Development of environmentally friendly lipase-catalysed kinetic resolution of (R,S)-1-phenylethyl acetate using aqueous natural deep eutectic solvents

Article abstract of DOI:10.1016/j.procbio.2020.12.001

Over the past decade, natural deep eutectic solvents (NADES) have become promising green solvent from both environmental and technological perspectives. The number of structural combinations encompassed by NADES is tremendous; thus, NADES with unique physicochemical properties can be designed for a particular purpose, such as for efficient biocatalytic processes. The aim of this work was to prepare several NADES based on renewable sources (biomass energy) and to apply these solvents in the industrially interesting process of lipase-catalysed (R)-1-phenylethanol synthesis. Based on experimental data on lipase behaviour in the tested NADES, choline chloride:glycerol (ChGly) was selected as the most promising solvent for the given reaction. The resulting (R)-1-phenylethanol was also successfully isolated and purified from the reaction mixture using liquid-liquid extraction with ethyl acetate, whilst ChGly was recycled and reused. (R)-1-phenylethanol and unreacted (S)-1-phenylethyl acetate were purified on silica gel columns with yields of 81.91 %. The biocatalyst was also successfully recycled and reused in 4 cycles. Based on laboratory-scale optimum conditions, (R)-1-phenylethanol synthesis performed in a 500 mL batch reactor achieved reaction conversions comparable to those obtained with millilitre-scale biocatalytic reactions. The downstream process was also performed on a preparative scale with a yield of 49.48 %.

Full text of DOI:10.1016/j.procbio.2020.12.001

Process Biochemistry 102 (2021) 1–9
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Development of environmentally friendly lipase-catalysed kinetic
resolution of (R,S)-1-phenylethyl acetate using aqueous natural deep
eutectic solvents
Manuela Pani ´c , Mia Radovi ´c , Izabela Maros, Ana Jurinjak Tu ˇs ek, Marina Cvjetko Bubalo*,
Ivana Radoj ˇc i ´c Redovnikovi ´c
Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000, Zagreb, Croatia
A R T I C L E I N F O
A B S T R A C T
Keywords:
Over the past decade, natural deep eutectic solvents (NADES) have become promising green solvent from both
environmental and technological perspectives. The number of structural combinations encompassed by NADES is
tremendous; thus, NADES with unique physicochemical properties can be designed for a particular purpose, such
as for efficient biocatalytic processes. The aim of this work was to prepare several NADES based on renewable
sources (biomass energy) and to apply these solvents in the industrially interesting process of lipase-catalysed
Green chemistry
Kinetic resolution
Natural deep eutectic solvents
Novozym 435
(
R)-1-phenylethanol
(
R)-1-phenylethanol synthesis. Based on experimental data on lipase behaviour in the tested NADES, choline
Stereoselective biocatalysis
chloride:glycerol (ChGly) was selected as the most promising solvent for the given reaction. The resulting (R)-1-
phenylethanol was also successfully isolated and purified from the reaction mixture using liquid-liquid extraction
with ethyl acetate, whilst ChGly was recycled and reused. (R)-1-phenylethanol and unreacted (S)-1-phenylethyl
acetate were purified on silica gel columns with yields of 81.91 %. The biocatalyst was also successfully recycled
and reused in 4 cycles. Based on laboratory-scale optimum conditions, (R)-1-phenylethanol synthesis performed
in a 500 mL batch reactor achieved reaction conversions comparable to those obtained with millilitre-scale
biocatalytic reactions. The downstream process was also performed on a preparative scale with a yield of
4
9.48 %.
1
. Introduction
Single-enantiomer drugs have become the standard in pharmaceu-
synthesised by traditional synthesis methods, such as oxidation of eth-
ylbenzene or reduction of acetophenone using organometallic com-
pounds as catalysts; however, the poor enantioselectivity and residual
metal catalysts in the final products makes these approaches rather
undesirable [4]. Enzymes, by contrast, display much higher enantiose-
lectivity and better regioselectivity than the organometallic catalysts
used in traditional organic synthesis. In the case of 1-phenylethanol
production, lipase-catalysed hydrolysis is a convenient method, as a
dehydrogenase-catalysed reduction must also be designed to regenerate
coenzymes.
tical companies when working with compounds featuring stereogenic
centres, and they have mobilised the chemical and biotechnological
industries to develop versatile methods for the production of biologi-
cally active chiral compounds of high enantiomeric purity. Enantiomers
of a single chiral drug may differ significantly in their biological effect
(
sometimes even having an opposite effect), as well as in their
bioavailability; rate of metabolism; metabolites; excretion; potency and
selectivity for receptors, transporters and/or enzymes; and toxicity [1].
