Cellulose as an efficient matrix for lipase and transaminase immobilization

Article abstract of DOI:10.1039/c5ra24976g

Immobilization of enzymes is important to improve their stability and to facilitate their recyclability, aiming to make biocatalytic processes more efficient. One of the important aspects is the utilization of cheap, abundant, and environmentally friendly carriers for enzyme immobilization. Here we report the use of functionalized cellulose for lipase and transaminase immobilization. High immobilization efficiencies (up to 90%) could be achieved for the transaminase from Vibrio fluvialis. For immobilized lipase CAL-B as well as the transaminase, good conversions and recyclability could be demonstrated in kinetic resolutions to afford chiral alcohols or amines. Moreover, such application of the immobilized transaminase enabled very high conversions in a continuous-flow process in the asymmetric synthesis of (S)-phenylethylamine (80% conversion, >99% ee).

Full text of DOI:10.1039/c5ra24976g

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Dawood, RSC Adv., 2015, DOI: 10.1039/C5RA24976G.
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Cellulose as an Efficient Matrix
for Lipase and Transaminase Immobilization
Stefânia P. de Souza, a Ivaldo I. Junior, b Leandro S. M. Miranda, a Marcelo F.
Santiago, c Frank LeungꢀYuk Lam, d Ayad Dawood, e Uwe T. Bornscheuer, e*
Rodrigo O. M. A. de Souza, a*
aꢀ Biocatalysis and Organic Synthesis Group, Chemistry Institute, Federal University of Rio
de Janeiro, Rio de Janeiro, Brazil;
bꢀ School of Chemistry, University of Rio de Janeiro, Rio de Janeiro, Brazil;
cꢀ Institute of Biophysics Carlos Chagas Filho (IBCCF), Federal University of Rio de Janeiro
(UFRJ), Rio de Janeiro, Brazil
dꢀ Department of Chemical and Biomolecular Engineering, The Hong Kong University of
Science and Technology, Hong Kong SAR, China
eꢀ Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis, Greifswald
University, Greifswald, Germany
Keywords: Lipase, transaminase, cellulose, immobilization, chiral alcohol, chiral
amine, recycling
Abstract: Immobilization of enzyme is important to improve their stability and to
facilitate their recyclability, aiming to make biocatalytic processes more efficient.
One of the important aspects is the utilization of cheap, abundant, and
environmentally friendly carriers for enzyme immobilization. Here we report the use
of functionalized cellulose for lipase and transaminase immobilization. Highest
immobilization efficiencies (up to 90%) could be achieved for the transaminase from
Vibrio fluvialis. For immobilized lipase CALꢀB as well as the transaminase, good
conversions and recyclability could be demonstrated in kinetic resolutions to afford

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chiral alcohols or amines. Moreover, such application of the immobilized
transaminase enabled very high conversions in a continuousꢀflow process in the
asymmetric synthesis of (S)ꢀphenylethylamine (80% conversion, >99% ee.).
Introduction
1
As already pointed out by Sheldon & Van Pelt, enzymes are Nature’s
sustainable catalyst. This statement is based on the mild conditions required for
performing the requested transformation in addition to biocompatibility and
biodegradability characteristics. 2 Producing large quantities of an efficient and stable
biocatalyst is still an important challenge even if since the 90’s advances in
biotechnology and protein engineering have provided researchers with better tools for
the discovery and improvement of enzymes by means of genetic manipulations. 3
The commercial large scale application of enzymes is not hampered by a lack
of immobilization protocols, since it is easy to find different methods for enzyme
4ꢀ11
immobilization in the literature,
but rather because most of them use expensive
carrier materials with prices being prohibitive for largeꢀscale operations. Hence,
finding a cheap and readily available support for enzyme immobilizations is crucial
for the development of new immobilized biocatalysts. Cellulose is the most abundant
biopolymer consisting of a linear polysaccharide chain composed of Dꢀglucose units
linked by β(1ꢀ4) glycosidic bonds, fully decorated with hydroxyl groups. Since the
discovery of cellulose in 1839 by Anselme Payen, this natural biopolymer has been
used for many different applications ranging from thermoplastic polymers to cellulose
nanofibers. 12ꢀ15
The use of cellulose as a matrix for enzyme immobilization dates from the
beginning of the seventies when Kennedy and coꢀworkers have modified cellulose for

