A fluorescence-based activity assay for immobilized lipases in non-native media

Article abstract of DOI:10.1016/j.ab.2019.01.005

A new method for the analysis of lipase activity in the immobilized state is developed. The fluorescence assay aims to quantify the potential of lipases for the application in organic solvents. As lipases are universally immobilized on polymeric carriers for the use in bioorganic synthesis, the assay includes an immobilization step on the walls of polymeric cuvettes. The activity of the immobilized lipase is probed by 4-methylumbelliferyl ester hydrolysis. The activity retention as a function of solvent concentration is used as a measure for the solvent resistance of the enzyme variant. The method is applied to two different lipases, Candida antarctica lipase B (CalB) and Bacillus subtilis lipase A (BSLA) in the presence of the solvents acetonitrile and ethanol. By comparison of the assay results with a commercial biocatalyst consisting of CalB on polymeric carrier (Novozyme 435) it is demonstrated that the assay allows a good prediction of the activity of the respective lipase as immobilisate on polymeric carriers. The assay surpasses the respective analysis in solution in terms of accuracy and precision.

Full text of DOI:10.1016/j.ab.2019.01.005

Accepted Manuscript
A fluorescence-based activity assay for immobilized lipases in non-native media
Kim N. Ingenbosch, Anna Rousek, Dennis Wunschik, Kerstin Hoffmann-Jacobsen
PII:
S0003-2697(18)31108-4
https://doi.org/10.1016/j.ab.2019.01.005
YABIO 13227
DOI:
Reference:
To appear in: Analytical Biochemistry
Received Date: 4 November 2018
Revised Date: 20 December 2018
Accepted Date: 14 January 2019
Please cite this article as: K.N. Ingenbosch, A. Rousek, D. Wunschik, K. Hoffmann-Jacobsen, A
fluorescence-based activity assay for immobilized lipases in non-native media, Analytical Biochemistry
(2019), doi: https://doi.org/10.1016/j.ab.2019.01.005.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
A fluorescence-based activity assay for immobilized lipases in non-native media
1,2,3
1,†
1,2,3
1,
Kim N. Ingenbosch
, Anna Rousek , Dennis Wunschik
, Kerstin Hoffmann-Jacobsen *
1
Niederrhein University of Applied Sciences, Department of Chemistry, Adlerstr. 32, 47798
Krefeld, Germany
2
Deutsches Textilforschungszentrum Nord-West gGmbH, Adlerstraße 1, 47798 Krefeld,
Germany
3
Institute of Physical Chemistry and CENIDE (Center for Nanointegration), Universität Duisburg-
Essen, Universitätsstraße 5, 45117 Essen, Germany
*
kerstin.hoffmann-jacobsen@hsnr.de
†
Current address: YMC Europe, Dinslaken
Abstract
A new method for the analysis of lipase activity in the immobilized state is developed. The
fluorescence assay aims to quantify the potential of lipases for the application in organic
solvents. As lipases are universally immobilized on polymeric carriers for the use in bioorganic
synthesis, the assay includes an immobilization step on the walls of polymeric cuvettes. The
activity of the immobilized lipase is probed by 4-methylumbelliferone hydrolysis. The activity
retention as a function of solvent concentration is used as a measure for the solvent resistance
of the enzyme variant. The method is applied to two different lipases, Candida antarctica lipase
B (CalB) and Bacillus subtilis lipase A (BSLA) in the presence of the solvents acetonitrile and
ethanol. By comparison of the assay results with a commercial biocatalyst consisting of CalB on
polymeric carrier (Novozyme 435) it is demonstrated that the assay allows a good prediction of
the activity of the respective lipase as immobilisate on polymeric carriers. The assay surpasses
the respective analysis in solution in terms of accuracy and precision.
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Keywords
Lipase activity, enzyme assay, organic solvent, enzyme immobilization, fluorescence
spectroscopy, solvent resistance
Abbreviations
ACN, acetonitrile; BSLA, Bacillus subtilis lipase A; CALB, Candida antarctica lipase B; EtOH,
ethanol; 4-MU, 4-methylumbelliferone; PMMA, polymethyl methacrylate; PS, polystyrene;
Introduction
Lipases are hydrolytic enzymes that act on carboxylic ester bonds. [1] Their physiological role is
to cleave water-insoluble fatty acid ester bonds by the reaction with water. Lipases have gained
a dominant role in biocatalytic organic synthesis, e.g. in the synthesis of enantiopure products.
