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Evaluation of immobilized enzymes for industrial

Cite this: Chem. Soc. Rev., 2013,
applications
42, 6236
Andreas Liese and Lutz Hilterhaus*

Received 12th December 2012
DOI: 10.1039/c3cs35511j
www.rsc.org/csr
In contrast to the application of soluble enzymes in industry, immobilized enzymes often oer
advantages in view of stability, volume specific biocatalyst loading, recyclability as well as simplified
downstream processing. In this tutorial review the focus is set on the evaluation of immobilized
enzymes in respect to mass transport limitations, immobilization yield and stability, to enable industrial
applications.

Key learning points

 Evaluation of an immobilized biocatalyst in view of the establishment of a bioprocess
 Assessment of mass transport limitations and their mathematical description
 Experimental methods to characterize immobilized enzymes
 Mathematical description of the deactivation of immobilized biocatalysts to estimate productivity
 Systematic characterization of an immobilization step with a focus on activity yield and mass specific yield

(8) Consequences for reactor selection

Introduction
For a successful industrial application of enzymes, these catalysts
must be stable and fully functional under process conditions.
The reaction conditions in an industrial surrounding dier quite
often from the natural environment of enzymes in view of e.g.
temperature, organic co-solvents and pH-value. Consequently,
methods of physical stabilization are applied, besides optimizing
the molecular structure of the enzyme itself by protein engineering.1
The major physical stabilization is achieved by binding the enzyme
to a heterogeneous support, also addressed as the carrier. In this
tutorial review the characterization of immobilized enzymes is
discussed in respect to activity, stability and mass transport
limitations to enable industrial applications.
The tutorial review is subdivided into the following sections:
(1) Motivation and recent developments
(2) Choice of carrier for enzyme immobilization
(3) Evaluation of mass transport limitation
(4) Integral and dierential analysis
(5) Evaluation of immobilization yield
(6) Surface analysis technology for immobilized enzymes
(7) Evaluation of biocatalysts deactivation
Institute of Technical Biocatalysis, Hamburg University of Technology, Denickestr. 15,
D-21073 Hamburg, Germany. E-mail: Lutz.Hilterhaus@tuhh.de;
Fax: +49 40 42878 2127; Tel: +49 40 42878 2890
(9) Conclusion
The topics of enzyme activity as well as deactivation
are independent of the biocatalyst preparation, soluble or
immobilized. However, these properties must be characterized
in detail and their meaning understood, since those are the
parameters used to evaluate and compare dierent biocatalyst
preparations.
Motivation and recent developments
From the chemists point of view an enzyme is nothing else
than a catalytic macromolecule soluble in water at a concen-
tration ranging from almost complete insolubility to values of
several hundreds of milligrams per milliliter. To retain a
dissolved enzyme ultrafiltration devices with an appropriate
cuto can be used. However, the use of membrane technologies
can be limited by membrane fouling and membrane fluxes. If
ultrafiltration is not applicable, one alternative is the use of
immobilization strategies, meaning the attachment of a soluble
molecule to a heterogeneous support with the aim of simplifying
the retention of this catalyst.2 In addition to enhanced reusability
immobilization can improve the stability of an enzyme.3
Thus, three major driving forces for immobilization exist:
 Improvement of enzyme stability
 Increase of volume specific biocatalyst loading

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Tutorial Review
 Simplification of biocatalyst recycling and downstream
processing
Dierent approaches were developed to immobilize
enzymes e.g. adsorption or covalent binding to a carrier,
encapsulation and entrapment or crosslinking (e.g. cross linked
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enzyme aggregates or cross-linked enzyme crystals).4 As no
straightforward approach for choosing the best immobilization
method for a given enzyme is known, this has to be determined
experimentally.5 However, the structure of the biocatalyst
should be considered to achieve a higher stability towards e.g.
organic co-solvents and temperature.1
Methods for enzyme immobilization are reviewed and published
in a large number of journals and books. Especially, Linqiu Cao
presents a comprehensive survey of the field of immobilization
procedures used in enzyme technology.2 Laws governing the
behavior of enzymes at the interface of protein and carrier are
explained in detail. Using a large variety of case studies, dierent
approaches for enzyme stabilization by immobilization are
presented within the textbook edited by Jose M. Guisan.4 Reasons
for the stabilization of enzymes by immobilization can be found
in the multipoint and multisubunit attachment of enzymes to
surfaces.69 Immobilization may also generate a favorable micro-
environment (e.g., promoting the partition of some deleterious
compounds) and can also prevent intermolecular interactions
(gas bubbles, other protein molecules, proteases). However, a
general prediction if enzyme immobilization will lead to a more
stable biocatalyst has not been possible up until now. There is no
universal approach to immobilize a given enzyme for maintaining
its maximum activity and stability. Therefore, for protocols
regarding enzyme immobilization and stabilization the reader is
referred to these sources.
To optimize these protocols, a deeper understanding of the
immobilization procedure is beneficial. Recently, Ulf Hanefeld
et al. published a tutorial review, addressing the understanding
of enzyme immobilization.10 Here, the structure-based development
of immobilization is reviewed. Especially, the understanding of
Andreas Liese carried out his
doctoral research at the
Research Center Julich (under
Prof. Dr Wandrey). From 1998
to 2003 Liese was an assistant
professor at the University of
Bonn and at the same time head
of the Enzyme Group within the
Institute of Biotechnology,
Research Center Julich. For a
sabbatical he joined Pfizer
Global Research & Development,
Andreas Liese San Diego, USA, in 2000. From
2003 to 2004 he worked as an
associate professor at the University of Munster, soon receiving a
full professorship for Technical Biocatalysis 2004 at the Hamburg
University of Technology (TUHH) as the head of the Institute of
Technical Biocatalysis.
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protein unfolding allows the knowledge based immobilization,
thus enhancing activity yield as well as the stabilizing eect.11
The interaction of molecular modeling, molecular biology and
biochemistry can lead to a more rational approach for the
stabilization of enzymes by immobilization. Depending on
the structure and mechanism, more controlled immobilization
methods will be developed, yielding higher activities of the
immobilized enzymes, as recently demonstrated by Novozymes
A/S.12 A broad range of immobilization and stabilization techniques
is investigated; however, the degree of understanding of the
molecular mechanisms still needs to be improved. Irrespec-
tively, for process engineering the extent of stability increase
achieved for a given process is a value of interest. Certainly,
dierent applications of immobilized enzymes in pharmaceu-
tical processing13 and fine chemical and commodity chemical
syntheses14 have dierent demands on stability.
Mostly, the process conditions determine the factors
influencing the enzyme. These are the physical parameters
(e.g. temperature and pressure), the catalyzed reaction (substrates,
products, byproducts), the solvent (organic solvent, ionic liquid,
supercritical fluid, etc.) and the reactor type (stirred tank reactor,
membrane reactor, fixed bed reactor, bubble column reactor).