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chymotrypsin A immobilization. 16ꢀ18 A few years later lipases were also immobilized
on cellulose by different methods and successfully used for hydrolytic and
19
esterification reaction. As a cheap and abundant biopolymer, cellulose occurs as a
very interesting matrix for enzyme immobilization representing an affordable
immobilized biocatalyst for largeꢀscale operations.
In continuation of our efforts on the development of carriers and protocols for
20ꢀ26
enzyme immobilization,
herein we report the functionalization of cellulose
followed by enzyme immobilization (exemplified for lipase and transaminase) via
covalent bonds and its application in the production of chiral alcohols and amines.
Materials and Methods
Materials:
Cellulose,
3ꢀaminopropyltriethoxysilane
(APTES),
(3ꢀ
Glycidyloxypropyl)trimethoxysilane (Glymo) and glutaraldehyde were purchased
from SigmaꢀAldrich. Immobilized Candida antarctica lipase B (Novozyme 435,
NZ435 – 30 mg/g of support) was purchased from Novozymes as well as free
Candida antarctica lipase B on phosphate buffer (CalꢀB, 5.7 mg/mL). GDH-105
(Lot# D12010) and LDH-101 (Lot# A10036) was purchased from Codexis Inc
(Redwood City, CA). All other materials were at least reagentꢀgrade.
Preparation of APTES functionalized cellulose: (3-Aminopropyl)trimethoxysilane,
APTES (5 mL) and tetrahydrofuran (50 mL) were mixed and 500mg of cellulose were
added. This mixture was stirred at room temperature for 3 h. Then the solvent was
evaporated and 10 mL phosphate buffer pH 7 (50 mM) was added to the product,
followed by 2 mL glutaraldehyde. This mixture was stirred at room temperature for
24 h. The product was filtered and washed repeatedly with distilled water to eliminate
unreacted residues.

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Preparation
of
Glymo
functionalized
cellulose:
(3ꢀ
Glycidyloxypropyl)trimethoxysilane, Glymo (5 mL) and tetrahydrofuran (50 mL)
were mixed and 500 mg cellulose were added. This mixture was stirred at room
temperature for 5 h. Then the solvent was evaporated and the product was filtered and
washed repeatedly with distilled water to eliminate unreacted residues.
Vibrius fluvialis transaminase production: Overnight cultures of VflꢀTAꢀWT (in
pET24b vector with kanamycin resistance) were used for inoculation of 150 mL TB
media containing 50 µg/mL kanamycin. When OD600 of 0.6 was reached IPTG was
added for induction in a final concentration of 0.2 mM. The expression occurs at 20°C
for 18ꢀ20 h. The harvesting of cells was performed at 4000 x g for 15 min and 4°C.
French Press at 1700 psi was used for cell disruption after reꢀsuspension in 50 mM
NaPP pH 7.5, 0.1 mM PLP and 300 mM NaCl. The subsequent centrifugation step
was done at 37 000 x g for 1 h and 4°C. The crude lysate was filtered with a 0.2 µm
filter and stored at 4°C.
Immobilization conditions. The support (functionalized cellulose) was subjected to
the following immobilization process:
Lipase immobilization protocol: 2 mL Lipase B from Candida antarctica enzyme
solution (5.7 mg/mL) was dissolved in 10 mL 0.025 mM phosphate buffer pH 7.0 and
added to support (2 g). The mixture was stirred for 24 h at 40 °C using a flask shaker
or thermomixer, before being filtered and dried under vacuum followed by drying
over night at ambient temperature. Immobilization efficiency was evaluated by the
difference between initial amount of enzyme added and that in the supernatant after
filtration of the immobilized enzyme.
Transaminase immobilization protocol: 5 mL Vibrius fluvialis transaminase solution
(8.8 mg/mL) was dissolved in 30 mL 50 mM phosphate buffer pH 7.5 and added to