This originates form the broad substrate spectrum and the high stereoselectivity.[2–5]
Importantly, lipases tolerate the presence of water-soluble organic solvents.[6] Many lipases are
even stable and active in neat organic solvents.[7,8] This allows biocatalytic transformations of
water insoluble reactants, opens new reactions paths and can lead to altered selectivities.[9]
Hence, lipases have been applied in biodiesel production [10], oleochemistry [11],
pharmaceutical production [12], food modification and so forth.
Availability of efficient and cost-effective biocatalysts and processes is a key factor for the
competitiveness of a biocatalytic process. Therefore, lipases are typically immobilized to form a
solid formulation for the application in organic solvents.[13] Enzymes can be immobilized by
binding to a support (carrier), entrapment (encapsulation) and cross-linking. This allows an easy
handling and enzyme recovery. A further benefit is the generally enhanced stability towards
denaturation by heat and/or organic solvents.[14,15] Polymeric carriers are available in a huge
variety of polymer backbone, surface groups and pore size distributions for diverse applications.
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Thus, these materials are preferred supports for enzyme immobilization. However, the initial
activity, specificity, selectivity and pH optimum of enzymes are routinely altered by the presence
of a solid carrier. The determination of the optimal biocatalyst for a specific reaction involves
both, the suited enzyme and the optimal immobilization. This problem evolves into a multi-
parameter problem including enzyme, carrier and the interdependency between the optimization
of the reaction system on the one hand and activity and stability of the biocatalyst under these
conditions on the other hand.
Because of its wide range of substrates and high thermal stability the enzyme Candida
antarctica lipase B, (CalB) [16] is the most widely used lipase to date in industrial applications
and scientific researches. It is commercially available as the solid biocatalyst “Novozyme 435”.
This biocatalyst consists of the enzyme adsorbed on a macroporous acrylic resin. However,
today 150 hydrolases are commercially available with new lipases being continuously isolated
from various microbes by researchers.
In order to assess the catalytic performance, the enzymatic activity is commonly determined by
hydrolysis assays.[17] Whereas the classical titrimetric method is too time consuming for
screening purposes, spectroscopic assays allow a high sampling speed. [18] Most methods rely
on the masking of the phenolic chromophores by the presence of the ester bond. The hydrolysis
product, e.g. p-nitrophenol, can be detected by UV spectroscopy. Applying fluorescence
spectroscopy with the respective fluorogenic esters, e.g. 4-methylumbelliferyl esters, improves
the sensitivity of the assay and the robustness in turbid solution.[19,20] Although these methods
give a good estimate on the enzymes hydrolysis performance in aqueous solution the
applicability of these results on the activity in organic solvents is questionable. In order to
examine lipase activity in organic solvents, several assays have been developed to measure
synthetic activity [21–24] or hydrolysis in the presence of solvent [25].
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In this study we present an activity assay for lipases in organic media which analyses
immobilized lipases. Lipases are used almost exclusively in immobilized form in organic
solvents. Hence, an activity screening of lipases in organic solvents that is carried out in the
immobilized state should allow a more realistic evaluation of the enzymes applicability for
biocatalysis in organic media than an assay in solution. Moreover, it should reduce the number
of optimization steps in biocatalyst development. In the assay presented herein, the enzymes
are adsorbed on the walls of plastic cuvettes. Polyacrylate and polystyrene are the dominant
carriers used for lipase immobilization and the material of plastic cuvettes and microtiter plates.
This coincidence is employed to develop a screening method for immobilized lipases in the
presence of organic solvents. 4-methyllumbelliferone butyrate serves as substrate and
hydrolysis rates are determined in the presence of polar water-miscible solvents. Two lipases
are investigated: the ubiquitously used lipase B from Candida antarctica (CalB) and lipase A
from Bacillus subtilis (BSLA). BSLA is the smallest lipase known to date [26], which is less
frequently applied in chemo-enzymatic synthesis [27].