Often many of these parameters are given and an adaption of
immobilized enzyme is desired to overcome phenomena like
diusion limitation, leaching, enzyme oxidation or mechanical
destruction. In the end the developed strategies to improve
activity and stability need to be quantified.
One interesting point which is rarely mentioned is the
solubility of an enzyme in comparison to the solubility of its
substrate. The solubility of the enzyme in an aqueous system
strongly depends on the amino acid residues expressed on the
surface of the protein determining the hydrophobicity of this
biopolymer. For a specific volume element a specific loading of
an enzyme can be obtained this is the maximal solubility of
an enzyme (e.g. approx. 10 mg mL1 for alcohol dehydrogenase
from Saccharomyces cerevisiae) in aqueous buer with a given
Lutz Hilterhaus has been head of
the MicroBioTechnology group
within the Institute of Technical
Biocatalysis at Hamburg
University of Technology (TUHH)
since 2009. He received his PhD
(2008) from the TUHH for
research on the development of
a reactor concept for the
enzymatic production of novel
ester oils under the supervision
of Professor Andreas Liese and in
Lutz Hilterhaus close cooperation with Evonik-
Goldschmidt. Afterwards he
carried out his postdoctoral research in the group of Professor
Uwe T. Bornscheuer at the Ernst-Moritz-Arndt University
Greifswald. In the summer of 2008 he received the Karl-Heinz-
Ditze Award for special achievements in engineering sciences.
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mass specific activity (e.g. 50 mmol (min mg)1) or turnover
frequency. This leads to the maximum volume specific activity
in this case 500 mmol (min mL)1. However, the solubility of the
substrate might be higher e.g. 20 mg mL1 (this equates to
0.16 mmol mL1 for 2-phenylethanol, which is a substrate with
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a very low solubility). These physical parameters dictate that
with this enzyme in the given volume element a minimal
reaction time of 0.3 min is necessary (enzyme kinetics and
inhibitions are disregarded). These parameters lead to a limita-
tion in spacetime yield in continuous processes. Here, immo-
bilization oers the possibility of overcoming the problem of
catalyst density by increasing the enzyme loading per volume
element. Alternatively, permanent feeding of substrates and in situ
product removal have to be applied. The use of co-solvents and
surfactants to increase substrate solubility is somehow limited,
because of their possible negative eect on enzyme stability.
Another approach in biocatalysis is the use of solvent-free bio-
transformations. Here, the pure substrates are used which might
have completely dierent dissolving power regarding enzymes
compared to aqueous buer systems. In such systems, dispersion
as well as retention of the biocatalyst is achieved in almost all cases
by immobilized enzymes.
Thus, depending on the catalyzed reaction one has to
judge the stability of enzymes and choose appropriate para-
meters to be discussed in the following for a decision on
immobilization.
Choice of carrier for enzyme immobilization
Depending on the reaction medium and its viscosity, easier
separation using an immobilized biocatalyst can be achieved. At this
point a decision has to be made on the particle size/shape, the
chemical nature, density and porosity as well as the mechanical
strength of such a carrier.2 A huge variety of procedures is published
for the immobilization of an even larger variety of enzymes and
carrier materials.10 When keeping the total mass of carrier constant,
the smaller the particle size gets the larger the surface gets for
enzyme immobilization. Alternatively, the use of porous particles
will allow increasing the area for the immobilization of enzymes.6
When determining the distribution of immobilized enzymes in a
particle the enzyme molecules are mainly found in the outer layers
of the particle, because of diusion limitation.15 Additionally, Mei
and coworkers suggest that a strong anity of the enzyme for the
matrix and the low anity or even repulsion between immobilized
enzyme molecules at the surface and dissolved enzyme molecules
limits the extent that dissolved enzyme reaches and adsorbs to the
internal portions of the particle.15 Therefore, in view of porous
particles there is only a certain penetration depth of the
enzyme. Nevertheless, a complete and uniform distribution of
the immobilized enzyme throughout the particle is only beneficial
if catalysis can occur throughout the bead volume.
Hence, small particle sizes favor high enzyme loadings in
both cases: porous and non-porous particles. However, separation
of the immobilized enzyme from the reaction medium might get
more dicult. Furthermore, fixed bed reactors operated with
small particle size might result in higher pressure drops in the
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Fig. 1 Pressure drop in a packed bed is dependent on particle size. Calculations
were made for a theoretical case study. Assumptions: fluid density 890 kg m3,
fluid velocity 0.00685 m s1, void fraction 0.543, length 0.274 m, Euler number
1755.16
reactor (Fig. 1). The flow resistance of a particle in a fixed bed at a
flow velocity u0,PB is represented by the Euler number EuPB:
4 DpV;PB dP e2
EuPB 2
(1)
3 LPB rf u0;PB 1  e
where DpV,PB is the pressure drop in the packed bed, LPB is the
length of the packed bed, dP is the diameter of the particle, rf is
the density of the medium and e is the void fraction of the
packed bed. The Euler number depends on the Reynolds number,
which depends on the viscosity of the medium. For a comprehensive
overview of the dierent parameters and their mathematical
description the reader is referred to Wijngaarden et al.17
The mechanical stability of the carrier is a crucial parameter
for many applications of immobilized enzymes. Next to the
catalytic activity and the storage, operational, and thermal
stability of enzymes the mechanical stability can limit the long
term application of the biocatalyst in the process. Depending
on the carrier material the eect of mechanical stress can cause
disintegration of the catalyst, complicating the downstream
processing.
The physical nature of the carrier, e.g. pore size, porosity and
pore-size distribution, particle size and morphology, influences
the mechanical stability of the final biocatalyst. Thus, the
selection of these physical properties must not only address
properties needed for certain reactor configurations and process
conditions but also for high immobilization yields and low
diusion limitations.
Within the pioneering work of Rumpf18 a first mathematical
description of agglomerate strength for grains of uniform size
was presented:
1  eg
s 6S cosy (2)
edP
where s is the maximum tensile strength in the granule, S is the
liquid pore saturation, e is the granule porosity, dP is the
diameter of the particles, g is the liquid surface tension and y
is the liquidsolid contact angle. Therefore the trend is that the
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number of broken particles always increases with the increase
of porosity, irrespective of the primary particle size. Schuberts
findings are in good agreement with Rumpfs predictions for
agglomerates of randomly packed monosized glass spheres:19
1  eF
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s (3)
dp 2
Here, e is the void fraction in the agglomerate, F the breakage force
and d the particle diameter. Therefore the porous structure
represents a high inner surface however, porosity, size distribution
and geometry of pores have a strong influence on the breakage
tendency of the granules.20 One possibility to enhance the
mechanical stability is the use of silicon-coating.21
Within the last few years the use of so-called nano-carriers
has been investigated.22 One argument for the use of such
carriers with diameters in the nanometer range is their larger
surface to volume ratio in comparison to non-porous carriers in
the micrometer range. Furthermore, comparing the structure of
classical porous carriers and the structure of nano-carriers, a
huge dierence is observed in the localization of the enzyme
(Fig. 2). On nano-carriers the enzyme binds to an openly
accessible surface, whereas in porous carriers the largest
amount of enzyme is found at the inner surface of the carrier.