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the support (1 g) The mixture was stirred for 24 h at 30 °C using a flask shaker or
thermomixer, before being filtered and dried under vacuum followed by drying over
night at ambient temperature. Immobilization efficiency was evaluated by the
difference between the initial amount of enzyme added and that in the supernatant
after filtration of the immobilized enzyme.
Esterification reactions. The immobilized lipase (10 mg of support in 1 mL reaction
media) was evaluated in an esterification reaction between oleic acid and ethanol (1:1,
100 mM in nꢀheptane) at different temperatures. The reactions were performed in
cryotubes at 200 rpm agitation on a shaker. Samples (10 ꢁL) were collected after 1 h.
For calculation of the initial reaction velocity, the reaction times were varied from 5,
10, 15, 20, and 30 min. For thermal stability, the reaction was investigated at 50–70
°C. Quantification of esters formed was performed by GCꢀMS analysis.
Kinetic resolution using immobilized lipase. racꢀ1ꢀPhenylethanol (1 mmol, 122
mg), vinyl acetate (1 mol. eq.) as acyl donor, and 18 mg (15% w/w) of the
corresponding immobilized enzyme were reacted in cyclohexane (3 mL) for 2, 4 and
5 h at 60 °C. Enantiomeric excess values (ee) were determined by chiral GC analysis
(chiral column Betadexꢀ325, for details as shown in the section of “GC analysis”).
Discontinuous acetophenone assay
For immobilized transaminases a 24 deepꢀwell plate was heated in a thermomixer to
30 °C. A total of 50 mg wet immobilized transaminase was used for each
measurement. The reaction was started by adding 5 mL of preꢀwarmed reaction
solution (HEPES buffer 50 mM, pH 7.5, 2.5 mm (±)ꢀαꢀphenylethylamine ((±)ꢀαꢀ
PEA), 5 mM pyruvate, and 0.5% DMSO). Then, 200 ꢀL samples were taken and the
absorbance was measured at 245 nm. The slope was determined and conversions
calculated.

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Asymmetric synthesis using immobilized transaminase
Asymmetric synthesis of (S)ꢀphenylethylamine was performed using the GDH/LDH
28
system for shifting the equilibrium and cofactor recycling.27,
Reactions were
performed in a 5 mL mixture using 50mM HEPES buffer (pH 7.5), 10 mM
acetophenone, 250 mM alanine, 10 % DMSO, LDHꢀGDH (88 U/mL and 15 U/mL,
respectively), 1 mM NADH and 150 mM glucose, at 30 °C. For batch experiments,
100 mg immobilized enzyme was used in a total reaction volume of 5 mL on a
thermomixer at 800 rpm. The starting mixture was stirred for 5 min while the
instrument Asia Flow Reactor was equipped with Omnifit (6.6 mm x 10 mm with
0.3421 cm2 base and height 10 cm column) containing 2 g of the immobilized
catalysts. The reaction solution was pumped using an Asia syringe pump at different
flow rates in order to achieve the desired residence time. Samples were collected and
analysed by HPLC.
GC analysis. All GCꢀMS measurements were carried out in duplicate using a DB 5
(Agilent, J&W Scientific®, USA) capillary column (30 m × 0.25 mm × 0.25 ꢁm). The
GCꢀMS samples were prepared by dissolving 10 ꢁl of the sample in 98 mL of heptane
and 10 ꢁl of MSTFA (NꢀMethylꢀNꢀ(trimethylsilyl) trifluoroacetamide). 1 ꢁL of this
sample was then injected into the Shimadzu CG2010 GC. The injector and detector
temperatures were 250 °C and the oven temperature was constant at 60 °C for 1 min,
and then increased by 10 °C/min to 250 °C, where it was held constant for 3 min. The
conversion and the selectivity were analyzed from the peak areas in the
chromatograms. GCꢀFID: chiral column Betadexꢀ325 capillary column; 1 ꢀl samples
were injected at 100ºC. The oven was heated at 15 ºC/min to 150 ºC, at 8 ºC/min to
200 ºC, at 2 ºC/min to 240 ºC and then maintained for 4 min. After this, the oven was
heated at 15 ºC/min to 300 ºC. Chiral GC analysis was performed on a Shimadzu GCꢀ

