Combinatorial library based engineering of candida antarctica lipase a for enantioselective transacylation of sec-alcohols in organic solvent

Article abstract of DOI:10.1002/anie.201410675

A method for determining lipase enantioselectivity in the transacylation of sec-alcohols in organic solvent was developed. The method was applied to a model library of Candida antarctica lipase A (CalA) variants for improved enantioselectivity (E values)

Full text of DOI:10.1002/anie.201410675

Angewandte
Chemie
DOI: 10.1002/anie.201410675
Biocatalysis
Combinatorial Library Based Engineering of Candida antarctica
Lipase A for Enantioselective Transacylation of sec-Alcohols in
Organic Solvent**
Ylva Wikmark, Maria Svedendahl Humble,* and Jan-E. Bꢀckvall*
Abstract: A method for determining lipase enantioselectivity
in the transacylation of sec-alcohols in organic solvent was
developed. The method was applied to a model library of
Candida antarctica lipase A (CalA) variants for improved
enantioselectivity (E values) in the kinetic resolution of 1-
phenylethanol in isooctane. A focused combinatorial gene
library simultaneously targeting seven positions in the enzyme
active site was designed. Enzyme variants were immobilized on
nickel-coated 96-well microtiter plates through a histidine tag
(His6-tag), screened for transacylation of 1-phenylethanol in
isooctane, and analyzed by GC. The highest enantioselectivity
was shown by the double mutant Y93L/L367I. This enzyme
variant gave an E value of 100 (R), which is a dramatic
improvement on the wild-type CalA (E = 3). This variant also
showed high to excellent enantioselectivity for other secondary
alcohols tested.
with broad substrate scope are required. One problem is that
such enzymes may not be available in the natural collection of
lipases. A solution to this problem is to modify natural lipases
through engineering/directed evolution.
Evolution of enzymes through the generation of large
libraries with subsequent screening and selection is the most
efficient method for obtaining new enzyme variants with
improved properties.[8–10] There are many examples in which
the stereoselectivity of lipases has been significantly
improved by using this approach.[8,11–18] Although a method
for microtiter-plate screening in organic solvent has been
reported, this method was not tested for enantioselectivity.[19]
To date, all screening studies on lipase libraries for increased
enantioselectivity have dealt with the hydrolysis of esters in
an aqueous medium. Consequently, library-based engineering
has never been used for improving the DKR scope for sec-
alcohols, since these substrates involve transacylation in
organic solvent. In order to produce the desired enzyme
variant, it is necessary to mimic the true reaction conditions
according to Arnoldꢀs statement: “you get what you screen
for”.[8a] There are several reported examples of solvent
dependence for enzyme selectivity,[20,21] and even reversed
enantioselectivity between water and organic solvent has
been described.[21]
Herein, we report a method that enables the screening of
a lipase library for enantioselective transacylation in an
organic solvent. Normally, supernatants containing the pro-
tein of interest are directly used in enzyme-library screening
assays. In this work, the use of a standard His6-tag immobi-
lization technique provided one-step simultaneous immobili-
zation and purification of the enzyme library on microtiter
plates. This method allows the solvent to be changed from
water to isooctane, as well as the screening of purified enzyme
preparations.
T
he general demand for stereoselective synthetic methods
and environmentally compatible industrial chemical process-
es is increasing. This has led to an increased demand for
enzymes that can be used as catalysts for various purposes.[1,2]
To date, lipases are among the most used enzymes for
industrial biocatalytic applications.[3,4] Lipases generally show
high substrate and reaction promiscuity in combination with
high stereoselectivity.
Our research group has been involved in the development
of dynamic kinetic resolution (DKR),[5–7] in which lipases are
combined with transition metals. Efficient systems have been
obtained that allow the transformation of racemic alcohols
(and amines) into enantiomerically pure products in close to
quantitative yields based on the racemate. In these reactions,
the lipase catalyzes a transacylation in an organic solvent and
the metal complex racemizes the slow-reacting enantiomer of
the alcohol (or amine).
