Continuous micro-production using enzymatic reaction and online monitoring

Article abstract of DOI:10.2533/chimia.2010.799

A micro-reactor coupled to a microfluidic system and an online UV/VIS spectrometer is described. The enzymatic reaction studied is the hydrolysis of the N-benzoyl-l-tyrosine ethyl ester (BTEE) to N-benzoyl-ltyrosine (BT) and ethanol, catalyzed by chymotry

Full text of DOI:10.2533/chimia.2010.799

SwiSS Biotech
CHIMIA 2010, 64, No. 11 799
doi:10.2533/chimia.2010.799
Chimia 64 (2010) 799–802 © Schweizerische Chemische Gesellschaft
Continuous Micro-Production Using
Enzymatic Reaction and Online Monitoring
Regula Moser, Thierry Chappuis, Ennio Vanoli, Simon Crelier, and Olivier Naef*
Abstract: A micro-reactor coupled to a microfluidic system and an online UV/VIS spectrometer is described.
The enzymatic reaction studied is the hydrolysis of the N-benzoyl-l-tyrosine ethyl ester (BTEE) to N-benzoyl-l-
tyrosine (BT) and ethanol, catalyzed by chymotrypsin. The production is online monitored with UV spectroscopy
®
at 256 nm. Three different immobilization methods of the enzyme are discussed: Eupergit C, controlled-pore
glass (CPG), and Sepharose.
Keywords: Chymotrypsin · Enzyme immobilization · Enzymatic reaction · Micro-production · Micro-reactor
Introduction
tive in this context. Micro-reactors have Materials and Methods
been used to carry out enzymatic reactions
Nowadays the placement of new products both with the enzyme in solution or im- Chemicals
on the market demands more and more mobilized on a support. Immobilization
In order to perform the chymotrypsin-
flexibility from industry. The competition is usually preferred, since the enzyme can catalyzed hydrolysis of N-benzoyl-l-ty-
to innovate becomes tougher, especially in be recycled and reused during the entire rosine ethyl ester (BTEE) to N-benzoyl-
the domains of chemistry and biochem- lifetime of its activity. Thus much research l-tyrosine (BT) and ethanol (Scheme 1),
istry. In this context, reducing the devel- effort has been invested in this field. In the a-chymotrypsin from bovine pancreas and
opment costs and optimizing production review published by Miyazaki and Mae- N-benzoyl-l-tyrosine ethyl ester were pur-
[
2]
processes are valuable ways of gaining a da, typical techniques for the preparation chased from Sigma-Aldrich and used with-
competitive advantage. of immobilized-enzyme micro-channel out further purification.
The development time of a chemical or reactors are listed. The most widespread For the immobilization of the a-chy-
®
biochemical process can be reduced signif- techniques for enzyme immobilization motrypsin, Eupergit C, Sepharose 6B
icantly by using a micro-reactor. Indeed, use particle entrapment or immobilization were purchased from Sigma and Control-
due to the low volumes involved, tests can on surfaces or membranes. Immobiliza- led Pore Glass (120–200 mesh and 200–
be carried out with small amounts of re- tion on surfaces or membranes is gener- 400 mesh) from Fluka. These supports
actants. Moreover, as micro-reactors have ally performed by covalent binding with a were used without further treatment prior
an advantageous surface-to-volume ratio, crosslinker or by adsorption.
the temperature of the reaction mixture In the present study, three different
to enzyme immobilization.
Methanol and hydrochloric acid were
can be controlled more efficiently than in methods to immobilize a-chymotrypsin purchased from Merck, potassium dihy-
larger scale reactors. Hence, the safety of in a micro-reactor are presented. Chymo- drogenphosphate was purchased from
the process is better guaranteed and, due to trypsin is a protease which is used, in this Riedel-de-Haën and disodiumhydrogen-
the continuous nature of tubular micro-re- example, to hydrolyze an ester to a carbox- phosphate from Fluka. Unless otherwise
action systems, steady-state conditions can ylic acid. In industry, chymotrypsin is used stated, all other chemicals were purchased
be achieved easily. Then, when the process for example to produce l-phenylalanine from Sigma-Aldrich. These chemicals
is sufficiently mature, further scale-up and from the racemate mixing or peptide map- were all used without further purification.