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2010 chromatograph equipped with a FID, an AOCꢀ20i autosampler and a chiral CPꢀ
ChirasilꢀDex CB (25 m X 0.25 mm ID) or a chiral βꢀDex325 (30 m X 0.24 mm ID)
column using hydrogen as carrier gas. Injector and detector temperatures were set at
220 °C. GCꢀFID temperature program for 1ꢀphenylethylamine (PEA) using βꢀDex325
column: 90 °C | 30 min → 180 °C, 40 °C/min |10 min.
HPLC analysis
For quantitative analysis 150 ꢀL samples of the reaction mixture were derivatized
with 15 ꢀL trifluoroacetic acid (TFA), centrifuged (13000 g, 5 min) and 15 ꢀL of the
supernatant were directly analyzed by HPLC (Hitachi LaChrom) using a reversedꢀ
phase C18 column (LiChrospher®). Detection took place with a UV detector set at
245 nm.
Thermogravimetric (TG) analysis. The TG curves were obtained in a
thermogravimetric module, coupled to a thermal analyzer, both manufactured by
Netzsch®. Thermogravimetric measurements were performed using a platinum sample
holder containing about 10 mg of each immobilized enzyme. Each sample was heated
from 35 to 600 °C at 10 °C minꢀ1, under atmosphere of synthetic air and N2, both at a
flow rate of 60 mL minꢀ1.
X-Ray Photoelectron Spectroscopy (XPS)
The XPS was conducted using a Kratos Axis Ultra DLD spectrometer. A
monochromatic Al source was used as Xꢀray source. The soft Xꢀrays emitted from
excitation of Al source generate photoelectrons from the surface layers of atoms in a
solid sample. The electrons thus emitted are analyzed according to their kinetic
energy and the spectrum produced is used to identify the elements present and their
chemical states.
Infrared analysis.

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Analysis by infrared spectroscopy used a Shimadzu 8300 FTIR spectrophotometer.
The spectrum was obtained with 32 scans and with 4 cmꢀ1 resolution. For the analysis,
10 mg of a sample was placed in the sample collector to form tablets of approximately
2 mm thickness and 5 mm diameter without KBr addition.
Scanning electron microscopy (SEM) analysis.
Cellulose, functionalized cellulose and supported enzyme had their structure analyzed
by scanning electron microscopy (SEM) using a Zeiss EVO® 50H microscope. All
micrographs were obtained using a Shimadzu® sputter equipment. For this, each
sample was placed in a sample holder on carbon tape and then they were metallized
under vacuum. After preparation, the samples were placed under the microscope and
bombarded by an electron beam interacting with the sample's atoms. From the
interaction between the electron beam and the sample, the radiation was possible to
form a magnified image of the sample.
Laser Confocal Microscopy Analysis (LCMS).
Prior to being analyzed, the immobilized enzyme samples were mixed with
fluorescamine solution (50 mg/mL, in acetone) for 3 min to form a highly fluorescent
product through the reaction between the primary amines in proteins and the
fluorescamine. Fluorescence intensity quantification of both biocatalyst containing
fluorescent lipase (GCelꢀCalB) and transaminase (ECelꢀVF) were analyzed by
confocal microscopy (Zeiss, LSM 510 META). For this purpose, slides were mounted
with VectaShield (Vector Laboratories) and covered with a glass lamina. Stack
images were analyzed using Image J software (NIH).
Results and Discussion

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First, cellulose was modified in order to introduce the epoxy (Glymo) and
amino (Aptes) groups for covalent bond immobilization protocols. Cellulose was
successively mixed with APTES and glutaraldehyde in order to deliver the
glutaraldehydeꢀfunctionalized cellulose (GCel), after 24 h. The epoxyꢀfunctionalized
cellulose (ECel), was also produced by the reaction between Glymo and cellulose for
5 h, using THF as solvent (Scheme 1).
Scheme 1: Glymoꢀ and Aptesꢀfunctionalized cellulose.
After drying the functionalized cellulose (GCel and ECel) at ambient
temperature for 48 h, the enzyme immobilization was performed. For the lipase
immobilization, 2 mL Lipase B from Candida antarctica solution was dissolved in 10
mL 0.025 mM phosphate buffer pH 7.0. The functionalized cellulose (GCel and ECel)
was added (2 g) and allowed to react for 24 hours at 40°C. Immobilization efficiency
was evaluated by the difference between initial amount of enzyme added and that in
the supernatant after filtration of the immobilized enzyme (Table 1). For the
transaminase immobilization procedure, 5 mL Vibrius fluviaris transaminase solution
was dissolved in 30 mL 50 mM HEPES buffer pH 7.5. The functionalized cellulose
(GCel and ECel) was added (1 g) and underwent the reaction for 24 hours at 30 °C.
No optimization was done in order to first verify the feasibility of using the
functionalized cellulose as a matrix for enzyme immobilization. All supports were
characterized by infrared (IR), thermogravimetry analysis (TG), Scanning electron