To further improve and extend the scope of DKR
reactions, more efficient and more enantioselective enzymes
Lipases A and B from Candida antarctica are two
biocatalysts that have found widespread applications in
organic transformations, and both enzymes work very well
in a range of organic solvents.[22] The enzyme Candida
antarctica lipase A (CalA) has been employed to a lesser
extent compared to Candida antarctica lipase B (CalB). CalA
catalyzes the transacylation of sec-alcohols in organic solvents
at activities comparable to those of CalB,[23] but shows low or
no enantioselectivity for small sec-alcohols such as 1-phenyl-
ethanol (E = 3).[24] CalA shows a naturally high thermo-
stability, even in its free form,[23–25] and it has a rather large
active site that allows the transacylation of tertiary alcohols.[26]
We have previously demonstrated that CalA can be modified
through engineering/directed evolution for the enantioselec-
tive hydrolysis of esters in water.[17,18,27] In the present work,
[*] Y. Wikmark, Dr. M. Svedendahl Humble, Prof. J.-E. Bꢀckvall
Department of Organic Chemistry, Arrhenius Laboratory
Stockholm University, 10691 Stockholm (Sweden)
E-mail: jeb@organ.su.se
Dr. M. Svedendahl Humble
Industrial Biotechnology, School of Biotechnology
Albanova University Center, Royal Institute of Technology (KTH)
10691 Stockholm (Sweden)
E-mail: maria.humble@biotech.kth.se
[**] This work was supported by The Swedish Research Council (VR).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201410675.
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we used CalA as a test enzyme for improvement of the
enantioselectivity of transacylation in an organic solvent.
As in all lipases, the active site of CalA is made up of two
main substrate binding pockets; an acyl binding pocket and
a nucleophile (or alcohol) binding site. The enzyme exposes
its active site through interfacial activation.[28] Crystal struc-
tures of CalA[29,30] reveal a narrow acyl binding tunnel, a wider
nucleophile binding cleft, and a flexible lid.[31] The wide
nucleophile binding pocket may be the reason why CalA
shows low enantioselectivity with small secondary alcohols.
Hypothetically, decreasing the size of the nucleophile binding
pocket should increase the enantioselectivity of the enzyme
with smaller secondary alcohols.
Large gene libraries created by random mutagenesis often
show low hit ratios. For improved enantioselectivity, muta-
tions around the active site have proven to be the most
influential and in these cases, focused combinatorial gene
libraries with simultaneous variation of a few amino acid
residues have proven to be successful.[9,11,17,18] We recently
reported an efficient improvement in the enantioselectivity of
CalA for the hydrolysis of bulky esters in buffer, where nine
amino acid positions around the active site were varied
simultaneously. This combinatorial method accounts for
synergistic effects. Using 2–4 amino acids at each site yielded
a library of 1024 enzyme variants.[17,32]
Figure 1. Close-up view of the active site of CalA. Amino acid residues
included in the CalA library (Table 1) are shown as turquoise stick
models, while the overall structure is shown as a gray ribbon diagram.
Tetrahedral intermediates (TI) of the product, 1-phenylethyl butyrate,
are shown in dark blue for the (R)-enantiomer and in yellow for the
(S)-enantiomer. The molecular graphics were created by using the
YASARA software.[33]
In the present study, this combinatorial approach[17] was
used to create a focused enzyme library. The library obtained
was immobilized on microtiter plates through chelation of
a His6-tag and then screened for enantioselective transacyla-
tion in an organic solvent. Seven amino acid residues in the
active site were targeted and varied simultaneously. The
combinatorial library design was guided by molecular model-
ing (Figure 1). The closed form of the crystal structure of
CalA (PDB ID: 2VEO) was previously opened by a 10 ns
molecular dynamics simulation.[18] By using the YASARA
software[33] starting from the catalytic Ser184, tetrahedral
intermediates of (R)- and (S)-1-phenylethyl butyrate were
built into the open structure. Amino acid residues within 5 ꢁ
of the tetrahedral intermediates were identified. Seven
positions were selected to be included in the focused
combinatorial library (Table 1). These positions were to be
varied to one or two other amino acids to yield an enzyme
library of 648 variants. The residues were chosen to modify
the space available in the active site.
a new PCR reaction with a gene–plasmid construct containing
a His6 affinity tag in the N-terminus as the mother template.
The product was transformed into Pichia pastoris X33, which
was then grown on agar plates.