Among chiral compounds, (R)- or (S)- 1-phenylethanol is of special in-
terest because it is a high-value intermediate for drug and fine chemical
production [2,3]. Enantiomerically pure 1-phenylethanol can be
Apart from the necessity for highly efficient chiral drug preparation,
the pharma industry typically generates significant amounts of waste,
by-products and pollutants (e.g. contaminated solvents, depleted re-
agents, air pollutants, etc.). Therefore, the incorporation of green
Abbreviations: CALB, Candida antarctica lipase B; ChGlc, Choline chloride:glucose; ChGly, Choline chloride:glycerol; ChEG, Choline chloride:ethylene glycol;
EGGlcFru, Ethylene glycol:glucose:fructose; GlcGly, Glucose:glycerol; GlcEG, Glucose:ethylene glycol; NADES, Natural deep eutectic solvents; SD, Standard devia-
tion; SorEG, Sorbose:ethylene glycol.
*
Corresponding author.
E-mail address: mcvjetko@pbf.unizg.hr (M. Cvjetko Bubalo).
https://doi.org/10.1016/j.procbio.2020.12.001
Received 10 September 2020; Received in revised form 9 November 2020; Accepted 1 December 2020
Available online 3 December 2020
1
359-5113/© 2020 Elsevier Ltd. All rights reserved.

M. Pani ´c et al.
Process Biochemistry 102 (2021) 1–9
chemistry approaches for environmentally sustainable preparations
have also become an imperative [5]. The basics of green chemistry
suggest that chemical products and processes should be designed to
avoid the application and creation of harmful and dangerous reagents,
catalyst and products, with the unique goal of protecting the environ-
ment by inventing new chemical processes that do not pollute, rather
than depending on clean-up. Numerous tools have been studied for
green chemistry, such as the use of alternative solvents and environ-
mentally benign raw (or renewable) materials, the use of alternative
energy sources and performing reactions by biocatalysis [6].
Sigma–Aldrich, Germany (purity of ≥99 %) and used without further
purification.
Reaction mixtures were analysed by gas chromatography on a Shi-
madzu QP2010PLUS instrument equipped with Varian CHIRASIL-DEX
CB capillary chiral column (25 m ×0.25 mm ×0.25
μm) and a mass
spectrum (MS) detector.
2
.2. Preparation and characterisation of NADES
◦
Choline chloride was dried at 60 C for 24 h in a vacuum concen-
An overview of the current literature on chiral drug production and
formulation reveals exciting, new, efficient and green approaches,
particularly involving the use of natural deep eutectic solvents (NADES)
as media for the biochemical preparation of chiral biologically active
compounds and as auxiliaries in the development of chiral drug delivery
systems [7,8]. NADES have become promising green solvent from both
environmental and technological perspectives. These solvents are mix-
tures of cheap, natural, non-toxic and readily available components
prepared by mixing quaternary ammonium salts (e.g. choline chloride)
and hydrogen bond donors based on natural products (e.g. alcohols,
organic acids, sugars, vitamins and amines), as well as water in some
cases, in a specific molar ratio. The end results is a homogenous solution
based on hydrogen bonds between the NADES components [9,10].
NADES fully represent green chemistry principles owing to their
specific properties, which include non-volatility, non-flammability and
stability, together with a low ecological footprint. For these reasons,
NADES are almost ideal solvents for a wide range of fields ranging from
biocatalysis, extraction and electrochemistry to carbon dioxide capture
and biomedical applications. NADES may be considered ‘designer sol-
vents’ due to their numerous structural variations and the possibility to
design their physicochemical properties for optimal performance in a
specific process [11]. NADES are produced from naturally occurring
molecules; therefore, they have an inherently low toxicity while also
providing a natural environment for proteins, enzymes and other bio-
logically active molecules [12].
trator (Savant SPD131DDA SpeedVac Concentrator, Thermo Scientific,
USA) prior to use. The following components, in specific ratios, together
with 10, 30 or 50 % (v/v) water, were placed in a round-bottomed glass
flask: choline chloride:glucose (ChGlc, 1:1), choline chloride:glycerol
(
ChGly, 1:2), choline chloride:ethylene glycol (ChEG, 1:2), glucose:
glycerol (GlcGly, 1:2), glucose: ethylene glycol (GlcEG, 1:2), sorbose:
ethylene glycol (SorEG, 1:2) or ethylene glycol:glucose:fructose
(
(
EGGlcFru, 2:1:1). The mixture of HBA (hydrogen bond acceptor), HBD
◦
hydrogen bonf donor) and water was stirred in a flask at 50 C for 2 h.
The pH value and polarity of the prepared solvents were measured ac-
cording to Pani ´c et al. [19].
2
.3. Lipase catalysed hydrolysis of (R,S)-1-phenylethyl acetate
For initial screening of NADES, the reaction was started by adding 5
mg Novozym 435 to 1.0 mL solvent (buffer or NADES) containing 0.05
ꢀ 1
mol L (8.2 mg) (R, S)-1-phenylethyl acetate. Reactions without the
enzyme were also performed. A separate reaction was conducted for
each measurement point.