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microscopy (SEM) and Xꢀray Photoelectron Spectroscopy (XPS) (see supporting
information for further details).
The immobilization efficiency obtained for CalꢀB on epoxyꢀcellulose (ECelꢀ
CalB), CalꢀB on glutaraldehydeꢀcellulose (GCelꢀCalB), VF transaminase on epoxyꢀ
cellulose (ECelꢀVF) and VF transaminase on glutaraldehydeꢀcellulose (GCelꢀVF) are
shown on Table 1.
Table1: Immobilization efficiency of Lipase B from Candida Antarctica and Vibrius
fluviaris transaminase into glutaraldehydeꢀcellulose and epoxyꢀcellulose.
Immob.
Biocatalyst
ECelꢀCalB
GCelꢀCalB
ECelꢀVF
Immob.
Efficiency (%)
3.5
Amount of Protein
(mg/g of support)
0.2
0.6
10.5
90.1
40.5
12.4
GCelꢀVF
27.7
* Immobilization efficiency was evaluated by the difference between initial amount of enzyme (5.7
mg/mL) added and that in the supernatant after filtration of the immobilized enzyme.
As shown on Table 1, the immobilization protocol developed did not present
efficient results for the immobilization of lipase B from Candida antarctica, leading
to very low protein incorporation. The VF transaminase leads to good to excellent
immobilization efficiencies on both epoxy (up to 90.1 %, Table 1) and glutaraldehyde
functionalized cellulose. In order to prove the concept of using functionalized
cellulose as a renewable support for enzyme immobilization, the two supports were
attempted where the immobilization efficiency was higher, CꢀB on glutaraldehydeꢀ
cellulose (GCelꢀCalB) and VF transaminase on epoxyꢀcellulose (ECelꢀVF).
13
The solid state C NMR spectroscopy of the functionalized cellulose clearly
demonstrates the incorporation of the APTES and glutaraldehyde in the cellulosic

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material. The presence of the imino as well as the silyl propyl groups can be inferred
by the presence of the signals in 163ꢀ164 ppm and the signals at higher field 10ꢀ50
ppm, respectively. When the same analysis was performed after the incorporation of
the enzyme into the cellulosic support, an increase in the number of signals in the
range of 164ꢀ180 ppm can be observed, which correlates to the presence of imino and
amide functions (see supporting information for further details). This result
corroborates the XPS analysis where a peak at the binding energy of 400 eV is
observed for the glutaraldehydeꢀfunctionalized cellulose (GCel) being an indicator of
nitrogen atoms in the sample. For the sample GCelꢀCalB there is a shoulder peak
shown at the binding energy of 402 eV. This shoulder peak is the main difference
compared to the GCel sample, meaning that the nitrogen atom is in a higher oxidation
state, which can be related to the protein binding to the support (see supporting
information for further details). In order to verify the presence of protein on the
functionalized cellulose, we decided to perform a confocal laser scanning microscopy
(LCMS) experiment to visualize the dispersion of protein into the support (Figure 1).
29, 30 As displayed in Figure 1a (ECelꢀVF) the white color reveals the presence of well
dispersed protein on the surface of cellulose granules. In addition, the LCMS analysis
of GCelꢀVF (Figure 1b) shows a punctual localization of protein, which reflects the
low immobilization efficiency compared to the epoxyꢀfunctionalized pattern (Table
1).