Materials and methods
Bacillus subtilis Lipase A was expressed in E.coli and purified by nickel affinity chromatography
as described elsewhere.[20] CalB (liquid) was kindly provided by Novozymes, Denmark.
Novozyme 435, 4-methylumbelliferone butyrate and 4-methylumbelliferone (4-MU) were
purchased from Sigma Aldrich, Germany. Purified oleic acid (min. 72%) was purchased from
VWR, Germany. Acetonitrile, ethanol, hydrogen peroxide (30%) and disposable fluorescence
cuvettes for the UV (PMMA) and Vis (PS) range were purchased from Carl Roth, Germany. All
assay chemicals were analytical grade.
Enzyme immobilization was performed in the fluorescence cuvettes at 21°C in a 10 mM
Na HPO /NaH PO phosphate buffer at pH 8 for one hour. The used enzyme concentration was
2
4
2
4
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0
.5 µM. After incubation each cuvette was rinsed 50 times with ddH2O. Each cuvette was used
for a single measurement only.
-Methylumbelliferonebutyrat hydrolysis kinetics were recorded with a Varian Cary Eclipse
4
Fluorescence Spectrometer as described previously.[20] The excitation wavelength was 327 nm;
emission was acquired at 449 nm. The substrate concentration was 100 µM. In order to convert
fluorescence intensity data into concentrations, 4-methylumbelliferone was used for calibration.
Calibration data were recorded before each measurement at the respective buffer and
instrument conditions. The initial turnover rates were determined by a linear fit to the time trace
in the initial linear regime. All measurements were taken in triplicate. The level of autohydrolysis
of the substrate was monitored for all conditions. For all data shown the level of autohydrolysis is
below the given experimental error.
A stock solution of 50 % v/v of organic solvent in buffer was prepared and degassed in an
ultrasonic bath. The respective solvent concentrations were produced by dilution of the stock
solution in degassed buffer. Residual activities in organic solvents are given as the ratio of the
initial rate at the given solvent concentration to the value obtained in buffer. A complete
screening of the solvent concentrations was routinely performed at the same day. Michaelis
Menten parameters were determined by substrate variation in the range of 10-100 µM in 10
steps. The Michaelis Menten parameters were determined by linear regression to the Hanes-
Woolf equation.
The epoxidation reaction was performed in a 25 ml round-bottom flask containing 6.5 mmol oleic
acid dissolved in 10 ml acetonitrile/water (15 %) solution. 0.1 g Novozyme 435 were supplied
2 2
and 26.0 mmol H O were added dropwise over 1 hour under continuous stirring. The reaction
was performed for 24 h under shaking at room temperature. The reaction was stopped by
filtration of the enzyme and washing the samples with MeOH. The solvent was evaporated and
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the product was dried under vacuum. The product was identified by IR spectroscopy and melting
point (T : 50°C)
m
-
1
IR (FI-ATR), ν [cm ]: 3300 (-OH), 3010-2850 (C-H), 1750-1745 (C=O), 1280-1230 (ring
stretching), 1170-1150 (C-O), 950-750 (C-O-C), 725-720 (C-C)
Results and discussion
Activity measurements were performed in standard disposable 3 mL cuvettes using the water
soluble fluorogenic ester 4-MU butyrate. The release of 4-MU by hydrolysis was detected by its
fluorescence emission. 4-MU is especially suitable for the development of an assay in polar
organic media as it does not show a solvent dependent Stokes Shift with the media under
investigation. As depicted in Fig. S1, neither the emission maximum nor the intensity of 4-MU
depends on the concentration of the used solvents in the studied concentration range.
Fig. 1. Left: Kinetic time traces of immobilized BSLA in the immobilized state in phosphate
buffer. The linear fits illustrate the determination of the initial rate at 100 µM substrate
concentration as activity measure. Right: Hydrolytic activity of the lipases BSLA and CalB after
immobilization on polymethyl methacrylate (PMMA) and polystyrene (PS) supports as deduced
from the initial rate.