The lipase-catalysed hydrolysis is represented as Eq. (1):
The synergistic use of NADES and biotechnological methods (i.e. the
use of enzymes as catalysts) fits logically with the efficient and sus-
tainable production of enantiomerically pure compounds. Biotechno-
logical approaches (e.g. biocatalysis) ensure the catalysis of otherwise
difficult transformations with high regioselectivity, chemoselectivity
and enantioselectivity under mild and cost-effective conditions, while
the use of NADES can provide strong green support to modulate/direct
the reaction route to obtain the desired product. Previous papers dealing
with the use of NADES as media for enantiopure compound preparation/
recovery have implied that the unique properties of these solvents make
them promising candidates for reactions catalysed either by isolated
enzymes or by whole cells. NADES use can improve substrate/product
solubility, enhance enzyme activity and stability, increase reaction
yields, provide the possibility of tailoring the reaction enantioselectivity
and regioselectivity and allow NADES recycling and reuse [13–18].
The present study presents the development of green lipase-catalysed
process for the production of an optically pure secondary alcohol (R)-1-
phenylethanol, through screening of the NADES, optimization of the
process, followed by isolation of the product and scale-up. The results
were also used to formulate postulates for the development and scale-up
of eco-friendly biocatalytic technology using NADES.
(
1)
At specified time intervals, the reaction was stopped, 1 mL water was
added and the reaction mixture was extracted with 9 mL n-heptane on
vortex shaker (3 min). The organic phase was analysed by gas chro-
◦
matography under the following conditions: injector (220 C); detector
◦
◦
◦
◦
◦
ꢀ 1
(200 C); column temperature 80 C for 2 min, then 80 C – 140 C (5 C
ꢀ 1
min ); and helium as the carrier gas at a flow rate of 96.9 mL min .
Substrates and products were identified with external standards and the
quantified using calibration curves of (R)-1-phenylethanol (0.409–35
ꢀ 1
mmol L ).
The efficiency of the proposed biocatalytic reaction was monitored to
determine the initial reaction rate (k ), conversion (X), enantiomeric
excess (ee) and volumetric productivity (V
), (Eq. 2–5). The initial re-
, mmoL L min ) was calculated from the linear parts of
0
p
ꢀ 1
ꢀ 1
action rate (k
o
the plots of product concentration vs reaction time, according to Eq. (2):
p
k
0
= a
(2)
t
2
. Material and methods
is the slope of the line (mmol L 1) and t is time.
ꢀ
where a
p
2
.1. General
The reaction conversion (X, %) was calculated according to Eq. (3):
X = cc × 100
A
(3)
Novozym 435 (lipase B from Candida antarctica; immobilised on
AT
macroporous polyacrylic resin beads) was obtained from Novozym
Bagsvard, Denmark). (R)-1-phenylethyl acetate, (S)-1-phenylethyl ac-
(
is the concentration of (R)-1-phenylethanol (mol L 1) and cAT
ꢀ
where c
A
etate, (R)-1-phenylethanol, (S)-1-phenylethanol, n-heptane, ethyl ace-
is the maximal theoretical concentration of (R)-1-phenylethanol (mol
ꢀ 1
tate and all chemicals for NADES preparation were purchased from
L ).
2

M. Pani ´c et al.
Process Biochemistry 102 (2021) 1–9
Enantiomeric excess (ee, %) was calculated according to Eq. (4):
In all 15 experiments, the reaction was stopped at specified time
intervals and the products were analysed by GCMS, as described in
section 2.3.
(
R
R
OH ꢀ SOH
OH + SOH
)
)
ee =
× 100
(4)
(
where ROH is the area under the curve of (R)-1-phenylethanol (mol L 1)
ꢀ
2
.6. Downstream process for enzyme and NADES recovery at the
and SOH is the area under the curve of (S)-1-phenylethanol (mol L 1).
ꢀ
laboratory scale
Productivity (VP, mmol L 1 min ) was calculated according to Eq.
ꢀ
ꢀ 1
(
5):
Upon completion of the kinetic resolution of (R,S)-1-phenylethyl
◦
c
p2 ꢀ cp1
acetate under optimal reaction conditions (50.4 C, 47.75 % of water in
V
P
=
(5)
ChGly and 5 h 52 min), the reaction medium containing the Novozym
t
4
35 was filtered under vacuum. The recovered enzyme was washed with
ꢀ 1
where cP1 is the concentration of (R)-1-phenylethanol (mol L ) at 0 h,
water (5 × 10 mL) and ethyl acetate (5 × 10 mL), dried in an oven at 40
P2 is the concentration of (R)-1-phenylethanol (mol L 1) at the end of
ꢀ
c
◦
C overnight and stored in a fridge until the next run. A 5 mg sample of
the reaction, and t is the time of the reaction (min).
the recovered Novozym 435 was weighed and used again for hydrolysis
of (R,S)-1-phenylethyl acetate in fresh ChGly with 50 % water. The re-
action was monitored and analysed as described in section 2.3. This
recycling and reuse of the enzyme was repeated for 5 cycles.
All experimental measurements were performed in triplicate, and the
average values were reported (at 95 % confidence intervals, the results
have no statistically significant differences).