However, when working with nano-carriers agglomeration of the
single particles is an often observed phenomenon, especially
when working with paramagnetic and ferromagnetic carriers.
Having a look at these agglomerates in comparison to the porous
carriers there might still be a benefit because of less diusion
limitation. The structure of the porous carrier is somehow
inverted. The same mass of carrier material can oer an even
larger accessible surface for enzyme immobilization. However,
pressure drop of packed bed as well as changed sedimentation
rates of such particles must be considered. The numbers of
studies applying paramagnetic nano-particles for enzyme
immobilization are rising23 and perhaps the acceptance as a
commercial enzyme support will get higher. In summary, the
choice of the carrier (the physical characteristics like pores size
and porosity as well as the mechanical strength) influences
the mechanical and chemical robustness of an immobilized
biocatalyst and its applicability in industrial processes and
therefore needs to be considered.
In all cases, the fast success of an immobilization procedure
depends on the analysis of the enzyme to be immobilized. The
surface properties of the protein need to be considered like
functional groups, ionic charge and hydrophobic groups. Then
Fig. 2 Comparison of a porous carrier (A) and nano-particles (B) for enzyme
immobilization.
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an appropriate combination of carrier material and immobili-
zation method has to be chosen. Porous systems with high
internal surface area for adsorption or covalent binding as
well as functionalization of the carrier surface are preferred.
However, when a high activity yield on nano-particles is
achieved, diusion limitations can be reduced.
Evaluation of mass transport limitation
To analyze and consequently optimize the performance of an
immobilized enzyme it is important to understand the dierent
factors contributing to the overall macroscopic activity that is
the basis for determination of the stability of immobilized
enzymes. Two major eects can be dierentiated altering the
specific activity of the enzyme itself:
 physico-chemical surface properties
 mass transport limitations
Depending on the physico-chemical parameters of the
immobilization surface like charge density, pH value and
hydrophobicity the microenvironment of the enzyme might
have dierent properties in contrast to the bulk phase (compare
the Evaluation of immobilization yield section for a discussion on
zeta-potential). A change in the local charge density influences the
local proton concentration and as a result the local pH value in
near proximity of the enzyme; e.g. on a cationic surface the pH
value is shifted in the alkaline area and because of repulsion the
local proton concentration is decreased. The adverse eect is
observed in the case of negatively charged surfaces leading to an
acidic microenvironment of the enzyme.
A second major factor influencing the macroscopic activity
of an immobilized enzyme is mass transport limitations that
can be dierentiated into four separate transport steps of the
reactants to and from the active site:24
(1) Film diusion: substrate passing through the boundary
layer (stagnant film) close to the surface
(2) Pore diusion: substrate passing from the surface of the
porous carrier to the active site
(3) Pore diusion: product passing from the active site to the
surface of the porous carrier
(4) Film diusion: product passing from the surface through
the boundary layer (stagnant film) to the bulk phase
They are dierentiated into external (film diusion) and internal
(pore diusion) mass transport. In the case of diusion limitation
any of the transport steps 14 dominate and limit the overall
activity of the immobilized enzyme. It is important to determine
in which step the major limitation is given to enable optimization.
For a detailed discussion and mathematical description of mass
transport limitations the reader is referred to standard text books
on enzyme reaction engineering.3,25,26 Also the orientation of the
enzymes active center contributes to mass transport limitations,
because if oriented to the support surface, this can generate some
additional diusion limitations. This phenomenon is independent
of protein loading, except, if the active center is oriented towards
the neighboring protein molecule.
The simplest model for external diusion limitation
describes the flux N (moles per unit time and per unit area)
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of a starting material S from the bulk phase through a stagnant film
with thickness d and the mass transfer coecient kS to the surface:
kS  
N cS;bulk  cS;surface (4)
d
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Increasing the stirring rate in a stirred batch reactor will reduce
external diusion, since external diusion increases inversely with
the thickness of the stagnant layer. However, particle fragmenta-
tion has to be observed, because of the energy input. This is at the
same time a quick test for external diusion limitation. If the
overall reaction rate is not a function of the stirring rate, then no
external diusion limitation is present. Or in other words: in a
packed bed reactor with immobilized enzyme kS increases with
increasing flow rate. Alternative approaches to enhance mass
transport by reducing film diusion are: reduction of particle size
to increase mass transport at a constant fluid velocity, increase of
particle strength to withstand high fluid velocity and keep the
particle size constant as well as increase of the flow rate to
minimize the thickness of the boundary layer.26
As a consequence of the external mass transfer limitation
not the real MichaelisMenten constant KM for the starting
material but rather an apparent one, KM(app), is determined. There
are dierent expressions for KM(app) found in the literature,26 e.g.
the most basic one:
kcat cE
KMapp KM (5)
kS
Alternative approaches to enhance mass transport by reducing
pore diusion are: reduction of particle size to reduce the diu-
sion pathways and KM(app), increase of pore size to enhance the
convective flow through the particle, optimization of pH and
charge density of the carrier material surface.
Integral and dierential analysis
For the characterization of immobilized biocatalysts dierent
experimental methods are available making use of slurry as well
as plug flow reactors (pfr) in the mode of packed bed reactors
(pbr). They are dierentiated into two fundamental approaches,
namely integral or, respectively, dierential operation of a reactor.
However, all these reactor types can be used to determine the
specific characteristics such as tn, ttn, t0.5, kd, leaching as well as
diusion limitation. Those in the following discussed methods
are independent of the nature of the heterogeneous catalyst and
therefore are applicable to bio- as well as chemocatalysts. These
fundamental experimental methods are:
Applying integral reactors for the:
 Determination of eectiveness factors
 Determination of diusion limitation
Applying dierential reactors for the:
 Determination of the largest possible particle diameter
 Determination of diusion limitation
Integral reactors
An integral reactor is either a stirred tank or a plug flow reactor
(pfr) whereby from a reaction engineering point of view both
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reactors are comparable.5 The stirred tank reactor is operated
in a non-stationary way. Assuming ideal mixing, the concen-
tration is the same in every volume element as a function of
time. With advancing time the substrate concentration
decreases and the product concentration increases. There are
also several alternative operation modes of the standard stirred
tank reactor like fed batch or repetitive batch that can be used
to increase the catalyst specific productivity (mass of product/
mass of immobilized enzyme). In the plug flow reactor the
product concentration increases over the length of the reactor
and at the outlet the maximum conversion is reached. In other
words, the dimension of time in the stirred tank reactor is
exchanged with the dimension of place in the pfr. The reactant
concentrations to be measured at the outlet of the pfr are the
sum of the individual concentrations changed in the individual
volume elements in series of the reactor.