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After incubation of the disposable polymethyl methacrylate (PMMA) and polystyrene (PS)
cuvettes with lipase solution and excessive rinsing, kinetic time traces could be obtained from
the lipase immobilized on the four inner walls of the cuvettes. Fig. 1 shows a representative
kinetic time trace of the 4-MU fluorescence intensity upon 4-MU butyrate hydrolysis by adsorbed
BSLA. Hydrolytic activity is obtained from the slope of a linear fit to kinetic traces in the initial
linear regime at substrate saturation. The reaction rate is not limited by mass transport, as the
activity does not depend on the stirring speed (Fig. S2).
In order to optimize the immobilization process, the enzyme concentration of the immobilization
solution was varied and the initial rate was measured. Here, a saturation behavior was
observed. For the results presented herein, the minimum concentration leading to the saturation
activity was used. Moreover, the saturation behavior indicates that the enzyme surface coverage
is limited. It was further excluded that desorbed and redissolved proteins were studied, as only
the first 30 seconds after exposure to the solvent were used for analysis.
The amount of adsorbed enzyme was estimated by comparison of the initial rates of the
adsorbed lipase in buffer with time traces acquired from lipase in solution. By a set of enzyme
dilution experiments the concentration of lipase giving similar initial rates as the adsorbed lipase
was determined. Hence, the adsorbed lipase layer can be estimated to correspond to ca. 33
pmol BSLA rsp. 7.6 pmol CalB in solution. A rough estimation of the moles of enzymes in a
densely packed monolayer of lipase on the walls of the cuvette yields 50 pmol BSLA and 14
pmol CalB. Hence, it is very likely that the enzyme activity results from single monolayer of
adsorbed enzymes. Fig. 1 depicts the activity of the adsorbed lipases, BSLA and CalB, in
buffer. A ca. 70 times higher activity of adsorbed BSLA as compared to CalB is observed. This
results, first, of the higher activity of BSLA in solution (ca. factor of 15) and, second, the higher
number BSLA molecules in a monolayer coverage (ca. factor of 5). The higher activity of BSLA
7

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is not a result of the application of a short chain substrate as a higher activity of BSLA is also
found using the long chain substrate 4-MU oleate (data not shown). It reveals, that the assay
results depend on the geometrical size of the enzyme and should be used preferentially to
compare identically shaped enzymes, e.g. mutants.
Disposable cuvettes are generally made of PMMA or PS. Fig. 1 (right panel) shows that the
hydrolytic activity of BSLA in buffer depends on the support, whereas CalB activity is
independent of the support material. It cannot be resolved whether this is an effect of increased
surface coverage or increased viability of the adsorbed enzyme. Yet, the target parameter of the
assay developed herein is the activity in the presence of organic media.
Fig. 2. Influence of the organic solvents on the hydrolytic activity of lipase BSLA (left) and CalB
(
right) in solution. Residual activity is given as the ratio of the initial rate at the given solvent
concentration in buffer to the initial rate in buffer.
8

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Solvent resistance is quantified by the solvent dependency of residual activity. Residual activity
is obtained by the hydrolysis rate at the given solvent concentration divided by the activity in
buffer. The solvent resistance plots of BSLA and CalB in solution are depicted in Fig. 2. It
reveals a stronger solvent resistance of BSLA in solution as compared to CalB. Moreover, BSLA
shows a higher tolerance towards ethanol than towards acetontrile, whereas the solvent
resistance of CalB is identical for both solvents probed.
Similar plots have been generated previously from lipases in solution. [28] On the contrary, the
respective analysis with the assay of immobilized enzymes gives different results. Fig. 3 shows
the solvent dependency of CalB adsorbed on polymethylmethacrylate (PMMA) and polystyrene
(
PS) in buffer containing ethanol (left) and acetonitrile (right). The adsorbed enzymes show a
siginificantly higher activity retention in the intermediate concentration regime than the enzyme
in solution (Fig. 2).
Fig. 3. Influence of the organic solvents (left: acetonitrile, right: ethanol) on the hydrolytic activity
of the lipase CalB adsorbed on PMMA and PS. Residual activity is given as the ratio of the initial
rate at the given solvent concentration to the initial rate in buffer. The error bars reflect the
standard deviation of min. triplicate measurements.