Enzyme reusability ( relative, %) was calculated according to Eq. (8):
η
2
.4. Storage stability of the enzyme
ηrelative (%) = ηrun (1+n)∙100, n = 1, 2, 3… .
(8)
ηrun (1)
The storage stability of Novozym 435 in the prepared solvents was
measured according to the procedure reported by Cvjetko Bubalo et al.
15]. Briefly, the enzyme was incubated in ChGly and ChEG with 10, 30
where
η
run(1) is the reaction yield with fresh enzyme and
η
run(1+n) is the
[
reaction yield with the enzyme after recycling once, twice...,five times.
NADES recycling and recovery was performed as a separate experi-
ment. The kinetic resolution of (R,S)-1-phenylethyl acetate in optimal
conditions was stopped after 5 h 52 min, the enzyme was filtered from
the reaction medium, and the reaction medium was placed in a sepa-
ratory funnel and extracted with ethyl acetate (5 × 10 mL). The organic
phases were collected and evaporated under reduced pressure to yield
crude products. The regenerated NADES were tested for all four rounds
of assays.
and 50 % water or in phosphate buffer at room temperature. At the
specified time intervals, the enzyme reaction was initiated by adding the
butyl acetate substrate and the reaction was monitored over time. The
results were presented as residual enzyme activity.
The residual activity (A, %) was calculated according to Eq. (6):
A = k1
(6)
k
0
where k
1
is the initial reaction rate obtained by the enzyme after incu-
NADES reusability (Vrelative, %) was calculated according to Eq. (9):
bation in a given solvent and k
0
is the initial reaction rate obtained
relative (%) = V
run (1+n)
V
∙100, n = 1, 2, 3…
run (n)
(9)
without prior incubation.
V
Inactivation rate constants (K
d
) were calculated according to first
order kinetics using StatSoft Statistica version 8.0 software.
where, Vrun(1) is the volume of NADES in the n-cycle and Vrun(1+n) is the
volume of the recycled NADES after each n-cycle.
2
.5. Optimisation of (R)-1-phenylethanol synthesis in NADES
The reaction conversion in the recycled NADES was calculated ac-
cording to Eq. (3).
The kinetic resolution of (R,S)-1-phenylethyl acetate in ChGly was
The (R)-1-phenylethanol and unreacted (S)-1- phenylethyl acetate
from the organic fraction were separated on a silica gel column (1.2 cm
optimised using the Box-Behnken design. The influence of the inde-
pendent variables of time (X
, v/v) and temperature (X
1
, 1–6 h), water content in ChGly (X
3
2
, 10–50
×
5 cm) using n-heptane:ethyl acetate (4:1, v/v) as the mobile phase.
◦
%
, 20–60 C) was studied on the dependent
Separation efficiency was calculated according to Eq. (10):
variables of the reaction conversion (Y, X, %).
m
k
The extraction process was optimised by performing 15 experiments
with 3 centre points per block, and the responses were analysed using
the numerical tools provided by Design Expert (Version 7.0.0., Suite
η
=
(10)
m
0
where m
k
is the mass of the (R)-1-phenylethanol after the silica gel
4
80, Minneapolis, MN 55,413). Each experiment was performed with 5
0
column separation and m is the mass of (R)-1-phenylethanol achieved
ꢀ 1
mg Novozym 435 and 1 mL ChGly containing 0.05 mol L (8.2 mg) (R,
S)-1-phenylethyl acetate. The responses were fitted with second-order
polynomial equation (response surface methodology [RSM] model), as
described by Sontakke [20],
by hydrolysis.
2.7. Preparative scale of kinetic resolution of (R,S)-1-phenylethyl acetate
in ChGly and isolation of (R)-1-phenylethanol
k
k
kꢀ 1
k
∑
∑
∑ ∑
2
Y = β +
βiX
i
+
β X +
βijX
i
X
j
i < j
(7)
0
ii
i
A 250 mg sample of Novozym 435 was added to 300 mL choline
i=1
i=1
i=1 j=2
ꢀ 1
chloride:glycerol in 47.75 % water containing 0.05 mol L (2.5 g) (R,
◦
where X
1
, X
, βj (i = 1, 2,.. ., k), βii (i = 1, 2,.. ., k), and βij (i = 1, 2,.. ., k; j =
, 2,.. ., k) are the regression coefficients for the intercept, linear,
2
,.. ., X
k
are the independent variables, Y is the dependent
S)-1-phenylethyl acetate. The reaction was run at 50.4 C for 5 h 52 min
variable, β
1
0
on an ES-20/60 Orbital Shaker-Incubator. The Novozym 435 was then
filtered from the reaction medium under vacuum. The corresponding
(R)-1-phenylethanol was isolated by extraction using ethyl acetate as the
renewable solvent. The extraction was performed in 5 cycles, and the
ethyl acetate fraction was collected each time. The (R)-1-phenylethanol
and unreacted (S)-1- phenylethyl acetate from the organic fraction were
separated on a silica gel column, as described in section 2.6.
quadratic and interaction terms, respectively, and k is the number of
variables.