Determination of eectiveness factors
There are two eectiveness factors that can be dierentiated,
the stationary and the operational one.3 The ratio of the initial
rates v0 of the immobilized enzyme to the free enzyme under
identical experimental conditions at a specific substrate
concentration is named the stationary eectiveness factor Z:
observed reaction rate with immobilized enzyme
Z (6)
observed reaction rate with free enzyme
Z is determined by size, shape and porosity of the support
matrix as well as the diusion of the compounds involved. A
limitation of this factor is its dependency on the substrate
concentration via enzyme kinetics and it is therefore also not
constant. As an alternative the operational eectiveness factor Z0
was introduced by Kasche (1983) to overcome this limitation.24 It
is defined as the ratio of the time required to reach the end point
of a process with the same initial substrate concentration and
the same amount of free and immobilized biocatalyst per reactor
volume unit (Fig. 3) under otherwise equal conditions:
tn;free
Z0 (7)
tn;immob
Determination of diusion limitation
There are two dierent experimental techniques applicable that
make use of plug flow reactors operated with dierent reaction
volumes, or dierent masses of catalyst preparation:
(A) Variation of the catalyst volume at constant residence
time t
(B) Variation of catalyst volume as well as residence time t
VR
t (8)
FV
with t [h] residence time, V [mL] reactor volume, and F [mL h1]
volumetric flow rate.
These methods are applicable because the specific reaction
rate constants of the enzyme itself are not a function of the flow
rate in contrast to the mass transfer coecient.
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Fig. 5 Alternative method to determine film diusion.

Fig. 3 Determination of the operational eectiveness factor.

Method A: the residence time in at least two parallel operated

packed bed reactors is kept constant, whereas the bed volumes
of the immobilized catalyst are dierent, demanding the
respective flow rates. At the outlet of the reactor the respective
conversion is determined based on the reactant concentrations.
For the analysis of a possible diusion limitation the determined
integral conversion is plotted as a function of the respective
volumetric flow rates F for the individual plug flow reactors
(Fig. 4). If at high flow rates the conversion is increasing at
constant residence time a film diusion limitation is given. If no
influence of the flow rate on the conversion is observed, the Fig. 6 Principal flow scheme of a dierential reactor as continuously operated
circulation reactor.
reaction is predominately controlled by catalyst kinetics.
Method B: the alternative method is operation of two pfr with
dierent catalyst bed volumes at several residence times. Here Determination of the largest possible particle diameter
it is important to note that prior to changing the residence time
the steady state of the operation point needs to be reached. If in The particle diameter plays a crucial role in minimization of
this case conversion is plotted as a function of the residence time diusion limitation. In view of applicability the particle diameter for
(Fig. 5), film diusion limitation can be identified, if the two use in packed bed reactors should be ideally between 200400 mm to
resulting curves are not superimposable. Higher conversion is ensure low backpressures. For applications in stirred tank reactors
reached in the reactor with the respective higher flow rate. the diameter can be reduced to Z10 mm still enabling separation by
filtration or sedimentation.3
Dierential reactor To determine the maximal particle diameter of an immobi-
lization matrix exhibiting the lowest possible pore diusion an
Here, a dierential reactor is a circulation reactor with a packed
easy experiment can be carried out. The catalyst particles are
bed of immobilized enzyme (Fig. 6) that is either operated as
ground to dierent diameters and subsequently packed as a
batch or continuously. Dierential means that in contrast to
fixed bed. Determination of the initial reaction v0 is carried out
the integral reactor there is only a very low concentration
in a continuously operated circulation reactor with a fixed bed
gradient over the length of the packed bed per cycle. Therefore
under initial rate conditions, meaning an overall conversion of
this is also often addressed as a gradientless circulation
5% is not exceeded to prevent an inhibitory eect by the
reactor. The characteristics of a continuously operated stirred
products. For dierent particle sizes of the catalyst the reaction
tank reactor (cstr) are established, if as a rule of thumb the
rates are determined until no increase in the reaction rate with
circulation flux is at least 20 times faster than the continuous
decreasing particle diameter is observed. For analysis the
flux entering and leaving the circulation reactor.
eectiveness factor for the diameter Zd is plotted as a function
of particle diameter (Fig. 7):
v0;particle diameter
Zd (9)
v0;smallest particle diameter

Determination of diusion limitation

Only this setup of a continuously operated circulation reactor
enables the variation of the flow rate in the packed bed at a
constant residence time for the overall reactor. In contrast to
the integral reactors only a short packed bed is needed because
of the high recycle stream (lower investment of immobilized
Fig. 4 Measurement of conversion as function of volume flow F. catalysts).

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Fig. 7 Eectiveness factor as function of particle diameter.
Fig. 8 Investigation of pore diusion limitation in circulation reactor.
If at constant residence time with increasing flow rate at the
individual catalyst particle the measurable individual residual
starting material concentration is decreased, pore diusion is
the reason. For two dierent residence times the flow rates
(circulation) are varied in respect to a large and a small particle
Reynolds number (ReP). The reaction rate v is calculated
according to:
cS;inlet  cS;outlet
v (10)
t
with t [h] residence time, cS,inlet [mM] starting material concen-
tration at the reactor inlet and cS,outlet [mM] starting material
concentration at the reactor inlet.
With increasing circulation flow rate the reaction rate
increases (Fig. 8) while the residual concentration of the start-
ing material decreases (increasing conversion at constant
residence time).
Evaluation of immobilization yield
The above-mentioned stability combined with the total turn-
over number is strongly dependent on the initial activity v0 of a
biocatalyst. When working on enzyme immobilization the
question for the quality of the immobilization needs to be
answered. Dierent activities can be determined in the labora-
tory: the activity in the stock solution [U mL1], the activity
which is found on the carrier material [U g1] as well as the
remaining activity in the supernatant [U mL1]. When judging
the immobilization and keeping the above-mentioned turnover
number in mind which is related to the amount of enzyme it
is quite obvious that the determined activities need to be
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related to the molar amount of enzyme. However, using these
parameters an activity yield Yact can be calculated meaning:
aimmo mimmo
Yact;immo (11)
astock Vstock
asupernat Vsupernat
Yact;supernat 1  (12)
astock Vstock
with Yact,immo [] activity yield based on the activity of the
immobilized enzyme, aimmo [U g1] activity of the carrier after
immobilization, mimmo [g] mass of the carrier, astock [U mL1]
activity of the stock solution, Vstock [mL] volume of applied
stock solution, Yact,supernat [] activity yield based on activity
loss in the supernatant, asupernat [U mL1] activity of the super-
natant after immobilization, and Vsupernat [mL] volume of the
supernatant.