To exclude variants lacking lipase activity, the agar plates
contained tributyrin [0.5% (v/v)]. The use of tributyrin
enables visible identification of colonies containing active
lipase through the formation of halos where the tributyrin is
hydrolyzed.[34] About 25–30% of all colonies showed lipase
activity after four days of incubation at 298C. This pre-
screening step reduced the library size substantially and the
accuracy of the selection was verified with a small fraction of
the library.
In the library screening, individual colonies were
expressed in 96-well deep-well plates (8 days, 298C,
250 rpm). The plasmid used, pBGP1, enables secretion of
the protein into the supernatant.[27,35]
The gene library was created through overlap-extension
PCR with seven small gene fragments. The gene fragments
were fused together through PCR to give one full-length gene
sequence. This polynucleotide was used as a megaprimer in
Supernatants were transferred to Nunc immobilizer Ni-
Chelate F96 microtiter plates from ThermoScientific. The
nickel coating of the microtiter plates enables the coordina-
tion of His6-tagged proteins to the walls of each well. After
enzyme immobilization on the plate, the supernatant was
removed. To remove residual water from the plate, each plate
was washed three times with isooctane and air dried.
A screening solution of isooctane containing vinyl buty-
rate (200 mm), 1-phenylethanol (20 mm), and dodecane
(20 mm, as internal standard) was added to the plates
containing the immobilized lipase variants (Scheme 1).
After 2.5 h, samples were transferred to a second 96-well
microtiter plate. The plate was then screened by chiral gas
Table 1: The amino acid (Aa) residues included in the CalA library.
Aaposition
AawildÀtype
Aalibrary
93
Y
Y
F
I
L
E
F
A or L or Y
Y or W
A or F
F or I or L
F or I or L
E or F or L
F or L
183
233
336
367
370
431
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Table 3: Enantioselectivity of wild-type CalA and the Y93L/L367I variant
determined for the kinetic resolution of various secondary alcohols by
transacylation in isooctane at 218C.[a]
Entry
1
Alcohol
Ewild-type
EY93L/L367I
Scheme 1. The model transacylation reaction applied to explore the
enantiospecificity of CalA variants in the kinetic resolution of 1-phenyl-
ethanol in organic solvent at 218C.
3
100
chromatography (GC-FID). The total time to screen through
one 96-well plate was around 24 h when using a method that
takes 14 min per analysis. Variants showing enantioselectivity
(eep) over 95% were determined to be hits. In total, 17 hits
were cultivated in 50 mL scale for further characterization
(Table S1 in the Supporting Information).
The 17 enzyme variants considered to be possible hits
were sequenced, but only three different variants were
identified (Y93A/F233A/L367I, Y93L/L367I, and Y93L/
L367I/G387E). However, the mutation G387E (G387 is
positioned on the enzyme surface) was spontaneous since it
was not included in the library.
Enzyme kinetics were measured for wild-type CalA and
the Y93L/L367I variant. Pseudo-one-substrate conditions
were applied for the model reaction by varying the concen-
tration of 1-phenylethanol in a microtiter plate. The method
involving His6-tag binding gives approximately equal amounts
of enzyme in each well, which gives a reliable comparison of
the activity of the variants. The Y93L/L367I variant showed
increased apparent KM and Ki values compared to the wild-
type enzyme. Wild-type CalA gives a low apparent Ki value of
30 mm and the corresponding value shown by the Y93L/L367I
variant is improved 2.5-fold (81 mm; Table 2). An improved
inhibition constant is beneficial for further applications in
DKR,[36,37] in which higher substrate concentrations are
preferred. Variant Y93A/F233A/L367I showed varying
results, thus indicating a mix of different enzyme variants.
This variant was not further characterized.
The enantiospecificity and substrate scope of variant
Y93L/L367I for a number of sec-alcohols were determined
and compared to those of wild-type CalA. Enzyme variant
Y93L/L367I showed improved enantioselectivity for six out of
the seven alcohols evaluated (Table 3). For 1-phenylethanol
(entry 1), p-substituted 1-phenylethanol (entries 3 and 4), and
1-phenylchlorohydrin (entry 7), a significant increase in the
E value was obtained.
2
3
4
5
20
>300
>300
30
70
5
6
7
70
32
4
100
23
100
[a] Reaction conditions: 200 mm vinyl butyrate, 20 mm alcohol, and
20 mm dodecane in isooctane at 218C. The enzyme was immobilized on
Nunc 96-well nickel-coated microtiter plates.