transfer to production can be envisaged by ping. The supports used to achieve enzyme
parallelization.[
1]
immobilization include Eupergit C and Homogeneously Catalyzed
®
The development of micro-reactors in- Sepharose polymers and controlled pore Hydrolysis Reaction
tegrating enzymatic catalysis is a topic of glass (CPG). The chymotrypsin is always
Before immobilization of the enzyme
current interest. The environment-friendly immobilized by covalent binding on the on different supports, the hydrolysis of the
[
3]
nature of enzymatic conversion is attrac- support.
BTEE to BT and ethanol was first stud-
Scheme 1. Hydrolysis
of N-benzoyl-l-tyrosine
ethyl ester (BTEE) to
N-benzoyl-l-tyrosine
(BT) and ethanol.
*
Correspondence: Prof. O. Naef
Ecole d’ingénieurs et d’architectes de Fribourg
Boulevard de Pérolles 80
CP 32
CH-1705 Fribourg
Tel.: +41 26 429 67 05
E-mail: olivier.naef@hefr.ch

800 CHIMIA 2010, 64, No. 11
SwiSS Biotech
ied in a batch setup in order to determine
the evolution of the absorbance of a 0.56
mM BTEE solution in the reaction buffer
as a function of time, with chymotrysine
used in solution as a homogeneous cata-
lyst. The reaction buffer is prepared as a
Scheme 2. Binding
reaction between
CGP particles and
a-chymotrypsin using
disuccinimidyl suberate
(DSS) as a crosslinker.
1
.42:0.9:0.08:0.1 solution of Tris/HCl
buffer at pH 7.8, methanol, calcium chlo-
ride and HCl. As a comparison, the evolu-
tion of the absorbance in a 0.56 mM BTEE
solution in the reaction buffer in the ab-
sence of chymotrypsin was also monitored.
®
Immobilization on Eupergit C
®
Eupergit C is an acrylic polymer with
porous structure vested by epoxy groups
on the surface. The immobilization takes
place by covalent binding with the epoxy
groups.
Fig. 1. (a) Micro-
The immobilization of a-chymotrypsin
reactor and (b) reaction
channel used to study
the chymotrypsin-
catalyzed hydrolysis of
N-benzoyl-l-tyrosine
ethyl ester (BTEE) to
N-benzoyl-l-tyrosine
[
4,5]
proceeded as described in the literature.
The immobilization was done in a phos-
phate buffer, 1 M at pH 7, room tempera-
ture, for 20 h. For the coupling reaction 1 g
®
of Eupergit C was reacted with 9 ml of
enzyme solution (concentration between
(BT) and ethanol.
1
–5 mg/ml) in a reaction tube.
The analysis of the activity (SIGMA
Quality control Test, Enzymatic Assay of
chymotrypsin EC 3.4.21.1) and of the re-
maining protein concentration (BIO-RAD,
TM
Quick Start Bradford Protein Assay) in
the reaction mixture permitted the control
of the process. The difference between
the concentration at the beginning and the 3 ml of HCl 0.1 mM and CaCl 10 mM so- After stirring for 30 min, the reaction was
2
concentration at the end can be considered lution (300 ml HCl 1 mM, 60 ml CaCl 2 M complete. At the end, the CPG is rinsed
2
as the concentration immobilized on Eu- and 2.64 ml of water) were added. Once with water, before storing in HCl 0.1 mM
®
pergit C.
the treatment was finished, the product was and CaCl 10 mM solution at 4 °C.
2
stored in the fridge at 4 °C.
®
Immobilization on Sepharose 6B
To follow the reaction, the activity and Backed-bed Reactor and Micro-
®
Sepharose 6B is a polysaccharide with the protein concentration was analyzed as reactor Systems
®
reactive epoxy groups, which are involved described above for Eupergit C.
in the immobilization process of chymo-
After immobilization of the enzyme
on different types of supports, the immo-
bilized enzyme was filled as a packed-bed
either in a tube (glass or stainless steel, in-
trypsin the same way as described above for Immobilization on Controlled Pore
Eupergit C supports. Sepharose 6B parti- Glass (CPG)
®
®
cles have a size distribution of 45–165 mm.