Sometimes, both of these procedures for the calculation of
the activity will not give the same result. This can have two
reasons:
 aimmo is lower/higher than theoretically expected
 asupernat is lower than theoretically expected
In the first case, the immobilization procedure itself
can have a negative eect on the enzyme activity. Chemical
modifications of the enzyme can aect the tertiary structure.
Furthermore the binding of the biological macromolecule to
the solid surface can change its flexibility. This is due to
multipoint attachment of the enzyme and accompanied rigidi-
fication. A certain amount of flexibility and the possibility of
changes in conformation are necessary to obtain the optimal
activity of an enzyme. Drastic changes of the structure will lead
to activity loss and subsequently to the deactivation of the
enzyme. One solution to this problem can be the control of the
immobilization position.27 Especially the eect that the calcu-
lated activity of an immobilized enzyme is higher than that of
an enzyme in solutions is surprising. This eect occurs when
co-immobilization of multiple enzymes is carried out.28 One of
the first examples is the use of hexokinase and glucose-6-
phosphate dehydrogenase reported by Mosbach and Mattiasson,29
who attributed this eect to the reduced way of diusion for the
substrate from one enzyme to the other.
The second case the activity of the supernatant is lower
than theoretically expected can occur when the (covalent)
immobilization procedure itself has a negative influence on the
activity of the dissolved enzyme. This might occur when a
coupling reagent reacts with the dissolved enzyme, especially
when the coupling of the enzyme is carried out in one step.
Furthermore, shear stress, thermal deactivation as well as
oxidation reactions during the incubation can lead to faster
deactivation. In general, this is the case if the conditions cause
inactivation of the dissolved enzyme. However, also due to
dilution that favors enzyme dissociation inactivation can occur.
To overcome these problems especially the covalent coupling
should be carried out in two steps. First the reaction of the
coupling reagent with the carrier followed by extensive washing
of the carrier and then the addition of the enzyme for immo-
bilization. Also a well optimized immobilization procedure can
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lead to discrepancies between the theoretically expected activity
yields and the experimentally measured values. To get a better
understanding of the immobilization process the information
regarding the activity yield has to be linked to mass specific
yield. This second value is defined by the mass of protein
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bound to the carrier:
csupernat Vsupernat
Ym;supernat 1  (13)
cstock Vstock
cimmo mimmo
Ym;immo (14)
cstock Vstock
with Ym,supernat [] mass specific yield based on protein mass
loss in the supernatant, csupernat [mg mL1] concentration of the
supernatant after immobilization, Vsupernat [mL] volume of the
supernatant, cstock [mg mL1] concentration of the stock
solution, Vstock [mL] volume of applied stock solution, Ym,immo
[] mass specific yield based on protein mass on the carrier,
cimmo [mg g1] protein mass on the carrier after immobilization,
and mimmo [g] mass of the carrier.
Here, the mass specific immobilization yield is based on the
protein concentration in the supernatant as well as in the stock
solution given in mg mL1. Using this method, the amount of
protein on the surface can be determined and in combination
with the activity yield one can calculate the mass specific
activity of an immobilized enzyme (in the case of applying a
purified enzyme solution for immobilization). Because immo-
bilization can result in a loss of active sites depending on the
applied method, information about the amount of immobilized
and active enzyme molecules is needed. Active site titration can
be used to compare dierent immobilization methods by
quantification of the recovery of active sites and catalytic
eciency. The method is based on the titration of the amount
of active sites present by employing an irreversible inhibitor.30
This value is the value of interest, when calculating the costs
for fermentation, purification and immobilization, in view of
economics. Comparing the number of active sites of the
dissolved and immobilized enzyme is possible which allows
the evaluation of the additional costs for the immobilization.
The eciency of enzymes binding on particles depends
besides the chemical properties of the enzyme and the particle
on both surface potentials. The majority of particles dispersed
in an aqueous solution exhibit a surface charge (compare
Fig. 9), basically either by ionization of surface groups, or
adsorption of charged molecules/ions. These surface charges
alter the distribution of the ions in a layer around the particle
(Stern layer), which is dierent from the bulk solution. The zeta
potential is the potential at the point in this layer, where it
moves past the bulk solution (slipping plane) and is one of the
major forces that mediate interparticle interaction. Therefore
the zeta potential can be considered as an indicator of the
eciency of enzyme binding, allowing the quantification of possible
electrostatic interactions between enzyme and particles.31 Fig. 9
illustrates the potential dierences as a function of distance from
the charged surface. An increasing binding eciency is expected
when decreasing electrostatic repulsion or increasing electrostatic
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Fig. 9 Schematic representation of zeta potential: ionic concentration and
potential dierences as a function of distance from the charged surface of a
particle suspended in a medium.
attraction between the interacting compounds. Schultz et al.
found a clear correlation between zeta potential and binding
eciency but no correlation between the particle related
specific activity and the zeta potential.32 This illustrates again
the importance of the correct 3D-structure and orientation of
the enzyme after immobilization to achieve a high activity yield,
in addition to the mass specific yield.
Therefore the measurement of the zeta potential can be used
as a diagnostic tool to estimate the electrostatic interactions
between enzymes and carriers. Despite the important role of
the zeta potential in enzyme immobilization, there are very few
reports considering its measurement before immobilization of
the enzyme onto the carrier. However, this technology allows
the reduction of the experimental work necessary and a more
straightforward and knowledge based immobilization of enzymes.
One experimental case study is found in Gomez et al.33
Finally, a theoretical example (compare Fig. 10) illustrates
that only a combined analysis of the before mentioned yields
and activities can give a comprehensive picture of the immo-
bilization process. The example describes the enzyme immobi-
lization on a porous carrier, the amount of enzyme in the stock
solution is increased stepwise, and the activity of the immobilized
enzyme is analyzed. This experiment clearly shows the limitation
when only observing a parameter like aimmo. Certainly, the activity
on the carrier will increase because of a higher protein loading
whereby the mass specific immobilization yield will be almost
constant. Therefore, one would calculate a high Ym (the specific
activity of the carrier increases). Nevertheless, the interesting
information is the activity yield coupled to the mass of immobi-
lized enzyme (which represents the yield of carrier bound activity).
This yield drops because the immobilized enzyme does not show
the same apparent activity as the corresponding amount of
enzyme in solution. From this calculation it becomes obvious
that a high mass specific yield does not mean automatically a
high activity specific yield. Also, these considerations illustrate the
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Fig. 10 Theoretical case study to illustrate non-linear dependency of protein
loading and activity yield. Assumption: exponential decay of activity yield
because of multilayer formation of enzymes.
great importance of surface analysis technologies on a mole-
cular level for the free as well as the immobilized enzyme to
understand immobilization in detail. An experimental case
study can be found in Ragnitz and Syldatk.34
Surface analysis technology for immobilized
enzymes
Traditionally, protein quantification techniques are UV-spectro-
photometric or fluorometric methods based on bicinchoninic acid
or the Bradford assay as well as assays such as Lowry, Micro
Pyrogallol Red and FluoroProfile. Depending on the individual
case quantification of enzyme in stock solution and the super-
natant as well as on the carrier should be carried out. Especially,
the quantification on the carrier might be challenging for a direct
spectrophotometric test. However, a highly sensitive fluorescence
reaction for amino acids using o-phthalaldehyde and 2-mercapto-
ethanol permits the detection of amino acids separated by HPLC.