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Figure 1: Confocal Laser Scanning Microscopy (CLSM) and Scanning Electron
Microscopy (SEM) analysis of ECelꢀVF and GCelꢀVF. (1) SEM analysis of cellulose
before functionalization; (2) SEM analysis of ECelꢀVF; (3) SEM analysis of GCelꢀ
VF; (4) CLMS analysis of ECelꢀVF; (5) CLMS analysis of GCelꢀVF.
With the characterized results, catalytic performances of the immobilized
enzymes at different reaction temperatures were evaluated in the designed reactions.
The immobilized lipase GCelꢀCalB was evaluated by the esterification reaction
between oleic acid and ethanol (1:1, 100mM in heptane and 200 rpm), at different
temperatures (50ꢀ70 °C) for 1 h. The behavior presented by GCelꢀCalB was compared
to the commercial immobilized lipase Novozyme 435 and the results are presented, as
shown in Table 2. A similar evaluation was made for ECelꢀVF immobilized enzyme,
in this case the kinetic resolution of αꢀphenylethylamine (PEA) was used as standard
reaction at temperatures ranging from 30 to 60 °C. Unfortunately, there is no
commercial transaminase that can be used as a positive control for comparison
purpose.

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Table 2: Temperature profile for the esterification reaction catalyzed by
GCelꢀCalB and comparison with the results obtained for Novozyme 435.
Entry
Immob. Enzyme
GCelꢀCalB
Temperature (oC)
Conversion (%)
1
2
3
4
5
6
50
60
70
50
60
70
85
97
84
84
80
81
Novozyme 435
Reaction conditions: GCelꢀCalB or Novozyme 435 (10 mg immobilized enzyme in 1 mL
reaction media) was evaluated in the esterification reaction between oleic acid and ethanol (1:1 – 100
mM in nꢀheptane) at different temperatures.
Table 3: Temperature profile of the ATA as determined by the acetophenone
assay for ECelꢀVF. 31
Entry
Immob. Enzyme
ECelꢀVF
Temp. (°C)
Conv. (%)
1
2
3
4
30
40
50
60
38
42
43
35
Reaction conditions: ECelꢀVF (50 mg immobilized enzyme in 5 mL reaction media) was
evaluated using the discontinuous acetophenone assay (5 mL 50 mM HEPES buffer, (
5 mM pyruvate, 0.5% DMSO and 800 rpm) at different temperatures.
±)ꢀPEA 2.5 mM,
A first look at the results presented on Table 2, can take the reader to the
wrong assumption that GCelꢀCalB and Novozyme 435 have the same behavior at
different temperatures, but it is worthy to note that Novozyme 435 has 50x more

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protein attached to the support compared to GCelꢀCalB, making this one much more
efficient than the commercial immobilized enzyme. GCelꢀCalB was also evaluated at
higher temperatures (80 °C) and the results obtained show a similar behavior, leading
to the desired product on 75% of conversion (see supporting information for further
details). The results for temperature profile of ECelꢀVF (Table 3) show good results at
temperatures ranging from 30 to 50 °C and a slight decrease is observed at 60 °C.
Especially for the transaminase, the pH profile was also evaluated in the range
between 7.5 and 9.5, where the case at pH 7.5 offered the best result.
At this stage we also evaluated the recyclability of the immobilized
biocatalysts GCelꢀCalB and ECelꢀVF. After each reaction the immobilized biocatalyst
was washed three times with buffer and dried at ambient temperature for 24 h and this
was repeated five times (Table 4).
Table 4: Recyclability of the immobilized enzymes under standard conditions.
Conversion (%)
Entry
Immob. Enzyme
Cycle 1
84
Cycle 2
71
Cycle 3
56
Cycle 4
26
Cycle 5
20
1
2
3
Novozyme 435 a
GCelꢀCalB a
85
60
54
51
43
ECelꢀVF b
43
39
39
40
35
a
Reaction conditions: GCelꢀCalB or Novozyme 435 (10 mg immobilized enzyme in 1 mL
reaction media) was evaluated in the esterification reaction between oleic acid and ethanol (1:1 – 100
b
mM in nꢀheptane) at 50 °C. ECelꢀVF (50 mg immobilized enzyme in 5 mL reaction media) was
evaluated using the discontinuous acetophenone assay (5 mL 50mM HEPES buffer, (
5 mM pyruvate, 0.5% DMSO and 800 rpm) at 30 °C.
±)ꢀPEA 2.5 mM,
The findings of the recyclability study show that using the GCelꢀCalB
immobilized lipase a decrease of the conversion was observed in the second cycle
compared to the commercial enzyme while Novozyme 435 continues to lose its