Controlled Pore Glass (CPG) is a new ternal diameters varying between 0.5 and
®
About 1 g of Sepharose 6B was rinsed packing material used for chromatography 5 mm) or in a micro-reactor. Eupergit® C
with deionised water until it swelled up columns. It is composed of almost pure and Sepharose® 6B carriers were only
three times its dry volume. To start the quartz glass. It is chemically inert and very tested in tubes while CPG-enzyme cataly-
®
reaction, the swelled Sepharose 6B was hard. Activated CPG has a functionalized sis was also tested in the micro-reactor.
transferred in a tube, and then 5 ml of the surface containing aminopropyl groups.
The tubular micro-reactor used to study
solution of chymotrypsin were added to Convalent binding between a-chymot- the chymotrypsin-catalyzed reaction pre-
®
the Sepharose 6B. The dispersion was rypsin and the CPG particles is achieved sented here is represented on Fig. 1a. This
shaken for 23 h at room temperature.
by using disuccinimidyl suberate (DSS) micro-reactor was manufactured at Uni-
After the immobilization of chymo- as a crosslinker. The binding reaction of versity of Applied Sciences Western Swit-
trypsin the remaining amine groups were a-chymotrypsin on CPG particles is illus- zerland (HES-SO) La Chaux-de-Fonds
deactivated by rinsing first with water, af- trated in Scheme 2.
(HE Arc, Switzerland) and further tested
First of all, the CPG was swelled to and characterized at HES-SO Fribourg
terwards 5 ml of ethanolamine 1 M were
added and the dispersion was shaken about three times its dry volume with (EIA-FR, Switzerland). The channel was
overnight (23 h) at room temperature. To distilled water. Afterwards, the CPG was produced from a silicon wafer etched by a
remove excess uncoupled ligands after transferred to a vial and a solution of 2 mg/ laser. Its dimensions are 0.3 mm in width
®
coupling, the Sepharose 6B was washed ml chymotrypsin in borax buffer pH 8 was and 1 mm in height. Holes were positioned
alternatively with high and low pH buffer added. This mixture was stirred for 15 min at each end of the channel to allow both
solution at least three times. Starting with to form intermolecular bindings. This was inflow and outflow of the liquid and the
Tris/HCl buffer pH 8, following with ace- followed by adding a solution of disuc- whole channel structure was covered by a
tate buffer pH 4. To store finally the Sepha- cinimidyl suberate (DSS) solution (3.6 mg glass plate fixed to the silicone structure
®
rose with the immobilized chymotrypsin, DSS in 100 ml N,N-dimethylformamide). by anodic bonding (Fig. 1b). The micro-

SwiSS Biotech
CHIMIA 2010, 64, No. 11 801
Fig. 2. Schematic
but the amount of enzyme immobilized
per gram of Sepharose is very low with a
poor yield of the immobilization reaction.
According to these results, the technique
of immobilizing chymotrypsin on Sepha-
rose 6B carriers was not carried forward
for tests with the hydrolysis reaction in
continuous flow tubes.
representation of the
process flow. 1 is the
pump, 2 is channel,
3
enzyme support, 4
is a filter and 5 is the
detector.
®
Table 1. Overview of the process flow
The amount of a-chymotrypsin immo-
1
2
3
4
5
®
bilized on Eupergit C proved to be larger
®
Pump
Channel
Enzyme support Filter
Detector
than on Sepharose 6B and Controlled
Pore Glass. While testing the hydrolysis
reaction in a continuous flow packed bed
tube, a conversion of 90% was achieved
after 0.3 min. of contact with the immobi-
lized enzyme.