9

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Hence, the assay reflects the generally accepted fact that lipase stability towards organic
solvents is enhanced by immobilization on polymeric carriers [29] and provides a quantification
thereof. The effect of the solvent on adsorbed CalB is stronger for acetonitrile as compared to
ethanol (Fig. 3), whereas both solvents show an almost identical effect on hydrolytic activity of
CalB in solution (Fig. 2). This reveals that the stabilization effect depends also on the specific
solvent.
It has been suggested that the decreasing activity of lipases with increasing alcohol
concentration in solution can be modelled with inhibition by the solvent and the thermodynamics
of non-ideal mixtures.[28,30] The impact of the solvent on enzyme activity is suggested to result
from various processes. First, the solvent can bind at the active site which leads to competitive
inhibition. This was reported for the interaction of CalB with methanol. [28] Second, the structure
and dynamics of enzymes are suspected to be altered by the interaction with organic solvents.
Here, the replacement of enzyme bound water is discussed as major effect. [31] Hence, the
activity decay is expected to be characteristic for each lipase, solvent and substrate and it is
determined by the mutual interaction between substrate, solvent and the individual lipase. A
Michaelis Menten analysis of the substrate dependent hydrolysis rate should give first
indications of the mechanism of the solvent lipase interaction. As depicted in Table 1, the
Michaelis Menten parameters of adsorbed CalB do not show a clear solvent dependency
pattern. The presence of increasing amounts of solvent affects both, the apparent K and vmax,
m
leading to reduced vmax/K values. This implies a mixed inhibition by the solvent, which is not
m
m
exclusively taking place at the active site. The most significant increase of K being found at the
lowest ethanol concentration could indicate the presence of competitive solvent binding. For the
assay development, these results reveal vmax being the parameter which is predominantly
altered by the presence of the solvent. This is a good validation for the usage of the initial rate at
substrate saturation as activity parameter in the rapid assay. Moreover, it demonstrates, that
Michaelis Menten parameters can be determined in the assay setting for mechanistic studies.
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The Michaelis Menten parameters of CalB in solution are provided in the Supplementary
Information (Table S1).
Table 1: Michaelis Menten parameters of CalB immobilized on PMMA: Michaelis Menten
m
constant K , maximum rate vmax. σ depicts the standard deviation of three independent analyses
by linearization.
Solvent
K
m
σ
[µM]
v
max
σ
v
max/ K
[1/s]
m
σ
[1/s]
[
%]
[µM]
[µM/s]
[µM/s]
Buffer
1
-1
1
-2
-4
-4
-6
1
00
2.5
∙
10
4.0
∙
10
1.7
∙
10
3.4
∙
10
6.7
∙
10
3.8
∙
10
ACN
1
1
-2
-3
-3
-4
-4
-5
-5
-6
1
0
4.8
∙
10
1.2
∙
10
1.1
∙
10
10
1.2
∙
10
10
2.4
∙
10
3.6
∙
10
1
2
0
7.7
∙
10
2.1
∙
10
1.5
∙
2.4
∙
2.0
∙
10
6.5
∙
10
EtOH
1
1
1
1
1
-3
-3
-4
-4
-4
-5
-4
-5
-6
-6
-6
-7
10
20
30
6.8
7.1
7.2
∙10
∙10
∙10
1.0
∙
10
9.5
5.2
3.8
∙
10
10
10
8.6
3.0
4.2
∙10
∙10
∙10
1.4
7.3
5.4
∙10
∙10
∙10
9.0
2.5
3.1
∙10
∙10
∙10
6.6
1.2 10
∙
∙
∙
1
1

ACCEPTED MANUSCRIPT
Fig. 4. Influence of the organic solvents (left: acetonitrile, right: ethanol) on the hydrolytic activity
of the lipase BSLA adsorbed on PMMA and PS. Residual activity is given as the ratio of the
initial rate at the given solvent concentration to the initial rate in buffer. The error bars reflect the
standard deviation of min. triplicate measurements.
In Fig. 4 the solvent dependent residual activity of the second lipase under investigation, BSLA,
in the immobilized state is shown. The solvent resistance plots of BSLA in solution and on the
surface coincide within the experimental error of the measurement in solution, which is
remarkably high. This reveals that in the case of BSLA the solvent resistance cannot be further
increased by adsorption on a polymeric carrier. As a consequence, the prominent solvent
tolerance of BSLA with respect to CalB is reduced after immobilization.
