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Catalysis Today
journal homepage: www.elsevier.com/locate/cattod

Advanced characterization of immobilized enzymes as heterogeneous

biocatalysts
Juan M. Bolivar a , Ingrid Eisl a , Bernd Nidetzky a,b,∗
a
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, A-8010 Graz, Austria
b
Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz, Austria

a r t i c l e i n f o a b s t r a c t

Article history: Like in chemical catalysis, there is a clear trend in biocatalysis to carry out synthetic transformations
Received 5 April 2015 at the manufacturing scale heterogeneously catalyzed. Recycling of insoluble catalysts is simplified, and
Received in revised form 5 May 2015 continuous reactor development thus promoted. Heterogeneous biocatalysis usually involves enzymes
Accepted 6 May 2015
immobilized on mesoporous solid supports that offer a large internal surface area. Unraveling enzyme
Available online xxx
behavior under the confinement of a solid surface and its effect on the catalytic reaction in heterogeneous
environment present longstanding core problems of biocatalysis with immobilized enzymes. Progress in
Keywords:
deepening the mechanistic understanding of heterogeneous biocatalytic conversions is often restrained
Heterogeneous biocatalysis
Immobilized enzymes
by severe limitations in methodology applicable to a direct characterization of solid-supported enzymes.
Mesoporous solid support Here we highlight recent evidence from the analysis of protein distribution on porous solid support
Imaging analysis using microscopic imaging methods with spatiotemporal resolution capability. We also show advance
Internal sensing in the use of spectroscopic methods for the analysis of protein conformation on solid support. Methods
Direct characterization of direct characterization of activity and stability of immobilized enzymes as heterogeneous biocatalysts
In operando are described and their important roles in promoting rational biocatalyst design as well as optimization
and control of heterogeneously catalyzed processes are emphasized.
© 2015 Elsevier B.V. All rights reserved.

1. Immobilized enzymes: great catalysts for chemical
process development
Modern chemical synthesis strives for synthetic routes that
are selective, atom- and step-efficient and inherently safe. Use of
enzymes as catalysts potentially enables all of these tasks to be
achieved at once [1–3]. Bio-catalysis has therefore been identi-
fied, and already serves, as key enabling technology for chemical
synthesis at the industrial process scale [3–6]. Like in chemical
Abbreviations: AFM, atomic force microscopy; CD, circular dichroism;
CLSM, confocal laser scanning microscopy; DLR, dual life-time referencing;
DRIFT, diffuse reflectance infrared Fourier transform spectroscopy; FESEM, field
emission scanning electron microscopy; FTIR, Fourier transformed infrared spec-
troscopy; IR, infrared spectroscopy; MIRS, mid-infrared spectroscopy; NIRS,
near-infrared spectroscopy; NMR, nuclear magnetic resonance spectroscopy;
PM-IRRAS, polarization-modulated infrared reflexion absorption spectroscopy;
QCM, quartz crystal microbalance; SECM, scanning electrochemical microscopy;
SIRMS, synchrotron infrared microspectroscopy; SPR, surface plasmon resonance;
STEM, scanning transmission electron microscopy; TEM, transmission electron
microscopy; XPS, X-ray photoelectron spectroscopy.
∗ Corresponding author at: Institute of Biotechnology and Biochemical Engineer-
ing, Graz University of Technology, NAWI Graz, Petersgasse 12, A-8010 Graz, Austria.
E-mail address: bernd.nidetzky@tugraz.at (B. Nidetzky).
catalysis, there is the fundamental choice between homogeneous
and heterogeneous biocatalysis [4,7–9]. Homogeneous biocatalysis
involves enzymes dissolved in aqueous liquid phase. Heteroge-
neous biocatalysis involves enzymes in a water-insoluble (solid)
form [9–11]. Clear trend to prefer heterogeneously bio-catalyzed
reactions in current industrial production processes is recognized,
in consequence of two main advantages. Firstly, separation and thus
recycling of the catalyst are simplified when enzymes are present
insoluble. Secondly, continuous biocatalytic process development
is supported ideally [4,7,8]. Benefits of continuous processing for
high-quality chemicals manufacturing can thus be exploited fully.
Different principles of heterogeneous biocatalyst preparation have
been described in almost countless varieties [9]. However, the
principle most widely used by virtue of overall practical effect is
immobilization of an initially soluble enzyme on a mesoporous
solid support [10,12–14]. The support is usually selected to offer
a high internal surface area accessible to and chemically suitable
for the enzyme to become attached physically, chemically or often
both [9–11,15–19].
Good choice of an immobilization requires that consider-
ations from the various underlying disciplines, including protein
chemistry and enzymology, materials and surface sciences, and
reaction engineering, are all integrated adequately. Designing an