The second-order polynomial coefficients were calculated by
applying analysis of variance (ANOVA) using Design Expert (Version
7
.0.0., Suite 480, Minneapolis, MN 55,413) software. The value of the
2
determination (R ) was used to predict the model capability.
The reaction yield was calculated according to Eq. 11:
3

M. Pani ´c et al.
Process Biochemistry 102 (2021) 1–9
Fig. 1. Flowchart of experimental design and results of development of an environmentally friendly method for lipase-catalyzed (R)-1-phenylethanol production in
natural deep eutectic solvents (NADES).
m1
economic and ecological points of view. For that purpose, the kinetic
resolution of lipase catalysis of (R,S)-1-phenylethyl acetate within
NADES was performed. For this research, the following major points
governed the design of the biocatalytic reaction involving NADES: (i)
screening of NADES for optimal enzyme performance; (ii) optimisation
of the biocatalytic protocol; and (iii) preparative-scale upstream and
downstream (R)-1-phenylethanol synthesis (Fig. 1).
η
=
(11)
is
mo
where m
1
is the mass of the (R)-1-phenylethanol after reaction and m
0
the mass of (R)-1-phenylethyl acetate before the biocatalytic reaction.
2
.8. Data analysis
All experimental results were statistically analysed using Statistica 8
software. Data in the text and tables are expressed as the mean ±
standard deviation (±SD), and error bars in the figures indicate the SD.
The differences between the means were analysed by ANOVA, followed
by a post-hoc Tukey’s test. A p value < 0.05 was considered statistically
significant.
3.1. NADES selection for optimal lipase-catalysed performance for (R)-1-
phenylethanol synthesis
This work examined the enantioselective hydrolysis of (R, S)-1-
phenylethyl acetate to (R)-1-phenylethanol by immobilised Candida
antarctica lipase B (Novozym 435) in 8 different cholinium-based or
cholinium-free NADES (Table 1S). Enantioselective hydrolysis was also
performed in 0.025 M potassium phosphate buffer as a reference sol-
vent. Blank experiments without the enzyme were also performed for all
tested NADES, and no conversion was observed within the reaction
3
. Results and discussion
The aim of this study was to develop a NADES-assisted protocol for
the synthesis of (R)-1-phenylethanol that would be of interest from both
4

M. Pani ´c et al.
Process Biochemistry 102 (2021) 1–9
Fig. 2. Initial reaction rates (A), conversion (X) (a) and productivity (Vp) (b) of
Novozym 435—mediated hydrolysis of (R,S)-1-phenylethyl acetate within
different natural deep eutectic solvents (NADES) (50 %, v/v) and buffer. Re-
action conditions: 0.05 M (R,S)-1-phenylethyl acetate; 5 mg Novozym 435; 25
◦
C; 1 mL. Each data point represents the mean of triplicate experiments; error
bars represent the standard deviation (SD). Every group of results followed by
nonidentical letters (a-e and A
–
E) indicate signifcant diff ;erence (p < 0.05)
determined by Tukey’s HSD test.
Fig. 3. Residual activity of Novozym 435 after incubation in choline chloride:
ethylene glycol (ChEG) (a) choline chloride:glycerol (ChGly) (b), choline
◦
times used in this study (data not shown).
chloride:glucose (ChGlc), and buffer (c) at 25 C. Reaction conditions: 0.1 mol
ꢀ 1
◦
The selected NADES were weakly acidic to neutral, since lipase is
well known to perform reactions under mild conditions (pH and tem-
perature), with remarkable chemoselectivity, regioselectivity and ster-
eoselectivity [6,21]. The price of using NADES was estimated based on
L
butyl acetate; 5 mg Novozym 435; 25 C; 1 mL of solvent. Each data point
represents the mean of triplicate experiments; error bars represent the standard
deviation (SD).
ꢀ
1
the cost of raw materials and varied from approximately 5.57
€
kg for
NADES (ChGly, ChEG and ChGlc) compared to the reference solvent
(buffer); however, the stability of the enzyme was significantly
enhanced in certain NADES (Fig. 2 and 3). The influence of NADES on
lipase-catalysed reaction mechanism was previously proposed by
Cvjetko Bubalo et. al. [15]. Authors suggested that lipase activity in
cholinium-based NADES containing polyols as HBD could be explained
by increase in nucleophilicity of the hydroxyl group of the serine residue
in active lipase site and by the fact that this solvent supports stabilization
of tetrahedral inter mediates through H-bonding, leading to decrease in
the activity energy barrier and therefore increase the reactivity. The
ꢀ
1
GlcGly up to 656.89
€
kg for SorEG, in agreement with the cost of
conventional solvents (Table 1S).