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activity for four consecutive cycles to only 20% conversion. The immobilized
transaminase ECelꢀVF presented a better stability affording the same conversion in
fours cycles and only a slight decrease in the last one.
Finally, the newly developed immobilized biocatalysts GCelꢀCalB and ECelꢀ
VF were used for the synthesis of chiral products. The immobilized biocatalysts
GCelꢀCalB was applied in the kinetic resolution of (R,S)ꢀαꢀphenylethanol using vinyl
acetate as acyl donor at 60 °C for 5 h (Table 5).
Table 5: Kinetic resolution of (R,S)ꢀαꢀphenylethanol catalyzed by GCelꢀCalB.
Productivity
Immob.
Enzyme
Reaction
Time
Entry
Conv. (%)
E
(g of product/ h . mg
protein)
1
2
3
4
Novozyme 435
2 hours
2 hours
4 hours
5 hours
49
35
40
42
>200
>200
>200
>200
0.053
1.80
1.08
0.91
GCelꢀCalB
Reaction conditions: (R,S)ꢀαꢀphenylethanol (1 mmol), vinyl acetate (1 mol. eq.) as acyl donor, and 18
mg (15% w/w) immobilized enzyme were reacted in cyclohexane (3 mL) for 2, 4, and 5 h at 60 °C.
Enantiomeric excess values (ee) were determined by chiral GC analysis.
Comparatively, GCelꢀCalB immobilized enzyme could not mimic the
efficiency shown by Novozyme 435, arriving to 49% of conversion after 2 h.
However a promising result could be obtained by the use of GCelꢀCalB with
conversions reaching 40% after 4 h. The most important feature of this immobilized

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biocatalyst is the fact that even a low protein loading can lead to very high
productivities (up to 1.8 g of product/ h * mg immob. enzyme), which could never be
reached by the commercial immobilized enzyme.
The immobilized transaminase ECelꢀVF was subject to an asymmetric
synthesis protocol using acetophenone as starting material. At this stage – since the
development of immobilized transaminases is an emerging area –the performance of
ECelꢀVF was evaluated in both, batch and continuousꢀflow reactors (Table 6).
Table 6: Asymmetric synthesis catalyzed by ECelꢀVF in batch and under
continuousꢀflow conditions.
Reaction/Residence
Entry
Conv. (%)
e. e. (%)
Time
1
2
3
4
5
6
48 h (Batch)
31
33
14
37
72
80
>99
>99
>99
>99
>99
>99
96 h (Batch)
15 min (Cont. Flow)
30 min (Cont. Flow)
60 min (Cont. Flow)
90 min (Cont. Flow)
Reaction conditions: 50 mM HEPES buffer (pH 7.5), 10 mM acetophenone, 250 mM alanine, 10%
DMSO, LDHꢀGDH, 1 mM NADH and 150 mM Glucose. For the batch experiments, 100 mg
immobilized enzyme was used in a total reaction volume of 5 mL on a shaker at 800 rpm at 30 °C. For
continuousꢀflow experiments, 2 g immobilized enzyme were packed arriving on a packed bed with a
total volume of 10 mL.

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As observed in Table 6, the asymmetric synthesis protocol under continuousꢀ
flow conditions is better than in batch mode since lower reaction/residence times are
needed and higher conversions could be obtained without compromising the
enantiomeric excess. Probably, the continuousꢀflow system enables to enhance mass
transfer and consequently leads to better conversions. The packed bed, after washing
for 3 h with HEPES buffer, was stored and reꢀused with the same behaviour presented
before.
Conclusion
In conclusion, we have developed one lipase (GCelꢀCalB) and one
transaminase (ECelꢀVF) immobilized onto functionalized cellulose for different
applications. Effects of temperature, pH and recyclability profiles were evaluated and
satisfactory results were presented. The GCelꢀCalB immobilized enzyme has shown
very good results in the kinetic resolution of (R,S)ꢀαꢀphenylethanol enabling high
productivities. ECelꢀVF immobilized enzyme demonstrates its excellent catalytic
activity in the kinetic resolution as well as in asymmetric synthesis where batch and
continuous flow protocols have been applied, reducing the reaction time from 48 h to
90 min and increasing the conversion from 30% to 80%.
Acknowledgment
The authors thank CAPES, CNPq, FAPERJ and DLR (Grant No: 01DN14016) and
the Alexander von Humboldt Foundation (Germany) for financial support.
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