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Fig. 1. Enzyme immobilization in porous carriers is shown. Influence of parameters related to the support, the enzyme and the mode of enzyme-on-surface deposition on
observable catalytic properties.
immobilization is complicated not only by the multidisciplinary
nature of the problem, but also because relevant effects occur
at vastly different length scales in the nanometer (molecular)
to millimeter range [10,15,16,20]. Therefore, this exacerbates the
selection of suitable methodology for monitoring of the immo-
bilization process and for characterization of the final enzyme
immobilizate. Preparation of immobilized enzymes for biocatalytic
use needs to be practical and cost-effective. Highly demanded fea-
tures of the final immobilizate are adequate loading of enzyme
activity relative to the unit mass of support as well as high-enough
stability of both the enzyme activity and the support under condi-
tions of use [9–11]. Fig. 1 illustrates important parameters of the
support, the enzyme and the mode of enzyme-on-surface deposi-
tion and it also shows how systematic variation of these parameters
affects outcome of the immobilization regarding criteria of activity
and stability.
2. Understanding the behavior of enzymes immobilized on
solid support
Characterization of solid-supported immobilized enzymes
nearly always involves comparison to the free enzyme in terms
of specific activity and stability [9,18,19]. Note: specific activity
is typically expressed as a reaction rate/unit mass of the protein
preparation used. Commonly used dimension is ␮mol/(min × mg).
Less often, the reaction rate is related to the moles of enzyme or
enzyme active site in which case the specific activity has the dimen-
sion of a turnover frequency (1/min). The immobilizate is normally
less active than the free enzyme, with a percentage of retained
activity anywhere between usually 5 to around 80% [9,18,19]. There
are two principal factors underlying the effect, of which one is the
direct consequence of structural distortions in the enzyme resulting
from attachment to the solid surface and another is indirect con-
sequence of enzymatic reaction taking place in a heterogeneous
environment [16,17,19,21] (Scheme 1a). Enzyme stability is often
positively affected by the immobilization [18,19]. The stabilizing
effect is dramatic in certain cases, but well-grounded mechanistic
interpretations based on conclusive direct evidence are typically
not available [18,19]. Unraveling enzyme behavior under the con-
finement of a solid surface and its effect on the catalytic reaction
in porous support present longstanding core problems of biocatal-
ysis with immobilized enzymes [16,21]. Progress in deepening the
mechanistic understanding of heterogeneous biocatalytic conver-
sions is often restrained severely by limitations in methodology
applicable to a direct characterization of solid-supported enzymes
[20,22]. Therefore, despite substantial efforts over decades, perfect-
ing an enzyme immobilizate to a specific activity approaching that
of the free enzyme (or another target value) remains an elusive task.
Lacking direct evidence, optimization of immobilized enzymes in
Please cite this article in press as: J.M. Bolivar, et al., Advanced characterization of immobilized enzymes as heterogeneous biocatalysts,
Catal. Today (2015), http://dx.doi.org/10.1016/j.cattod.2015.05.004
regard to activity and stability is mostly addressed empirically and
is not well predictable in its outcome [17,17,20,21].
Fig. 2 depicts a productive cycle of characterization of immo-
bilized enzymes that moves from initial evaluation of basic
parameters to advanced direct examinations at different levels of
resolution under test conditions as well as in real (in operando) stud-
ies. Note: the term in operando as herein used is distinguished from
the mere in situ in implying realistic conditions of immobilized bio-
catalyst application. Suggestion from Fig. 2 is that running through
the cycle in an iterative manner would constitute a paradigmatic
approach of systematic development and optimization of immobi-
lized enzymes. This review describes where we stand in the efforts
to close the development cycle for heterogeneous biocatalysts.
Opportunities from an emerging set of imaging methods with spa-
tiotemporal resolution capabilities are emphasized and research
needs to overcome current limitations are identified. An optimal
design of heterogeneous biocatalysts would be built on evidence
from advanced characterization of biocatalysts (Scheme 1b), which
ideally provided a comprehensive and detailed understanding of
the relationship between reaction kinetics and structural features
of the catalyst elucidated at the relevant length scale.
3. Enzyme loading in high capacity and high quality for
immobilized biocatalyst preparation
For practical and economic use, heterogeneous biocatalysts are
required to exhibit a specific activity that is as high as possible.
Unlike specific activity of the enzyme as soluble or immobilized
preparation (see above), the specific activity of the heterogeneous
biocatalyst is normally related to the unit mass of solid support
and its dimension therefore is ␮mol/(min × gsupport ). In the first
instance, the specific activity is determined by the quantity of
enzyme mass that can be loaded onto the support and maximiz-
ing this amount presents a clear strategy for catalyst optimization
[4,9,23]. The internal surface area of the immobilization support
accessible to the enzyme via pores of suitable geometry is evi-
dently of high importance [10,11]. The interaction between enzyme
and solid surface is another key parameter [9,17,19]. It provides
a lot of room for optimization regarding protein-binding capac-
ity through molecular engineering of the enzyme, the surface or
both. Balance between surface hydrophilicity and hydrophobic-
ity, surface charge, functional/reactive surface group density and
distribution are all critical aspects in the selection of a suitable
support [10,15,17]. The presence of covalent attachment sites on
the surface is another significant feature to be considered. Covalent
coupling is usually not a main factor of the protein binding capacity
but it ensures the protein attachment to become quasi-permanent.
Instead of modifying the support, targeted modification of the sur-
face of the enzyme presents a fully complementary possibility of
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Scheme 1. Conventional and advanced characterization of heterogeneous biocatalysts are compared. Observable specific activity of heterogeneous biocatalysts depends on
different features that overlap in the information provided by observable parameters. Advanced characterization provides tools to dissect the individual critical parameters
governing catalyst performance.