The results indicated that lipase exhibits a strong R-stereopreference,
regardless of the solvent choice (ee~99 %). This stereopreference is
advantageous, as it allows the direct production of the desired R-alcohol
without further chemical manipulation. Rios et al. [2] also noted
enantioselectivity of a lipase to hydrolyse only (R)-phenylethyl acetate
in a racemic mixture of this ester. Conversely, the measured initial re-
action rates (k
o
), conversion (X) and productivity (V
, X and V
p
) showed a
ranged from
◦
dependence on the NADES used. The values for k
o
p
storage stability of Novozym 435 was monitored at 25 C for 28 days in
ꢀ
1
ꢀ 1
0
.1323–0.369 mmoL L min , 11.69–41.14 % and 0.00456–0.01905
ChGly, ChEG, ChGlc and buffer (Fig. 3). ChGlc with 10 % of water was
too viscous for manipulation, so no experiments were performed in that
solvent. In summary, no differences were observed in Novozym 435
stability in the various solvents at room temperature.
ꢀ 1
ꢀ 1
mmoL L min , respectively (Fig. 2). The highest k
the conventional solvent, potassium-phosphate buffer.
No improvement was observed in the X and V values in the optimal
o
was observed in
p
5

M. Pani ´c et al.
Process Biochemistry 102 (2021) 1–9
Table 1
Table 2
Deactivation constants (K
d
) of Novozym 435 under different inactivation sol-
Experimental matrix and values of observed response. The numbers in bracket
are level factorials of experimental matrix.
◦
vents on 25 C during 30 days.
ꢀ
1
◦
Solvent
Abbreviation
water content in
solvent [%, v/v]
K
d
[day
]
Run
t [h]
w
(H2O,NADES) [%, w/w]
T [ C]
X [%]
1
3.5(0)
3.5(0)
3.5(0)
6(+1)
3.5(0)
1(-1)
10(-1)
50(+1)
50(+1)
50(+1)
30(0)
20(-1)
60(+1)
20(-1)
40(0)
20.02
50.52
24.90
42.03
35.99
36.65
41.06
27.11
35.71
21.44
24.20
35.75
31.58
55.28
19.74
0
0
.0244 ±
2
1
0
.00699
3
Choline chloride:
ethylene glycol
0.0428 ±
4
ChEG
30
0.00113
5
40(0)
0
0
0
0
.0519 ±
.0103
6
30(0)
60(+1)
60(+1)
20(-1)
40(0)
5
1
0
0
7
3.5(0)
6(+1)
3.5(0)
1(-1)
10(-1)
30(0)
.015 ±
.00125
8
9
30(0)
Choline chloride:
glycerol
0.0249 ±
10
11
12
13
14
15
10(-1)
50(+1)
30(0)
40(0)
ChGly
ChGlc
30
0.00840
1(-1)
40(0)
0
0
0
.0401 ±
.00959
3.5(0)
6(+1)
6(+1)
1(-1)
40(0)
5
3
5
0
0
0
10(-1)
30(0)
40(0)
.0215 ±
60(+1)
20(-1)
Choline chloride:
glucose
0.0303
30(0)
0.0388 ±
*t -time, w(H2O,NADES)- water content in NADES, T- temperature.
0
0
0
.0422
.0317 ±
.0116
buffer
Table 3
Analysis of variance (ANOVA) of the modelled responses.
The only exception was ChGly, where immobilised Candida
antarctica lipase B showed a much higher stability (ꢀ 80 % of initial
activity, 28 days) compared to buffer (ꢀ 40 % of initial activity, 28 days)
Source
Conversion (Quadratic)
Sum of squares
Mean square
F-value
p-value
(
Fig. 3). This finding implied that NADES could serve as a stabilising
Model
1640.42
364.08
94.82
1052.04
14.79
31.72
5.25
182.27
364.08
94.82
1052.04
14.79
31.72
5.25
906.56
1810.83
471.63
5232.61
73.57
< 0.0001
< 0.0001
< 0.0001
< 0.0001
0.0004
X
X
X
X
X
X
X
X
X
1
2
3
1
1
2
2
1
2
2
2
3
medium for this lipase. Similarly, when monitoring the stability of
Candida antarctica lipase B immobilisation with cross-linking aggre-
gates, Guajardo et al. also noted a higher stability of the enzyme in
ChGly than in other NADES (ChEG and ChU) [23]. These results
regarding storage stability of Novozym 435 in ChEG are in agreement
with the results of Cvjetko Bubalo et al. [15], who found an approxi-
mately 50 % decrease in the initial lipase activity after a 3 day incuba-
tion of the enzyme in ChEG. Nevertheless, extending the incubation time
up to 15 days did not result in any significant enzyme activity loss [15].
In the present study, NADES stabilised the enzyme. Zhou et al. used
circular dichroism spectroscopy to examine the effect of DES (deep
eutectic solvents) on the structural integrity of Candida antarctica B for a
deeper understanding of the stability of the enzyme [24]. They
concluded that the increased stability of the enzyme was due to the
preservation of its structural integrity in DES.
X
2
X
3
X
3
157.76
26.13
< 0.0001
0.0037
27.20
39.90
9.41
27.20
39.90
9.41
135.29
198.47
46.81
< 0.0001
< 0.0001
0.0010
Residual
Lack of fit
Pure error
Total
1.01
0.20
0.96
0.32
13.81
0.0683
0.046
1641.42
0.9994
0.02
2
R
p< 0.01 highly significant; 0.01 ≤ p < 0.05 significant; p ≥ 0.05 not significan.