The reasons for the dierent activities obtained after immo-
bilization and also the dierent stabilities for immobilized
enzymes are found in the microenvironment of the enzyme on
the surface as well as in the degree of multipoint attachment. The
major problem with enzyme immobilization is the high complex-
ity of the enzyme in combination with the three-dimensional
structure and the structural variability necessary for a correct
function. To optimize the attachment itself information about the
folding/unfolding of the protein caused by the interaction of the
protein with the surface as well as the interaction of the protein
with other proteins is mandatory. It is obvious that the informa-
tion about the mass specific activity somehow correlates with the
surface of the carrier. Therefore, two central topics need to be
addressed: first, the enzyme loading per surface (mg m2) is
important. Second, the orientation of the enzyme to the surface as
well as the influence of the surface on the three-dimensional
structure and the structural variability needs to be investigated.
This molecular level can be investigated using Forster resonance
energy transfer (FRET) analysis,35 atomic force microscopy (AFM)
and scanning electrochemical microscopy (SECM), quartz crystal
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Fig. 11 Eect of the density of the active functionality on the immobilized
enzyme and analytical tools: (A) and (E) rigidification and unfolding of a protein
because of multiple attachment; (B) single point attachment; (C) conformational
limitations because of proteinprotein interaction; (D) diusion limitations
because of multilayer formation.
microbalance (QCM)36 and time-of-flight secondary ion mass
spectrometry (TOF-SIMS)37 which are illustrated in Fig. 11.
Enzymes undergo a large number of conformational
rearrangements to fold into their specific structure and func-
tional state. At this juncture, this phenomenon has not been
completely understood. The prediction of an enzyme structure
from its amino acid sequence remains one of the key challenges
in life science. When addressing the interaction of an enzyme and
a surface, conformational changes and refolding of the enzyme
can occur. This change in its three-dimensional structure can
strongly influence the resulting activity of enzymes. Therefore,
information about the structure of an enzyme bound to a surface
is of high importance. FRET is a structure sensitive probe, which
is used in single molecule fluorescence spectroscopy. Here, the
analysis of the fluorescence emission from individual labeled
proteins is carried out.38 FRET is a sensitive tool for examining
structural and dynamic properties of individual molecules on an
atomic level. Especially, time trajectories of folding pathways and
transient intermediates can be characterized.35
The evaluation of enzymes on surfaces, such as distribution
or orientation, is one important issue for the development of
sophisticated biocatalysts. The ability to orient enzymes on surfaces
will enhance a wide range of applications in biotechnology.39 As
pointed out above the orientation and three-dimensional structure
of immobilized enzymes are a key to assure their high performance
in view of activity and stability. One tool to analyze enzymes on
surfaces is the TOF-SIMS. This method allows the submicron-scale
mapping of surfaces. TOF-SIMS has been already used in the steric
analysis of proteins.37 The sampling depth of TOF-SIMS is less
than 2 nm, whereas the size of most proteins is larger. Therefore,
TOF-SIMS provides the chemical structure of the surface side of an
immobilized enzyme indicating the enzyme orientation. However,
TOF-SIMS has till now not come into widespread use in the field
of enzyme immobilization because of its complicated spectrum
interpretation. In the future not only the orientation is to be
analyzed but also the protein structure and structural changes
because of dierent media or surface binding.40
Characterization technologies for the study of enzyme
immobilization on surfaces are needed especially at the nanometer
level. This characterization of immobilization processes should
include the visualization of the immobilized enzymes. Among the
technologies that have been employed AFM has been shown to be
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useful to achieve morphological and mechanical information at
the nanometer level.41 The way in which enzymes are adsorbed
rather than the amounts elicits the specific biological function.
Therefore AFM studies can contribute to improve the immobiliza-
tion process. Additionally, AFM can also provide information on
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proteinprotein and proteinsurface interactions, through force
spectroscopy measurements, and on surface-induced conforma-
tional changes of enzymes.42 However, these very precise measure-
ments tolerate only a certain roughness of the surface. Finally, AFM
does not provide information about the catalytic activity of the
immobilized enzyme. A complementary technology is SECM. SECM
provides insights into the remaining activity of the immobilized
enzyme, allowing the determination of the influence of the immo-
bilization procedure on the catalytic activity.36 Spatially resolved
information on a wide range of electrochemical processes occur-
ring at interfaces can be obtained. For detailed information on
this technique the reader is referred to Bard and Mirkin.43 Lei
et al. have applied SECM successfully to obtain local enzyme
activity images and visualized the localized enzymatic activity
using glucose oxidase as a model enzyme.44 Additionally, SECM
is able to evaluate kinetic parameters and diusion profiles of
reactants. The combination of AFM and SECM can provide informa-
tion without destruction of the enzyme under real conditions.
The above introduced techniques allow studying the surface
and the enzyme bound to the surface at a specific time. For
information on the dynamics of protein immobilization further
methods are required. One method is the QCM which can be
used as a highly sensitive mass detector and allows the in situ
measurement of mass on the nanogram level.36 Kinetic infor-
mation on the adsorption process as well as on the surface
coverage of the resulting adsorbed layer is accessible. However,
these very precise measurements are based on a quartz crystal
and the piezoelectric eect observed via a pressure on quartz.
Therefore, this technology can be applied to investigate certain
immobilization protocols and the eect on enzyme loading and
activity on a (modified) quartz crystal, only. Afterwards, the
gathered information must be transferred to porous carriers for
real time application.
The analysis of the orientation of the enzyme to the surface
as well as the influence of the surface on the three-dimensional
structure and the structural variability are of great importance
because of the impact on activity and stability. Next to static
analysis also the flexibility and possibility of conformational
changes of the enzyme for correct function need to be kept
in mind.
Evaluation of biocatalysts deactivation
In the past decades, the number of biotechnological processes
in the chemical and pharmaceutical industry is permanently
increasing.45 Fermentation processes, whole cell catalysis as
well as enzymatic syntheses are applied. However, for all of
these biocatalytic routes the costs for catalysts and processing
need to be compared to chemical routes. In addition to the raw
material costs required for organic synthesis the costs for the
preparation of the catalysts have to be additionally considered.