Fig. 2. Standard and advanced methodologies of characterization of immobilized enzymes are shown. Conventional catalyst characterization would be ideally supported by
structural characterization and in-operando measurements.

increasing protein binding capacity [24–26]. Chemical conjugation
of the enzyme with small molecules or polymers can be used for
that purpose but chemo- and site-selectivity in the derivatization of
amino acid residues on the protein’s surface can be a problem [26].
Inactivation of the enzyme to a substantial degree is often an unde-
sirable consequence of chemical conjugation. Substitution of one
or more surface-exposed amino acids by alternative amino acids
can be achieved using protein “mutagenesis”. However, redesign
of the protein surface to create alternative programmable patterns
of positive/negative charge or hydrophilic/hydrophobic groups is
not an easy task due to often unpredictable effects of the substitut-
ions on the overall protein folding, hence on activity and stability
[24]. Use of modules in the form of surface group-binding peptides
or small protein domains that can be flexibly appended to the N-
or C-terminus of the enzyme presents an alternative that is often
preferred due to broad applicability and attenuated interference
with the enzyme’s native conformation [24,25]. As a rough guide
for practical immobilization, minimally several tens of milligrams
of enzymes should be loaded on each gram of dry matter sup-
port. Loadings of several hundred milligrams have been achieved
in certain cases however.
The specific activity of a heterogeneous biocatalyst is deter-
mined as the product of two factors: the enzyme amount loaded
(quantity) and the intrinsic specific activity of the bound enzyme
(quality). We use intrinsic here to indicate the absence of effects of
the heterogeneous environment on the observable enzymatic reac-
tion rate. The possibility of development of concentration gradients
inside the porous support and the consequent impact on enzyme
behavior need to be kept in mind however [10,15,19,27]. Effective
concentrations inside the support, including that of the proton,
can be dramatically different from the corresponding concentra-
tions in bulk [27–29] (Scheme 1a). Enzyme catalysis is potentially
affected by the internal environment in various ways as will be dis-
cussed later. Loss of intrinsic enzyme activity in consequence of the

