nucleophilic Ser-residues approach from the back, both His are located at the
inside, with the oxy-anions pointing outside. Both active sites have suitable
space for the large and medium-sized substituents of the sec-alcohol moiety
(red). The mirror-image orientation of the catalytic center favors opposite
enantiomers, which is exemplified by the hydrolytic kinetic resolution of an
α-chiral indolyl propionic ester using the (R)-selective Mucor sp. lipase and the
(S)-selective protease α-chymotrypsin (Scheme 2.46, bottom) [393]. The activated 2-chloroethyl ester was used to ensure enhanced reaction rates.
• Substrate-type IV represents the general structure of a lesser used ester type for
lipases. For type-IV substrates, the alcohol moiety R
3 should preferentially
consist of a long straight-chain alcohol such as n-butanol. For esters of type IV
the stereochemical preference is often (S) (Scheme 2.45) but the predictability is
less accurate than with type-III substrates [372].
A large variety of different lipases are produced by bacteria or fungi and are
excreted as extracellular enzymes, which makes their large-scale production
particularly easy. The majority of these enzymes are created by the organisms
in two isoforms (isoenzymes), usually denoted as type A and B. Both are closely
related and usually show the same enantiopreference, but slight structural differences do exist, leading to certain differences in enantioselectivity. Crude
technical-grade lipase preparations usually contain both isoforms; the only notable exception is Candida antarctica lipase, for which both pure isoforms A and
B have been made available through genetic engineering. In contrast to esterases,
only a minor fraction of lipases are isolated from mammalian sources such as
porcine pancreas. Since some lipases from the same genus (for instance, from
Candida or Pseudomonas sp.) are supplied by different commercial sources, one
should be aware of differences in selectivity and activity among the different
CO 2 H
N
CO 2 H
N
CO 2 -(CH 2 ) 2 Cl
N
O
O
Ser221
O
R
H
His64
HN
NH
O
O
Ser209
O
R
H
His449
NH
HN
E = 71
E = 62
Buffer
Mucor sp. Lipase
Buffer
α-Chymotrypsin
rac
L, M = large and medium-sized substituent at alcohol moiety (red); R = acid moiety in transition state (blue)
(S)-selective
Subtilisin
(R)-selective
Candida rugosa lipase
L
H
R
S
H
H
M
M
L
Scheme 2.46 Mirror-image orientation of the catalytic machinery of Candida rugosa lipase and
the protease subtilisin and enantiocomplementary ester hydrolysis using Mucor sp. lipase and
α-chymotrypsin
86
2 Biocatalytic Applications
inside, with the oxy-anions pointing outside. Both active sites have suitable
space for the large and medium-sized substituents of the sec-alcohol moiety
(red). The mirror-image orientation of the catalytic center favors opposite
enantiomers, which is exemplified by the hydrolytic kinetic resolution of an
α-chiral indolyl propionic ester using the (R)-selective Mucor sp. lipase and the
(S)-selective protease α-chymotrypsin (Scheme 2.46, bottom) [393]. The activated 2-chloroethyl ester was used to ensure enhanced reaction rates.
• Substrate-type IV represents the general structure of a lesser used ester type for
lipases. For type-IV substrates, the alcohol moiety R
3 should preferentially
consist of a long straight-chain alcohol such as n-butanol. For esters of type IV
the stereochemical preference is often (S) (Scheme 2.45) but the predictability is
less accurate than with type-III substrates [372].
A large variety of different lipases are produced by bacteria or fungi and are
excreted as extracellular enzymes, which makes their large-scale production
particularly easy. The majority of these enzymes are created by the organisms
in two isoforms (isoenzymes), usually denoted as type A and B. Both are closely
related and usually show the same enantiopreference, but slight structural differences do exist, leading to certain differences in enantioselectivity. Crude
technical-grade lipase preparations usually contain both isoforms; the only notable exception is Candida antarctica lipase, for which both pure isoforms A and
B have been made available through genetic engineering. In contrast to esterases,
only a minor fraction of lipases are isolated from mammalian sources such as
porcine pancreas. Since some lipases from the same genus (for instance, from
Candida or Pseudomonas sp.) are supplied by different commercial sources, one
should be aware of differences in selectivity and activity among the different
CO 2 H
N
CO 2 H
N
CO 2 -(CH 2 ) 2 Cl
N
O
O
Ser221
O
R
H
His64
HN
NH
O
O
Ser209
O
R
H
His449
NH
HN
E = 71
E = 62
Buffer
Mucor sp. Lipase
Buffer
α-Chymotrypsin
rac
L, M = large and medium-sized substituent at alcohol moiety (red); R = acid moiety in transition state (blue)
(S)-selective
Subtilisin
(R)-selective
Candida rugosa lipase
L
H
R
S
H
H
M
M
L
Scheme 2.46 Mirror-image orientation of the catalytic machinery of Candida rugosa lipase and
the protease subtilisin and enantiocomplementary ester hydrolysis using Mucor sp. lipase and
α-chymotrypsin
86
2 Biocatalytic Applications