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In particular, the fermentation yield in terms of final achievable
cell concentration and the expression level as well as the produc-
tion scale are crucial for decreasing the total cost contribution of
biocatalysts.46 Filtration as well as cell disruption and protein
purification might be necessary to purify the enzyme from the
cultivation medium. Therefore, coupling of immobilization to
purification might reduce the total work eort and costs.6 However,
there is still high interest in the eective use of enzymes because of
the numerous steps necessary to obtain a purified protein.47
The eectiveness in using enzymes is represented by the
turnover number. The turnover number is the amount of
product produced per amount of catalyst used:
np 1
tn   (15)
nc vp 
Here, tn is the turnover number; np is the amount of product
synthesized; vp is the stoichiometric factor for the product; and
nc is the amount of catalyst. The tn is independent of the
substrate concentration and directly corresponds to the produced
amount of a specific product. Regarding the above-mentioned
costs for a biocatalyst the tn should be as high as possible to
reduce final product costs and to make the implementation of
biocatalytic reactions in industrial processes more feasible. The
importance of tn gets obvious plotting the relative catalyst costs
versus the turnover number (Fig. 12).
For a general consideration Janssen et al. introduced two
critical turnover numbers for catalysts:48
 Critical turnover number 1: if a catalyst has a too low
turnover number, due to low activity or due to a limited
lifetime, it makes no sense to immobilize it. The additional
costs for modification and recycling facilities cannot be
compensated.
 Critical turnover number 2: if the catalyst activity and
lifetime are very high (meaning very high turnover number)
and/or the added value of the products is high, so that the total
catalyst costs are o0.05% of the added value, there is no
economic need for recycling. Still, improved product purity
and quality might be an argument.
Therefore, if the enzyme costs are between these two critical
turnover numbers one should consider immobilization as a
valuable tool.
Furthermore, the contribution of catalyst costs to the product
costs can be estimated at a given ttn (total turnover number).
The ttn gives the moles of product formed or substrate converted
per mole of catalyst consumed. The ttn should be >1000 for
expensive products being produced on a small scale and >50 000
for large-scale or less expensive products.49 To determine a ttn,
dierent experimental setups can be applied. This might be the
use of a repetitive batch system applying e.g. a filtration unit
between each batch or it might be a continuously operated
reactor with catalyst retention.
In all cases, the process needs to be operated long enough to
observe catalyst deactivation (or e.g. leaching). This is crucial
because the reliability of the calculated costs is only given, if the
half-life of a catalyst is determined correctly.50 The half-life
time is the limiting parameter for the ttn and strongly depends
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Fig. 12 Eect of the turnover number on the relative catalyst costs (loglog
plot).
on the reaction parameters which need to be named when deter-
mining this value: temperature, pH-value, substrate and product
concentration, co-solvents etc. The time necessary for halving the
initial activity v0 [U mg1] is expressed as the half-life time:
vi 0:5v0 (16)
vi v0 expkd ti  t0  (17)
ln 2
t0:5 (18)
kd
where t0.5 is the half-life of the catalyst [h]; vi is the catalyst
activity [U mg1] at time ti [h]; and kd is the deactivation
constant [1 h1]. The activity usually shows a typical exponential
decay. Therefore, the half-life time gives the extent of the catalyst
deactivation independently of the considered time dierences.
Recent reviews highlighting stabilization of enzymes by both
chemical and biological means at higher temperatures or in
organic solvents are found in ref. 5154. When addressing the
stability of an enzyme, the mechanism of protein folding and
unfolding is a fundamental question.55 Proteins are highly
complex biopolymers, exhibiting a substantial degree of structural
variability in their properly folded, native state. In the presence of
denaturants, this heterogeneity is enhanced, and fluctuations take
place among vast numbers of folded and unfolded conformations
via dierent pathways.35 An experimental case study on the partial
thermal unfolding of D-amino acid oxidase as well as the dissocia-
tion of the cofactor flavin adenine dinucleotide shows that the
overall inactivation rate is composed of several steps.56 Therefore
one has to consider that the loss of activity is not directly
connected to unfolding of the protein structure. Furthermore,
deactivation and unfolding for a certain enzyme are even
often uncoupled.57 However, unfolding of the protein will cause
massive changes in its activity. Although complex pathways of
unfolding and deactivation of enzymes were found a simple
approach to calculate the half-life, which is often sucient for
bioprocess engineering, can be applied.
For theoretical estimations based on a preferably small
number of experiments the combination of deactivation and
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turnover number is of interest.58 However, to get reliable values,
kd should not only be determined by end point measurements of
the activity, but rather at dierent time points applying progress
curve analysis. The integration of the curve shown in Fig. 13 will
give the total amount of product produced. With respect to the
known amount of catalyst the total turnover number can be
calculated being discussed later on.
Within the introduction a possible improvement of enzyme
stability by immobilization of an enzyme was mentioned. Such
improvement in view of stability is presented in Fig. 13. The
decrease of the deactivation constant by a factor of 2 will lead to
a non-linear increase of the amount of product produced.
Clearly, the exponential decay of enzyme activity is the reason.
Interpreting this theoretical estimation, a major benefit of
immobilization is the possibility of an increase of the total
turnover number.
When investigating enzyme stability under process condi-
tions deactivation and inhibition need to be dierentiated.
Villela Filho et al. investigated the influence of dierent organic
solvents on the stability of alcohol dehydrogenases applying
two phase systems and the loss in activity was measured over
time.59 For all investigated enzymes the measured residual
activity v0i can be described by considering the contributions
of time dependent deactivation kdt and instantaneous inhibi-
tion I at t0 = 0:
v0i v0 I expkd ti  (19)
To determine I, the enzyme activity in the water phase before
and shortly after addition of the solvent must be compared. Its
magnitude depends on the specific interaction between organic
solvent and enzyme. The time dependent deactivation is
described as already illustrated above by a first-order exponen-
tial decay of activity. Therefore, applying this model instanta-
neous inhibition and deactivation can be dierentiated.
The choice of operating conditions especially the tempera-
ture involves the consideration of two competing processes.
The rise in catalytic activity with increased temperature comes
Fig. 13 Mathematical simulation of enzyme deactivation over time for dierent
deactivation constants and half-life times. The areas under the curves correlate
directly with the produced amount of product. The horizontal line represents the
reduction of activity to 50%.
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along with the loss of structural integrity of the enzyme. Therefore,
the optimum temperature is a compromise between activity and
stability. The integration of the curve shown in Fig. 13 gives as
already mentioned the total amount of product produced or in
other words the yield of the process. Rogers and Bommarius
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published a simple procedure to predict the total turnover number
based on catalytic activity and half-life time.60 The process yield of
a biocatalyst is equivalent to the integral of the instantaneous rate
vmax with respect to time, from zero to infinity:
Z1
yield vmax tdt (20)
0
vmax is given by
vmax t kcat;obs cE;0 expkd;obs t (21)
The non-varying terms kcat and cE,0 may be taken outside the
integral:
Z1
yield kcat;obs cE;0 expkd;obs tdt (22)
0 (1) Thermal deactivation
And integrations will give a term for the overall yield:
 
1 (4) Gasliquid or liquidliquid interfacial areas
yield kcat;obs cE;0 (23)
kd;obs
Dividing by the concentration of enzyme the total turnover num-
ber can be calculated:
kcat;obs
ttn (24)
kd;obs
Both parameters kcat and kd need to be measured under the same
conditions in view of temperature, pH-value and buer content.