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immobilization is caused by reversibly or irreversibly disrup-
tive effects on the protein’s native structure [21]. Unproductive
binding of the enzyme in false orientation or in a too rigid con-
formation (such that the catalytic cycle is no longer run through
efficiently) also leads to a decrease in intrinsic activity. Control-
ling the enzyme’s orientation on the solid surface through the
exploitation of specific binding interactions is a promising strategy
to optimize immobilizations for highest possible enzyme quality
[17,18,24,30]. Biological affinity might seem an obvious choice in
an effort of creating specifity of binding. However, other types of
also highly specific, pseudo-affinity interactions are often prefer-
ably used for reason of overall practical effect [24,30]. Binding to
immobilized (chelated) metal ions or ionic adsorption to negatively
charged surfaces are interesting examples.
Conventional characterization of heterogeneous biocatalysts
involves determination of the enzyme loading, that is, the pro-
tein and activity amounts loaded onto the solid surface, typically
from an end-point balance with the liquid phase once the bind-
ing has reached an apparent equilibrium [9,16,20,23]. The specific
activity of the biocatalyst thus obtained is also determined. How-
ever, many effects are convoluted in this specific activity and the
lumped nature of the observable parameter complicates targeted
optimization. Advanced characterization of heterogeneous biocat-
alyst strives for systematic deconvolution of the experimentally
determined specific activity into its underlying factors, as sum-
marized in Scheme 1b. The productive cycle of characterization
would therefore involve a direct quantification of the enzyme load-
ing, ideally in real time. It would allow for protein imaging in solid
materials, and it would also provide insights into the structure of
the immobilized enzymes (Fig. 2).
Table 1 shows a set of emerging techniques that are applied to a
direct quantification of the protein amount attached to the solid
phase [20–22,31–41]. Measurements from within solid material
are increasingly used and they complement the traditionally used
measurements from the liquid phase. However, the information
obtained is mostly qualitative. Typical application is investiga-
tion of protein loading limits of carriers. Active-site titration is a
useful procedure to determine the amount of accessible enzyme
active sites that were immobilized. Well-known problems, how-
ever, are that convenient procedures of active titration have been
developed only for limited subset of enzyme classes and that the
relationship between the number of active sites as titrated and the
actual specific activity of the immobilized enzyme is not really clear
[15,20,42,43]. Reasons to favor the indirect measurements from
solution are practicability and simplicity.
Irrespective of whether the loading onto the solid support is
determined directly or indirectly, the evidence obtainable from
these measurements is necessarily incomplete in characterizing the
immobilization process fully. Important problems and complexi-
ties recurring often in the development of immobilized enzymes
are the following. There is a complex (nonlinear) relationship
between the enzyme loading and the specific activity of the hetero-
geneous biocatalyst [16,19,21]. The maximum amount of enzyme
loaded and the time needed for maximum loading vary strongly
across varying immobilization conditions and they do so in an often
difficult to rationalize manner [9,10]. Also, compared at equiva-
lent effective loading, the behavior of heterogeneous biocatalysts in
conversion studies can vary strongly in dependence on the immo-
bilization parameters used, and it is often not clear why [10,20].
Improved understanding of the factors at play will require direct
evidence featuring adequate resolution not only in space but also
in time. The dynamics of immobilization processes has previously
not always received the attention deserved [44–49].
Fig. 3 shows that inhomogeneous (heterogeneous) distribution
of enzymes in porous supports could cause effects in conven-
tional characterizations of solid biocatalysts that might easily be
Please cite this article in press as: J.M. Bolivar, et al., Advanced characterization of immobilized enzymes as heterogeneous biocatalysts,
Catal. Today (2015), http://dx.doi.org/10.1016/j.cattod.2015.05.004
misinterpreted. In panel a, for example, it is shown the dependence
of the immobilization yield, that is, the percentage of the initially
applied enzyme protein or activity that was bound to the support,
on the enzyme amount loaded. Leveling out of the curve to reach
a constant value at high loadings could be taken to indicate satu-
ration of the support. Under conditions of inhomogeneous enzyme
distribution, however, effects such as enzyme aggregation and pore
clogging could produce highly similar dependencies of the observ-
able parameter despite the fact that the surface of the support is
not at all saturated. Protein crowding on the surface and inho-
mogeneous intra-support distribution of the enzyme might affect
the overall activity and thus the reaction kinetics [21,49]. Inter-
play between physical diffusion and enzymatic reaction is affected
by heterogeneity of the enzyme distribution inside porous support
[49,50]. Time courses of conversion can therefore be markedly dif-
ferent for situations of homogeneous and inhomogeneous surface
coverage with bound enzyme, as shown in Fig. 3a.
4. Direct visualization of protein distribution in
solid-supported biocatalysts
Table 1 summarizes the analytical methods for measurement
of the distribution of bound proteins on surfaces and more
specifically inside porous supports [22,31–36,51–58]. Level of
application, strengths and limitations are pointed out. Minimally
invasive procedures are preferred, and methods can also be
distinguished according to the spatial resolution they provide.
Fluorescence microscopy is most widely used (Table 2). Intrinsic
protein fluorescence due to tryptophan residues can be applied
for detection, with the caveat that the fluorescence emission is
usually not very stable [21]. When present in the enzyme studied,
cofactors such as FAD and FMN present useful fluorescent reporter
groups [59]. Labeling through normally covalent attachment of
fluorescent groups is a general approach that is convenient and
also widely used [50]. Choices of the fluorescent label and the
conjugation chemistry involved, however, must be made with
some caution and also the stage of the immobilization process
at which the labeling is carried out needs to be given thought.
Generally, labeling in a post-immobilization step appears to be
advantageous. Possible effect of labeling on enzyme activity needs
to be considered however [21,22].
Early efforts of protein imaging analysis used laborious micro-
tome sectioning of the solid supports [60,61]. Layers thus prepared
were then analyzed microscopically. Confocal laser scanning
microscopy (CLSM) has significantly advanced the analysis of pro-
teins in intact porous supports (Table 2) and researchers in protein
chromatography were forerunners in this respect [62–70]. CLSM
offers spatial resolution at a length scale well suitable for the char-
acterization of enzyme immobilizates and it is conveniently used
even by nonspecialists. However, poorly transparent (“opaque”)
supports present a problem. Soaking the support with liquid of
comparable refractive index is a possible solution to enable direct
measurements also in such cases [12,62,63]. Evidence from CLSM
studies on the interaction of proteins with chromatographic sup-
ports can be translated almost directly to enzyme immobilization
[50,62,71–83]. Relationships between the dynamics of protein
adsorption and the external and internal geometrical features
of the support were established [84–86]. They provide a useful
basis supporting process optimization and control that can be
applied in related fields, including enzyme immobilization (Table 2)
[49,72,84–86]. It was shown, for example, that in addition to
choice of adequate support (e.g. material, internal architecture),
controlling the rate of enzyme adsorption was also important
when considering homogeneous distribution of bound enzymes in
porous supports [87–89]. Moreover, the degree of reversibility of
the adsorption also affected the final distribution of enzyme within
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Table 1
Structural studies of immobilized biocatalysts.

