in organic solvents. While biocatalyst
engineering aims to optimize the function of the biocatalyst by modifying the biocatalyst
structure, intermediate engineering in the context of biocatalysis in an aqueous solvent
involves modifying the environment around the biocatalyst (Laane, 1987). So the first gross
intermediate engineering rule is implied: non-polar solvents are better than polar because the
former provide a better microenvironment for proteins / enzymes.
 Biocatalyst Engineering

Just as enzyme immobilization and protein engineering have been attempted to

increase the efficiency / stability of catalysis in water, similar attempts to improve catalyst
performance in organic solvents have been made. The term 'umbrella' 'biocatalyst
engineering' has been used by Laane (1987) for this and seems quite appropriate. 
Tanaka and Kawamoto (1991) recently reviewed immobilized enzymes in organic
solvents. As they show, apart from the usual (i.e. in the case of catalysis in aqueous medium)
the advantages of presumed stabilization and reusability (given that enzymes do have some,
albeit limited, solubility in organic solvents), the added advantage of the advantageous
sharing of those available water (and its retention) with suitable support and dispersion of
enzyme molecules over a larger surface area with a concomitant increase in accessibility
from the active site to the substrate can be enumerated. First, limited data are available that
prove in quantitative fidshion the superiority of immobile enzymes over free ones. enzymes
(in organic solvents) by comparing each show under the same conditions. . Halling (1987)
has mentioned that, in the case of lipases, lipases tend not to move to give a faster reaction
rate compared to simple powdered enzymes.
 Arnold and co-workers have contributed significantly to the application of a
promising protein engineering approach to improve enzyme function in organic
solvents. Arnold (1988, 1990) has advocated a set of rules for design proteins in non-
aqueous solvents.

  This is largely based on this group's work with crambins (Arnold,

1988). Crambins are small plant proteins that are soluble in organic solvents. The rules
suggested by Arnold (1988, 1990) emphasize the importance of increasing the
conformational stability and compatibility of the enzyme surface with organic
solvents. The recent protein engineering work of this group (Chen et al., 1991) tends to
emphasize the discretionary principles of this method.
 The old picture of enzymes as fragile biocatalysts is and is changing. They can
even be used in most organic media. However, for most enzymes, the organic solvent
must be non-polar (insoluble in water). An important, unresolved problem is why only
some of them, such as xanthine oxidase and porcine pancreatic lipase, can function
even in water-soluble organic solvents (Khmelnitsky et al., 1988).

 Subtylisin functions even in dimethylformamide anhydrous (Riva et al.,

1988). Nevertheless, intermediate engineering and biocatalytic engineering are likely
to be increasingly used in a useful way to optimize enzyme performance in low water /
organic media. The seemingly simple approach of this waterless enzyme has
undoubtedly made enzymes even more powerful tools in the field of organic synthesis
and analysis.
Thanks!