The value of kcat can be calculated from the more prevalent specific
activity [U mg1] applying the molecular mass of the enzyme per
active site [g mol1]. Thus, the following formula gives kcat [1 s1]
considering that the activity is given in units [mmol min1]:
specific activity  enzyme molecular mass
kcat;obs (25)
60000
Hence, based on short-term experiments calculation of the ttn
becomes possible and the process yield during the lifetime of a
biocatalyst can be estimated.
In all cases, one needs to keep in mind that the accuracy of
the measured activity decrease influences the reliability of the
determined deactivation constant. Therefore, it might be advisable
to investigate a time range td which shows at least a residual
activity vd of 80% of the initial activity v0 as well as the activity after
dierent time points. After these investigations one has to decide if
stability as well as the turnover number are high enough to enable
an economically feasible process.
In an early stage of bioprocess development assessing
process (or operational) stability of biocatalysts is necessary
for informed decisions on their rational use for production
processes. This operational stability needs to be dierentiated
from the often reported storage stability. The conditions for
biocatalyst storage are mostly optimized in view of high activity
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retention after a longer period of time. Therefore, buer salt
concentrations, pH-value and temperature are adjusted in view
of low deactivation constants. Certainly, these conditions are
completely dierent from the conditions applied in bioprocesses.
Here, depending on the catalyzed reaction, temperature and
pH-value will be adjusted for optimal performance and the use
of salts is reduced to the absolutely necessary level. Furthermore,
substrates, products and byproducts as well as solvents with
varying concentration influence the stability of the enzyme. In
summary, the half-life time under process conditions is of great
importance, to assess the stability for a given biotransformation.
Consequences for reactor selection
Most procedures to characterize the stability of an immobilized
enzyme determine the apparent macroscopic activity of the
biocatalyst preparation. However, for a specific batch of immo-
bilized enzyme it is sometimes dicult to ascertain the reason
for activity loss during a running process. There are several,
often independent, factors contributing to activity loss:
(2) Oxidation
(3) Organic solvents or high concentration of reactants
(5) Chemical instability of the support
(6) Deactivation by a product
(7) Deactivation by a starting material
(8) Abrasion due to shear forces, stirrer and particleparticle
collision
(9) Desorption of metal ions required for activity and/or
stability
(10) Leaching of the enzyme from the carrier
In the case of immobilized enzymes often the immobilization
matrix contributes to decreased stability because of an increased
concentration of products in the stagnant film and the pore
volume. Factors (1)(5) depend on the reaction system and must
be addressed during setting of the reaction conditions.
In the case of deactivation by a product (6) the application of
a continuously operated dierential reactor as a circulation
reactor should be prevented. In this mode operating under
outflow conditions, always the highest product concentrations
possible under the given reaction conditions are reached in
every volume element. Also a fed batch regarding the starting
material is not advisable, since here the maximum product
concentration is reached and even exceeded. Advisable is the
application of a standard batch (or in repetitive batch mode) or
a plug flow reactor where the product concentration increases
with time or length of the reactor, respectively. Alternatively,
the integration of in situ product removal like extraction,
distillation, adsorption or crystallization does provide a low
stationary concentration.
In the case of deactivation by a starting material (7) either a
fed batch is to be established or in continuous mode a
continuously operated dierential reactor (cstr). This reactor
maintains the lowest possible starting material concentration in
the steady state because of the outflow conditions. A standard
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batch or standard continuously operated plug flow reactor is not
advisable, because of the high starting material concentrations
at the start of the reaction in the batch or inlet of the pfr.
In the case of deactivation because of abrasion due to shear
forces, stirrer and/or particleparticle collision (8) the application
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of stirred reactors like slurry or fluidized/expanded bed reactors in
batch or continuous mode is to be prevented.
Desorption of metal ions required as eectors (9) or leaching
of the protein itself (10) in the case of adsorptive immobilization
or entrapment in a polymeric matrix are dicult to determine in
the first place. These eects are predominantly observed in
continuously operated reactors where a high number of residence
times is established in the production process. In both cases a
concentration of the reactor outflow by means of distillation
(metal ions) or ultrafiltration (enzymes) is the key for analysis.
Metal ions can be analyzed via atom spectroscopy and enzymes
via activity measurement in the case that exclusively leakage and
no deactivation are observed. Alternatively for enzymes, if no
nitrogen is present in the reaction itself, nitrogen analysis can
be carried out. Leakage of metal ions can be reduced if a certain
concentration thereof is established in the bulk volume shifting
the adsorptiondesorption equilibrium. Alternatively, establishing
a covalent immobilization procedure or coating of the whole
biocatalyst particle with a polymer can reduce leakage of
adsorbed enzymes from the support. Ansorge-Schumacher et al.
demonstrated a very successful industrial example, enhancing
mechanical and leaching stability.21 By deposition of silicone
coatings, available from cheap silicone building blocks under
simple reaction conditions, the stability of an immobilized lipase
(Novozyme 435) could drastically be increased. Activity yields in
the coating process of more than 92% were demonstrated.
Conclusion
Many parameters influence the structure of an immobilized
enzyme within a biocatalytic process. Often, the reaction to be
catalyzed by the enzyme and the reaction conditions (e.g.
temperature, solvent as well as the desired substrate and
product concentration) are given and adaption of immobilized
enzymes is required. For straightforward and knowledge based
stabilization of the immobilized enzyme the folding of the
enzyme on the surface needs to be understood in detail. Using
tools from molecular biology and chemistry to optimize the
attachment of the enzyme on the surface combined with
modern analytical technologies strategies to improve activity and
stability can be developed. Furthermore, transport phenomena of
substrates and products must be addressed by solid process
engineering and optimized geometry to yield a biocatalyst with
both high activity and high stability.
Notes and references
1 S. Lutz and U. T. Bornscheuer, Protein engineering handbook,
Wiley-VCH, Weinheim, 2012.
2 L. Cao, Carrier-bound immobilized enzymes. Principles, applications
and design, Wiley-VCH, Weinheim, 2005.
6248 Chem. Soc. Rev., 2013, 42, 6236--6249
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3 K. Buchholz, V. Kasche and U. T. Bornscheuer, Biocatalysts
and enzyme technology, 2nd edn, Wiley-Blackwell, Weinheim,
2012.
4 J. M. Guisan, Immobilization of enzymes and cells, 2nd edn,
Humana Press, Totowa, N.J., 2006.
5 A. Liese, K. Seelbach and C. Wandrey, Industrial biotransfor-
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