Application Method Carrier Information gained Application Comments Ref.

Assessment of Quartz crystal Modified quartz crystal Protein incorporation Low, due to material Set-up is highly [20,22]
immobilization microbalance surfaces at nanogram level restrictions. specific
Quantitative
information
N2 absorption Porous carriers Assessment of protein Indirect character. Challenges with [22]
incorporation Quantitative low protein loading
information
Thermogravimetric Diverse carriers Mass incorporated into Indirect character. Challenges with [22]
analysis solid carrier Quantitative low protein
information loading, wide
potential
application
Zeta potential Colloidal suspensions, Modification of Indirect character. Invasive sample [22]
charged surfaces electrostatic properties Qualitative information preparation
of enzyme carriers
UV-spectroscopy Non-opaque carriers Modification of Indirect character. Challenges with [41]
adsorption spectra due Qualitative information low protein loading
to protein
incorporation
AFM Non-porous carriers Quantitative Direct character. Limited to [21,22,31–36]
assessment of surface Spatial resolution. non-porous
coverage and protein materials or need
distribution of invasive sample
preparation
Infrared spectroscopy Diverse carriers Chemical surface Indirect character. Challenges with [37–41]
modification due to Qualitative information low protein loading
protein incorporation
Catalyst CLSM Diverse carriers Protein distribution Direct visualization, Fluorescence See Table 2
distribution visualization widely used labeling needed.
imaging Corrections for
(partially) opaque
carriers needed
Low-temperature Diverse carriers Protein distribution Low Comparative [52,53]
field-emission visualization and results to CLSM
scanning electron internal morphology of
microscopy material
(Cryo-FESEM)
Light microscopy Diverse carriers Protein distribution Generally applicable Need of particles [54]
sectioning and
protein labeling
TEM Inorganic carriers Localization of protein Applied in silica-based Dried samples [55]
incorporation carriers needed
Raman spectroscopy Diverse carriers Alteration of Raman Increasing use, High background [55,56]
spectra qualitative assessment for organic carriers
Spherical aberration Inorganic porous Protein distribution Low (new technique) STEM combined [57,58]
(Cs)-corrected STEM carriers visualization, with high angle
localization at annular dark field
molecular level detector and
electron energy
loss spectroscopy
Structural Infrared spectroscopy Diverse carriers Secondary structure Widely used Difficult for certain See Table 3
elucidation of elucidation carriers. Sample
immobilized preparation
enzymes (drying) needed
CD Diverse carriers Secondary structure Widely used Difficult for certain See Table 3
elucidation carriers
Raman Diverse carriers Secondary structure Increasing use, difficult High background See Table 3
elucidation for certain carriers for organic carriers