X
1
:time; X
2 3
: water content in ChGly; X : temperature.
2
5], whereas lower amounts of water (approx. 30 %) could negatively
d
The inactivation rate constants (K ) were also calculated (Table 1).
influence hydrolysis. Apart from water, other well-known process pa-
rameters that need optimisation are the reaction temperature and the
substrate(s) concentration [26–28]. It is well known that raising tem-
perature up to the temperature optimum speeds up enzyme-catalyzed
reactions, whereas high temperatures (depending of enzyme origin
and type) cause an enzyme to lose its active shape. Adjusting reaction
temperature in DES-mediated biocatalytic reactions is especially
important as elevation of temperature reduces viscosity of DES, enabling
better mass transfer in such viscous environment and consequently
higher reaction rate [22,29]. Based on our preliminary data and the
current literature, the following independent variables for RSM analysis
were selected in this study: reaction time (1–6 h), water content in
The rate of enzyme inactivation was the highest in ChEG with 50 %
ꢀ 1
water (0.0519 day ) and the lowest in ChGly with 10 % water (0.0150
day 1). The measured K
ꢀ
d
values decreased with a higher content of
NADES. The dependence of Novozym 435 deactivation on the water
content in NADES was also reported in a previous study of the influence
of water content in ChGly for the Candida antarctica lipase B-catalysed
reaction of acetic anhydride with 1-butanol to give the butyl acetate
ester [15].
Based on the experimental data on lipase behaviour in the tested
NADES and the enzyme stability in NADES, ChGly was selected as the
most promising solvent for the given reaction; therefore, it was used for
further optimisation.
◦
ChGly (10–50 %) and temperature (20–60 C). The enzyme-to-substrate
ratio was fixed at 1.3 according to our preliminary results (data not
shown).
3
.2. Laboratory scale process optimisation
These process parameters were subsequently optimised using an
RSM-based experimental design (Box-Behnken design). The software
package experiments were designed using Design Expert 7.0.0, the re-
sults were evaluated and the conversion of kinetic resolution of (R,S)-1-
pnenylethylacetate to (R)-1-phenylethanol was optimised. The obtained
reaction conversion varied from 19.09 to 55.28% (Table 2.). The lowest
The biocatalytic process has two parts which should be optimised:
the upstream (product development) and downstream (product purifi-
cation) parts. After choosing the optimal NADES and optimisation of the
pH, polarity and viscosity of the medium, the next step was optimisation
of the biocatalytic protocol. A search of the literature revealed that the
amount of water in the NADES, the temperature, the enzyme-to-
substrate ratio, the pH and the reaction time all influenced the
enzyme activity [6]. The presence of water in the NADES (>60 %, w/w)
may damage the strong hydrogen-bonding network of the NADES [24,
conversion for the investigated response was obtained using ChGly with
◦
1
0 % water, a time of 3 h 30 min and a temperature of 20 C.
The RSM model was evaluated and analysis of variance (ANOVA)
6

M. Pani ´c et al.
Process Biochemistry 102 (2021) 1–9
highly significant.
The experimental data acquired were used for the creation of sup-
plementary Fig. 1S and 2S, which showed that the hydrolytic conversion
of (R,S)-1-phenylethyl acetate to (R)-1-phenylethanol increased with the
reaction temperature and reaction time. Conversely, the water content
in the ChGly had a double effect on each response. An increase in water
content from 10 % to 45 % in the ChGly resulted in an increased con-
version, whereas a further increase led to a decreased conversion. This
once again indicates the importance of optimising the water content
when applying NADES as reaction solvents. The developed RSM model
showed that the optimum conditions for conversion were a time of 5 h
◦
5
2 min, a water content of 47.74 % and a temperature of 50.4 C. The
model predicted a conversion of 50.14 % as optimum. After performing
the experiment under these optimal conditions, the value of X was 52.30
±
1.11 %, which closely agreed with the predicted value.
The major cost in a biocatalytic process in industry lies in the
downstream purification [30]; therefore, the last steps in designing an
efficient biocatalytic process involving NADES are product isolation and
purification and enzyme and NADES recycling and reuse. For this
reason, downstream separation of (R)-1-phenylethanol from ChGly was
performed. When performing biocatalysis in an organic solvent, the
solvent is usually evaporated and the final product is further purified.
However, the low vapour pressure of NADES makes these solvents very
difficult to evaporate, which may pose a problem for industrial appli-
cations [31]. Only a few research groups have reported a downstream
separation of a product after a biocatalytic process in NADES [14,32,
3
3]. In the present research, the obtained R-alcohol and unreacted
(
S)-1-phenylethyl acetate were isolated by extraction using ethyl acetate
as the renewable solvent. Novozym 435 was removed by filtration, and
the (R)-1-phenylethanol and unreacted (S)-1-phenylethyl acetate from
the organic fraction were separated on a silica gel column using
n-heptane:ethyl acetate (4:1) as the mobile phase (Fig. 4). The yield of
the separated product was 81.24 %.