The comparison of Fig. 2 and Fig. 4 reveals another advantage of the presented assay. The
statistical error of the residual activity is substantially decreased in the adsorbed state assay. It is
well known, that organic solvents, especially alcohols, can lead to lipase aggregation followed by
inactivation and sedimentation.[32] BSLA shows an exceptionally high aggregation tendency.[20]
Enzyme aggregation leads to the loss of active enzyme available in solution and thus to the
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detection of decreased activity values. Immobilization of enzymes on a solid surface inhibits
aggregation. Aggregation is a non-equilibrium process. Hence the amount of aggregated
enzyme is suspected to vary from one analysis to another, which will lead to scattered activity
data. Suppression of aggregation is suggested to be the root cause for the lower statistical error
of residual activities obtained with the presented assay.
The Michaelis Menten parameters of immobilized BSLA are depicted in Table 2. Due to the high
experimental errors in solution, Michaelis Menten analysis of BSLA kinetics was only feasible
after immobilization. Again the data indicates mixed inhibition. In contrast to CalB, no indication
of competitive binding of the solvents is found. We conclude that vmax decreases in both solvents
with increasing solvent concentration.
Table 2: Enzyme specific Michaelis Menten parameters of BSLA immobilized on PMMA:
Michaelis Menten constant K
m
, maximum rate vmax. σ depicts the standard deviation of three
independent analyses by linearization.
Solvent
Km
[µM]
σ
[µM]
vmax
[µM/s]
σ
vmax / Km
[1/s]
σ
[1/s]
[
%]
[µM/s]
Buffer
1
-1
-3
-3
-5
1
00
6.4∙10
3.8
1.2
∙
10
5.3
∙
10
1.9
∙
10
3.1∙10
ACN
1
1
-2
-2
-3
-5
-3
-4
-5
-6
1
0
0
6.5
∙
10
2.8
8.9
∙
10
10
2.1
∙
10
10
1.4
∙
10
10
2.6
∙
10
-1
2
5.7
∙
10
2.9∙10
2.5
∙
7.3
∙
4.4
∙
2.8
∙
10
EtOH
1
1
1
-2
-2
-2
-3
-3
-3
-3
-3
-4
-5
-5
-5
1
2
3
0
0
0
4.1
5.3
7.3
∙10
∙10
∙10
3.1
2.3
7.6
5.7
3.0
∙
10
10
10
2.5
1.1
3.5
∙
10
10
10
1.9
1.1
4.1
∙
10
10
10
7.4
2.6
2.6
∙10
∙10
∙10
∙
∙
∙
1
1.4∙10
∙
∙
∙
1
3

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Finally, the effect of the different supports will be discussed. The solvent resistance of
immobilized BSLA shows only a weak preference for PMMA immobilization. CalB does not show
any preference for the polymer material used for immobilization. As the two different polymer
supports analyzed herein do not influence strongly the activity data in organic solvents, we
suggest that the comparison of supports should be performed when needed in a subsequent
testing step. Hence, we consider the immobilization of lipases on PMMA sufficient for the activity
screening of solvent resistant lipase variants.
Fig. 5. Residual activity of Novozyme 435 (N435) and CalB immobilized on PMMA (CalB PMMA)
at the given solvent concentration for acetonitrile (left) and ethanol (right).
The transfer of the assay results to real catalysts is studied by comparison with the commercial
product Novozyme 435. It is a biocatalyst prepared of CalB immobilized on a porous PMMA
resin. In Fig. 5 the solvent resistance of Novozyme 435 is juxtaposed to the results of CalB
immobilized on PMMA. The solvent resistance of Novozyme 435 towards ethanol is identical to
the assay data reported herein. In acetonitrile, the assay results coincide with the activity
retention of the real biocatalyst in the low solvent concentration regime. At higher acetonitrile
concentrations, the solvent resistance of Novozyme 435 exceeds the assay results. This
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discrepancy is suggested to result from the different surface structure of the carrier. The cuvette
surface does not provide a porous structure as the commercial carrier. However, the assay is
developed for screening applications and the carriers’ geometry will have to be probed
separately.
