The molecular reason for this phenomenon is a conformational rearrangement
within the enzyme [352]. A freely dissolved lipase in the absence of an aqueous/
lipid interface resides in its inactive state [Enz], because a part of the enzyme
molecule – the ‘lid’ – covers the active site. When the enzyme contacts the interface
of a biphasic water-oil system, a short α-helix is folded back. Thus, by opening its
active site the lipase is rearranged into its active state [Enz]
6 ¼ .
To ensure optimal activity, lipase-catalyzed hydrolyses thus should be
conducted in a biphasic medium. It is sufficient to employ the substrate alone at
elevated concentrations, such that it constitutes the second organic phase, or,
alternatively, it may be dissolved in a water-immiscible organic solvent such as
hexane, a dialkyl ether, or an aromatic solvent. Due to the presence of an interface,
physical parameters influencing the mass-transfer of substrate and product between
the aqueous and organic phase (such as stirring or shaking speed) have a marked
influence on the reaction rate of lipases. Triacylglycerols such as triolein or -butyrin
are used as standard substrates for the determination of lipase activity, whereas for
esterases p-nitrophenyl acetate is the classic standard.
The fact that many lipases have the ability to hydrolyze esters other than
glycerides makes them particularly useful for organic synthesis [386, 387]. Furthermore, some lipases are also able to accept thioesters [388, 389]. In contrast to
esterases, lipases have been used for the resolution of racemates more often than for
the desymmetrization of meso-compounds. Since the natural substrates are esters of
a chiral alcohol, glycerol, with an achiral acid, it may be expected that lipases are
most useful for hydrolyzing esters of chiral alcohols rather than esters of chiral
acids. Although this expectation is true for the majority of substrates (see substrate
type III, Scheme 2.45), some lipases also display high selectivity through recognizing the chirality of an acid moiety (substrate type IV).
substrate concentration [S]
CMC
substrate concentration [S]
Lipase
Esterase
substrate
activity
soluble
insoluble
saturation
activity
Enz
[EnzS]
-
[Enz]
-
organic phase
aqueous phase
Enz = inactive lipase (closed lid conformation)
[Enz]
- = active lipase (open lid conformation)
Enz
[Enz]
-
[EnzS]
-
+ S
[Enz]
- + P
interface
S = Substrate; P = Product
S
P
Fig. 2.12 Esterase and lipase kinetics
84
2 Biocatalytic Applications
within the enzyme [352]. A freely dissolved lipase in the absence of an aqueous/
lipid interface resides in its inactive state [Enz], because a part of the enzyme
molecule – the ‘lid’ – covers the active site. When the enzyme contacts the interface
of a biphasic water-oil system, a short α-helix is folded back. Thus, by opening its
active site the lipase is rearranged into its active state [Enz]
6 ¼ .
To ensure optimal activity, lipase-catalyzed hydrolyses thus should be
conducted in a biphasic medium. It is sufficient to employ the substrate alone at
elevated concentrations, such that it constitutes the second organic phase, or,
alternatively, it may be dissolved in a water-immiscible organic solvent such as
hexane, a dialkyl ether, or an aromatic solvent. Due to the presence of an interface,
physical parameters influencing the mass-transfer of substrate and product between
the aqueous and organic phase (such as stirring or shaking speed) have a marked
influence on the reaction rate of lipases. Triacylglycerols such as triolein or -butyrin
are used as standard substrates for the determination of lipase activity, whereas for
esterases p-nitrophenyl acetate is the classic standard.
The fact that many lipases have the ability to hydrolyze esters other than
glycerides makes them particularly useful for organic synthesis [386, 387]. Furthermore, some lipases are also able to accept thioesters [388, 389]. In contrast to
esterases, lipases have been used for the resolution of racemates more often than for
the desymmetrization of meso-compounds. Since the natural substrates are esters of
a chiral alcohol, glycerol, with an achiral acid, it may be expected that lipases are
most useful for hydrolyzing esters of chiral alcohols rather than esters of chiral
acids. Although this expectation is true for the majority of substrates (see substrate
type III, Scheme 2.45), some lipases also display high selectivity through recognizing the chirality of an acid moiety (substrate type IV).
substrate concentration [S]
CMC
substrate concentration [S]
Lipase
Esterase
substrate
activity
soluble
insoluble
saturation
activity
Enz
[EnzS]
-
[Enz]
-
organic phase
aqueous phase
Enz = inactive lipase (closed lid conformation)
[Enz]
- = active lipase (open lid conformation)
Enz
[Enz]
-
[EnzS]
-
+ S
[Enz]
- + P
interface
S = Substrate; P = Product
S
P
Fig. 2.12 Esterase and lipase kinetics
84
2 Biocatalytic Applications