encompassed 2800 active variants. Among the latter, the G105A and E188D mutants
showed significantly enhanced enantioselectivities for both substrates (E > 100). An
E188W/M193C double mutant even showed inverted enantiopreference (E ¼ 64) for
the trifluoromethyl substrate [349]. In order to avoid the undesired modification of the
substrate by a chromogenic reporter group, a second-generation screening method was
employed based on a commercial test kit: Thus, the acetate formed during ester
hydrolysis was activated into acetyl-CoA catalyzed by acetyl-CoA synthase (at the
expense of ATP). In a subsequent step, the acetate unit is transferred from acetyl-CoA
onto oxaloacetate yielding citrate (catalyzed by citrate synthase). The oxaloacetate
required for this reaction is formed by oxidation of L-malate (catalyzed by L-malate
dehydrogenase) under consumption of NAD
+ yielding an equimolar amount of
NADH, which can be spectrophotometrically monitored at 340 nm [350].
O
O
O
O
NO 2
O
O
NO 2
O
NO 2
colourless
>51
1.1
E
+
Buffer
Lipase (mutants)
Pseudomonas
R or S
yellow
aeruginosa
Lipase (mutant)
wild type
Method
mutant A
low rate epPCR (4x) at wild type
11
mutant B
saturation mutagenesis at #155 of mutant A
20
mutant C
low rate epPCR at mutant B
25
mutant G
CMCM #160-163 at wild type
30
mutant D
3.0
mutant E
6.5
high rate epPCR at wild type
high rate epPCR at wild type
mutant J
CMCM with mutants D & E
epPCR = error-prone polymerase chain reaction
CMCM = combinatorial multiple-cassette mutagenesis
R
S
*
both enantiomers tested separately
Scheme 2.43 Screening for Pseudomonas aeruginosa lipase mutants showing enhanced
enantioselectivities using pure enantiomers of a chromogenic surrogate substrate
OAc
R
OH
R
OAc
R
ACS = Acetyl CoA synthase; CS = Citrate synthase; L-MDH = LMalate dehydrogenase
LMDH
L-Malate
Citrate
Oxaloacetate
Acetyl-CoA
ACS
AMP + PP i
ATP
Enantioselectivity (E)
43
>100
>100
45
56
5
E188D
G105A
wild type
+
+ HOAc
buffer / DMSO
esterase (mutants)
Bacillus subtilis
rac
ATP = adenosine 5'-triphosphate; AMP = adenosine 5'-monophosphate;
PP i = inorganic diphosphate
CH 3
R
CF 3
H 2 O
CS
HOAc
NADH
NAD
+
CoA
Scheme 2.44 Enantioselectivities of wild-type Bacillus subtilis esterase and mutants acting on
tert-alcohol esters using a multienzyme acetate assay
2.1 Hydrolytic Reactions
79
showed significantly enhanced enantioselectivities for both substrates (E > 100). An
E188W/M193C double mutant even showed inverted enantiopreference (E ¼ 64) for
the trifluoromethyl substrate [349]. In order to avoid the undesired modification of the
substrate by a chromogenic reporter group, a second-generation screening method was
employed based on a commercial test kit: Thus, the acetate formed during ester
hydrolysis was activated into acetyl-CoA catalyzed by acetyl-CoA synthase (at the
expense of ATP). In a subsequent step, the acetate unit is transferred from acetyl-CoA
onto oxaloacetate yielding citrate (catalyzed by citrate synthase). The oxaloacetate
required for this reaction is formed by oxidation of L-malate (catalyzed by L-malate
dehydrogenase) under consumption of NAD
+ yielding an equimolar amount of
NADH, which can be spectrophotometrically monitored at 340 nm [350].
O
O
O
O
NO 2
O
O
NO 2
O
NO 2
colourless
>51
1.1
E
+
Buffer
Lipase (mutants)
Pseudomonas
R or S
yellow
aeruginosa
Lipase (mutant)
wild type
Method
mutant A
low rate epPCR (4x) at wild type
11
mutant B
saturation mutagenesis at #155 of mutant A
20
mutant C
low rate epPCR at mutant B
25
mutant G
CMCM #160-163 at wild type
30
mutant D
3.0
mutant E
6.5
high rate epPCR at wild type
high rate epPCR at wild type
mutant J
CMCM with mutants D & E
epPCR = error-prone polymerase chain reaction
CMCM = combinatorial multiple-cassette mutagenesis
R
S
*
both enantiomers tested separately
Scheme 2.43 Screening for Pseudomonas aeruginosa lipase mutants showing enhanced
enantioselectivities using pure enantiomers of a chromogenic surrogate substrate
OAc
R
OH
R
OAc
R
ACS = Acetyl CoA synthase; CS = Citrate synthase; L-MDH = LMalate dehydrogenase
LMDH
L-Malate
Citrate
Oxaloacetate
Acetyl-CoA
ACS
AMP + PP i
ATP
Enantioselectivity (E)
43
>100
>100
45
56
5
E188D
G105A
wild type
+
+ HOAc
buffer / DMSO
esterase (mutants)
Bacillus subtilis
rac
ATP = adenosine 5'-triphosphate; AMP = adenosine 5'-monophosphate;
PP i = inorganic diphosphate
CH 3
R
CF 3
H 2 O
CS
HOAc
NADH
NAD
+
CoA
Scheme 2.44 Enantioselectivities of wild-type Bacillus subtilis esterase and mutants acting on
tert-alcohol esters using a multienzyme acetate assay
2.1 Hydrolytic Reactions
79