However, in biocatalytic organic synthesis, the choice of the organic solvent is not only
determined by enzyme stability and activity in the respective medium. Organic synthesis design
usually considers reactant and product solubility, stabilization of intermediates etc. for the
selection of an appropriate solvent. Enzyme and support stability are just additional factors to be
considered in biocatalysis. The presented assay provides a method to screen the enzymes
tolerance for polar water miscible solvents selected by synthesis arguments. Hence, it aims to
reduce the number of optimization steps but does not replace completely the parameter
optimization of a specific reaction.
In an exploratory study, we aimed to demonstrate the applicability of the assay in biocatalytic
synthesis design. We used the epoxidation of unsaturated fatty acids as model reaction because
this reaction is commonly performed in solvent/aqueous hydrogen peroxide solution, typically in
toluene and alcohols. [33] Our assay results indicate a significant activity and stability of CalB on
PMMA at 15 % acetonitrile/buffer solution. This suggests that immobilized CalB (Novozyme 435)
can be applied at these conditions. Therefor we performed the epoxidation of oleic acid for the
first time at the given solvent/buffer mixture with Novozyme 435 which lead to an excellent yield
of 85 %. This is in line with a previous report of the epoxidation of citronellol by urea hydrogen
peroxide with CalB in solution revealing best yields in acetonitrile. [34]
The presented assay is the first assay known to date that includes the properties of lipases on
polymeric surfaces into the analysis of solvent tolerance. It is designed to mimic enzyme
stabilization by immobilization in a standardized, rapid and automatable one pot method. The
low standard deviations of repeated measurements demonstrate that the immobilization step is
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performed in a reproducible manner. The assay takes completely place in the measurement
vessel, which enables automation. In contrast to other documented methods for activity analysis
in organic media [25,24], the assay does not include an extraction step. We suggest that these
factors add to the high precision arising from the analysis of the ordered state on the surface.
We claim that the assay provides a more reliable performance indicator for the screening of
solvent tolerance than methods working with dissolved lipases in solvent solutions. Without
resolving whether the solvent tolerance is an inherent property of the enzyme in solution or a
result of a high stabilization by immobilization the method gives a direct indicator for the
applicability of the respective enzyme as solid biocatalyst in non-native media. Yet, we
emphasize that the application range of the method is restricted to water miscible solvents. For
nonpolar solvents complementary probe reactions, e.g. [21], will have to be included in the
concept. Hence, the method represents an effective component of the required toolbox of
activity analysis providing a reliable estimate of the lipase performance in organic media, which
strongly exceeds the respective measurement in solution in terms of precision and predictive
power for further applications.
Conclusion
In this study, we have developed a method for the activity screening of lipases which includes
immobilization in the analysis method. We have shown that this method can be used to analyze
the solvent resistance of lipases in a screening process to predict the lipase behavior after
adsorption on a typical polymeric carrier. Thus, the development process of new lipase
biocatalysts, which commonly includes the steps enzyme screening, subsequent immobilization
and final application screening can be accelerated with the present method. The assay is fully
compatible with a high-throughput format, and should be automatable by labs featuring
automatic pipetting station coupled to multi-well plate reader.
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Acknowledgements
We thank the internal research funding of the Niederrhein University of Applied Sciences for
financial support.
Supplementary Information
Fig. S1. Fluorescent spectra of 4-methylumbelliferone in phosphate buffer and in buffer mixtures
containing 30% acetonitrile (ACN) or 30% ethanol (EtOH)
Fig. S2. Dependence of the measured activity on the stirring speed
Table S1 Michaelis Menten parameters for CalB in solution.
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A fluorescence-based assay for determining the activity of immobilized lipases in
non-native media
Highlights
•
•
•
•
A new assay for the analysis of the solvent resistance of lipases is presented. The assay
uses in-situ adsorption of enzymes.
Fluorescence spectroscopy provides the sensitivity to analyze the activity of a single
lipase monolayer adsorbed to the walls of the cuvette.
The assay is a screening tool established to reduce the number of activity tests required
in enzymatic biocatalyst development
Areas of application of the lipase variant in organic solvents can be predicted by this
screening method.