Peristaltic pump Glass tube
CPG 120–200
Fibreglass
UV/VIS
mesh
Eupergit
spectrometer
with flow-
through cell
®
C
HPLC pump
HPLC pump
Stainless steel
tube
CPG 120–200
mesh und 200–
HPLC column
filter, Poren Ø
0.5 mm
HPCL
Spectrometer
UV/VIS
In spite of a larger immobilization
yield, the amount of a-chymotrypsin im-
mobilized on Controlled Pore Glass is
about 30% lower than that measured for
4
00 mesh
®
Eupergit
C
Micro-reactor
CPG 200–400
mesh
Fibreglass
HPCL
spectrometer
UV/VIS
®
Eupergit C. During the hydrolysis reac-
tion, chymotrypsin immobilized on Con-
trolled Pore Glass enabled a 95% conver-
sion of BTEE to BT when the continuous
tubular reactor was packed with a bed
structured channel was then sandwiched in action could be determined. In addition to length of 2.4 cm.
a housing made of stainless steel. the yield, the stability of the chymotrypsin
Fig. 4 shows the comparison of the BT
The liquid flow across the system was could be determined by calculating the ra- concentration profiles measured during
forced by an HPLC pump and a UV/VIS tio of the activity to the total protein con- the hydrolysis reaction for different con-
detector was positioned at the outflow end centration. If the enzyme is not degraded, tacting times between the liquid flow and
of the micro-reactor. This setup was used this ratio should reach a stable value during the immobilized phase. Here, the reaction
to analyze the hydrolysis of N-benzoyl-l- the reaction. The amount of chymotrypsin tube is packed with varying length beds
®
tyrosine ethyl ester (BTEE) to N-benzoyl- immobilized on the support and the yield of both Eupergit C and CPG. The results
l-tyrosine (BT) and ethanol (Fig. 2). The of the immobilization reaction is reported demonstrate good stability of the chymot-
®
reaction was analyzed at 256 nm. Fig. 2 in Table 2 for Eupergit C, Sepharose® 6B rypsin during the experiments. Moreover,
and Table 1 show a conceptual overview and Controlled Pore Glass.
of the experimental setup. As an enzyme, As reported in Table 2, it was possible clearly more active than the enzyme im-
the chymotrypsin immobilized on CPG is
®
a-chymotrypsin from bovine pancreas was to immobilize chymotrypsin on Sepharose, mobilized on the Eupergit C support.
chosen. The advantages of a-chymotrypsin
are a good thermal- and activity stabil-
ity (storage at 4 °C allows the conserva-
tion of the activity during a maximum of
months) and quick reactivity at 20 °C.
Fig. 3. Activity
and enzyme
2
concentration of the
supernatant liquid
during chymotrypsin
immobilization on
Results and Discussion
®
Eupergit C.
Immobilization of the Enzyme
Chymotrypsin was immobilized on
three different supports to compare the ac-
tivity of the immobilized enzyme with con-
tinuous flow hydrolysis of BTEE to form
BT and ethanol. In order to monitor the
immobilization reaction, the liquid super-
natant was analyzed to determine the re-
maining activity of enzyme (Sigma Qual-
ity Control Test Procedure, Enzymatic As-
say of chymotrypsin, EC 3.4.21.1) and the
protein concentration (Quick Start Brad-
ford Protein Assay, Instruction manual,
Bio-Rad) in the solution. These measures
for the immobilization of chymotrypsin on
Table 2. Amount of a-chymotrypsin immobilized on each support and yield of the immobilization
reaction.
Support
Chymotrypsin on support
[mg/g]
Yield of the immobilization
reaction [%]
®
Eupergit C are presented as a function of
time on Fig. 3. As seen in Fig. 3, enzyme
activity and concentration in the liquid su-
pernatant evolve in parallel. Thanks to the
results, the yield of the immobilization re-
Sepharose
4.6
46
94
50
Controlled Pore Glass
Eupergit C
9.3
13.5

802 CHIMIA 2010, 64, No. 11
SwiSS Biotech
Fig. 4. BT
cost design and fabrication of customized
microchannel-based reactors are currently
in development and have the potential to
pave the way for faster process develop-
ment and reduced scale-up and optimiza-
tion costs.
concentration profiles
for the hydrolysis
reaction carried
out in a continuous
tube packed with
varying length beds
®
of Eupergit C and
Controlled Pore Glass
1
20–200 mesh at 20 °C Conclusion
and pH 7.8.