The effect of each mutation in the Y93L/L367I variant on
the enantioselectivity of kinetic resolution in the model
reaction was explored by applying variants with single point
mutations (Y93L and L367I). These variants were created by
site-directed mutagenesis, cultivated, immobilized on 96-well
nickel-coated microtiter plates, and screened with the model
reaction (Scheme 1). The screening results show that the two
mutations are synergistic and that the Y93L mutation has the
larger influence on the enantiospecificity.
The effects of the mutations on enantioselectivity were
further explored through molecular modeling by using
a tetrahedral intermediate model (Figure S1 in the Support-
ing Information). The structure was prepared as described
above (by using the YASARA software). Double mutant
Y93L/L367I and the two single mutants (Y93L and L367I)
were created by swapping the specific amino acid residues,
followed by energy minimizations. The (R)- or (S)-configu-
rations of the tetrahedral intermediate in the different variant
structures were compared through alignment to the corre-
sponding configurations in the wild-type enzyme by using
MUSTANG.[38]
Table 2: Apparent kinetic parameters for wild-type CalA and the Y93L/
L367I variant determined for the kinetic resolution of 1-phenylethanol
(model reaction) under pseudo-one-substrate conditions at 218C.[a]
CalA variant
KM,app[b] [mm]
Ki,app[b] [mm]
E[c]
Wild-type
Y93L/L367I
22
45
30
81
3
96
The alignments showed that the mutations induce move-
ment of the phenyl groups as a result of steric hindrance.
These movements were measured and the resulting structures
compared to the wild-type structure (Table S3). Mutation
Y93L mainly affects the phenyl group of the tetrahedral
intermediate in the (S)-configuration, while mutation L367I
mainly affects the corresponding phenyl group in the (R)-
configuration. According to the molecular modeling results,
[a] The concentration of vinyl butyrate was kept constant at 500 mm
while varying the concentration of 1-phenylethanol (3-200 mm). Unde-
cane or dodecane (20 mm) was used as an internal standard. [b] The
kinetic constants were derived from non-linear regression using the
Michaelis–Menten equation with substrate inhibition. [c] Mean values of
3–6 reactions with 20 mm 1-phenylethanol for the wild-type and 50 mm
1-phenylethanol for Y93L/L367I.
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ees [%]
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Conversion [%]
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L367I
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25
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4
[a] Reaction conditions: 200 mm vinyl butyrate, 20 mm 1-phenylethanol,
20 mm dodecane in isooctane at 218C. The enzyme was immobilized on
96-well nickel-coated microtiter plates.
In conclusion, the reported method is
a powerful
approach for discovering new enantioselective biocatalysts
for the transacylation of alcohols in organic solvents. By using
the standard His6-tag binding technique, simultaneous immo-
bilization and purification of the whole enzyme library was
achieved. The method is general and should be applicable to
other enzymes for improving enantioselectivity and activity.
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performed within a reasonable time, and is not labor
intensive. The whole screening procedure is done in 28.5 h/
plate: 2 h (immobilization) + 2.5 h (biocatalysis) + 24 h (GC
measurement).
By using this screening method, a lipase variant showing
improved enantioselectivity in the transacylation of 1-phenyl-
ethanol was found. The enantioselectivity increased from 3
(R) to 100 (R). In addition, this enzyme variant showed
increased KM and Ki values compared to the wild-type
enzyme. The improvement in the Ki value, from 30 to
85 mm, makes the enzyme more useful for synthetic applica-
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enantioselectivity for several secondary alcohols. The dra-
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molecular modeling. The highly combinatorial approach for
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Angew. Chem. Int. Ed. 2015, 54, 1 – 6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

.
Angewandte
Communications
Communications
Biocatalysis
Y. Wikmark, M. Svedendahl Humble,*
J.-E. Bꢁckvall*
&&& — &&&
Combinatorial Library Based Engineering
of Candida antarctica Lipase A for
Enantioselective Transacylation of sec-
Alcohols in Organic Solvent
Try the library: A method is reported for
screening lipase libraries for the enantio-
selective transacylation of sec-alcohols in
organic solvents. This could be useful for
creating enantioselective lipase variants
for synthetic applications such as the
dynamic kinetic resolution of alcohols
and amines. The method could also be
applied to other His6-tagged enzymes
and other reactions.
6
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!