Fig. 4. Natural deep eutectic solvents (NADES) reusability and reaction con-
version in recycled NADES (a) and enzyme reusability (%) (b), of (R)-1-phe-
nylethanol synthesis in choline chloride:glycerol (ChGly) with 47.75 % of
water. Reaction conditions: 0.05 M (R,S)-1-phenylethyl acetate; 5 mg Novozym
After removing the product and unreacted substrate, the NADES was
recycled. The ChGly with 47.74 % water was also recycled and reused
for another round of (R)-1-phenylethanol production (Fig. 4). The yield
of the NADES ChGly after recycling five times decreased by 35 %
compared to the freshly synthesised solvent due to the downstream
procedures. Hydrolysis was also performed in the recycled NADES, and
the conversion and enantioselectivity in the recycled ChGly with 47.75
% water was similar to that achieved with freshly synthesised NADES.
After the isolation and purification of (R)-1-phenylethanol under the
RSM-optimised conditions (at a millilitre scale), Novozym 435 was also
recycled and reused. Novozym 435 was washed with ethyl acetate and
water, dried, and used again for the kinetic resolution of (R,S)-1-phe-
nylethyl acetate in (R)-1-phenylethanol in fresh ChGly. The procedure
was repeated four times (Fig. 4). At the end of the fifth cycle, the relative
reaction yield of Novozym 435 was 81.91 %, while the ee (>99 %) did
not decrease in any of the five repeated batches. Liang et al. also noticed
a similar effect when studying recycling of Novozym 435 in a buffer-
based media, as the ee value did not change through 11 cycles and the
relative activity was 70 % after the eleventh cycle [34]. By contrast, a
slightly larger decline in Novozym 435-activity was reported, at 47 %
after just five cycles of recycling in 92 % (v/v) ChGly [35].
◦
4
35; 40 C; 1 mL. Each data point represents the mean of triplicate experiments;
error bars represent the SD. Every group of results followed by nonidentical
letters (a-b and A
C) indicate signifcant diff ;erence (P < 0.05) determined by
Tukey’s HSD test.
–
was used to calculate the statistical significance of the model. The model
for the independent variable was selected according to this determina-
2
tion and the adjusted coefficient (R ) and the model p value (Table 3).
The second-order polynomial equations for conversion were as fol-
lows:
Conversion (% ) =
=
35.81 + 6.75∙X
1
+ 3.44∙X
2
+ 11.47∙X
3
+ 1.92∙X
1 2
X
2
2
+
+
2.82∙X
1.60∙X
1
X
2
3
+ 1.15∙X
2
X
3
ꢀ 2.71∙X
1
ꢀ 3.29∙X
2
3
1 2 3
where X is time, X is the water content in ChGly and X is the
temperature.
The results show that the independent variables had a significant
effect (p < 0.001). All four variables also had a positive coefficient,
whereby an increase in a specific variable led to an increased conver-
sion. One important point to mention is that ANOVA also showed sta-
tistical significance of both the quadratic and interaction factors.
ANOVA also showed good agreement between the data predicted by
the model and the experimental data, with a determination coefficient of
3
.3. Preparative scaling (up and down)
After laboratory scale optimisation, the biocatalytic reaction was
performed in a 500 mL batch reactor under the optimum conditions
determined at the laboratory scale, followed by the downstream process,
again following the conditions determined at the laboratory scale.
The preparative-scale upstream experiment proceeded as follows.
2
2
R = 0.9994, R adjusted = 0.9983 and with no significant lack of fit at p >
.05. The determination coefficient indicated that the proposed model
0
was able to explain 99.94 % of the results. The results also showed that
the model used to fit the response variable was significant (< 0.0001)
and suitable for description of the relationship between the independent
variables and the response. The F-test suggested that the model had a
very high model F-value (F = 906.56), indicating that this model was
ꢀ 1
Novozym 435 was used for hydrolysis of 0.05 mol L (R,S)-1-phenyl-
ethyl acetate in an overall reaction volume of 300 mL ChGly with 47.75
◦
%
water at 50.4 C for 5 h 52 min. The reaction conversion was 54.57 %,
similar to that achieved at the laboratory scale. The enantioselectivity
7

M. Pani ´c et al.
Process Biochemistry 102 (2021) 1–9
Table 4
Acknowledgements
Summary of up- and down-stream results of biocatalytic process on laboratory
and preparative scale.
The work was supported by the Croatian Science Foundation (Grant
No. 9550and7712).
Upstream
Downstream
X
η
ChGly
η(R)-1-phenyethanol purification [%]
Appendix A. Supplementary data
[
%]
[%]
Laboratory scale
Preparative scale
50.14
54.57
100ꢀ 65.5
81.24
49.48
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.procbio.2020.12.001.
97
was the same as for the laboratory scale experiment (Table 4). The
available literature indicates that the scalability of NADES-assisted
biocatalysis has been performed only for a preparative scale (up to
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