In the present article, the realization of
the enzyme-catalyzed hydrolysis of BTEE
to BT and ethanol in a continuous flow mi-
cro-reactor has been presented. Immobiliz-
ing the enzyme on a solid support clearly
appearedtobethelimitingstep, sinceitwas
proved to be both support- and enzyme-
®
dependent. In particular, Eupergit C was
This can be seen from the much larger rate potential advantages of immobilizing an able to retain more chymotrypsin but with
of formation of BT with Controlled Pore enzyme on a solid support directly packed a reduced activity compared to Control-
®
Glass.
in the reactor. As seen with Eupergit C led Pore Glass (CPG). Hence only the lat-
Fig. 4 also shows that the length of the and Controlled Pore Glass (CPG), a larger ter immobilization technique was used to
®
Eupergit C with chymotrypsin has almost amount of enzyme immobilized on Eu- pack the micro-reactor with the catalyzing
®
no influence on the kinetics of the kinetics pergit C did not necessarily result in a substance. Micro-production experiments
of BTEE to BT.
higher activity of the immobilized system. demonstrated interesting conversions at
Figs 5 and 6 depict the conversion and Indeed, the activity of an enzyme also de- steady-state and stable productivity up to
the productivity of the BTEE to BT hy- pends on its binding site. If the active site more than 18 mg/h of BT.
drolysis reaction implemented in a micro- is distant enough from the binding site, the
Acknowledgements
reactor using enzyme immobilization on enzyme is likely to retain its activity. How-
The project is funded by the University
1
5
20–200 mesh Controlled Pore Glass. Fig. ever, if the distance between these sites is
shows a slight decrease of the conversion too short, the binding of the enzyme to
of Applied Sciences of Western Switzerland
(
HES–SO) through the HES–SO CALL 2007.
as the flow rate of the reaction mixture in- the support could alter the activity of the
creases. However, the conversion remains catalyzing site. Hence, the method used for
over 80% for most tests.As a consequence, enzyme immobilization in a micro-reactor
immobilization of chymotrypsin in the is both support- and enzyme-dependent. It
micro-reactor allowed a linear increase of is probably the most limiting step for such
Received: September 6, 2010
[
1] K. Geyer, J. D. C. Codée, P. H. Seeberger,
Chem. Eur. J. 2006, 12, 8434.
productivity as a function of the linear ve- a micro-reactor to be of widespread use for [2] M. Miyazaki, H. Maeda, TRENDS Biotechnol.
2
006, 24, 463.
locity of the liquid phase.
real-life chemical and biochemical devel-
[
3] R. Nicoli, S. Rudaz, C. Stella, J.-L. Veuthey, J.
Chromatogr. A 2009, 1216, 2695.
Optimization still has to be performed opments involving enzyme catalysis. Once
withthemicro-reactorinordertomaximize the immobilization step is mastered with
the productivity in a micro-production maximized activity of the active substance,
system such as the micro-channel device further development with the micro-reactor
described here. In this study, the reaction proved to be very flexible, allowing steady
and the enzyme were chosen as a model production with high conversion on a con-
able to demonstrate the feasibility and the tinuous basis. Solutions for fast and low-
[
4] C. Mateo, O. Abian, R. Fernandez–Lafuente, J.
M. Guisan, Enzyme Microb. Technol. 2000, 26,
5
09.
[
5] M. J. Hernaiz, H. G. Crout, Enzyme Microb.
Technol. 2000, 27, 26.
Fig. 5. Conversion of BTEE to BT as a function of the rate of the flowing
phase through a continuous micro-reactor packed Controlled Pore
Glass.
Fig. 6. Productivity of a continuous flow micro-reactor with packed
Controlled Pore Glass for the chymotrypsin-catalyzed hydrolysis of
BTEE to BT.