the support [46,90]. It was also shown that degree of homogeneity
of enzyme adsorption had a decisive influence on the specific activ-
ity of the heterogeneous biocatalyst [87,88] (Table 2). Up to now,
distribution analysis in solid supports was mainly done for popu-
lations of the same enzyme. However, with the advent of more
complex catalytic systems, where two or more types of enzymes
are combined to promote synthetic cascades, the precise localiza-
tion and spatial distribution of the individual enzyme types within
the porous support become even more important [87,89]. Abil-
ity to achieve controllable protein patterns within the support
is highly desirable [89]. The same notion applies to immobilized
chemo-enzymatic systems where a chemical catalyst and an
enzyme are made to perform together.
5. Analysis of protein conformation in heterogeneous
biocatalysts
The underlying notion is that the lowered intrinsic specific
activity of an immobilized enzyme preparation compared to the
corresponding soluble enzyme reflects conformational changes
in consequence of the attachment of the enzyme onto the solid
support [17–19] (Fig. 2b). Methods capable of revealing the confor-
mation of proteins in solution and on solid surface would therefore
be required to test and eventually establish correlations between
the degree of conformational distortion and the residual specific
activity of the immobilized enzyme. In a similar manner, stability
of immobilized enzymes could be analyzed where the degree of

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Table 2
Fluorescence microscopic techniques for protein visualization in solid carriers.
Method Protein and carrier system
Fluorescence microscopy Microtome-sectioned
CNBr-activated Sepharose 6B
containing leucine aminopeptidase
Fluorescence tomography Diverse enzyme-carrier pairs
CLSM IgG antibodies immobilized on
CNBr-activated agarose beads
Protein A adsorbed onto IgG
Sepharose 6 Fast Flow
Lysozyme and human IgG
adsorbed on SP Sepharose Fast
Flow and SP Sepharose XL
Human IgG and bovine serum
albumin (BSA) adsorbed on two
different exchange adsorbents
BSA adsorbed on SP Sepharose FF
Egg white albumin (EWA)
encapsulated in alginate beads
BSA adsorbed on a cation
exchanger, SP Sepharose FF
Lysozyme, BSA and IgG adsorbed
on an agarose gel
Different proteins adsorbed on
Sepharose 6 FF
BSA adsorbed onto nanopore silica
particles
Trypsin on porous glycidyl
methacrylate (GMA–GDMA)
␤-Galactosidase on silica–alumina
mesoporous particles
Glucose oxidase (GOx)
encapsulated in alginate
microspheres
Lipases encapsulated in sol–gel
silica
Sterol esterase on
polyacrylate-based
epoxy-activated carriers
Penicillin acylase immobilized
onto epoxy-activated Sepabeads
ECEP303
Amidohydrolase immobilized onto
Sepabeads EC-EP5
Fructosyltransferase encapsulated
in dried alginate
Lipase immobilized into
macroporous poly(methyl
methacrylate) (PMM) and
polystyrene (PS) carriers
Lipase encapsulated into
mesoporous silica
Lipase and trypsin coencapsulated
into mesoporous silica
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Information gained
Homogeneous protein distribution
Protein distribution visualization
via the spatial resolution from
integral measured signal
Homogeneous ligand distribution
at increasing loading
At low sample amounts, Protein A
had been adsorbed to a thin outer
layer.
Preferential immobilization on
outer layer of beads at low protein
loading.
Direct observation of a two
component diffusion process
within an adsorbent support
Need of image processing to
compensate the use of partially
opaque carriers
Need or image processing to
compensate the use of partially
opaque carriers
Diffusion model validation for
uptake rate
Hindered diffusion of the protein
adsorption. Model validation for
protein adsorption
Identification of hindered protein
diffusion. Calculation of
intraparticle diffusion coefficient
Determination of the minimum
pore size required for efficient
intraparticle adsorption
Homogeneous protein distribution
Homogeneous or heterogeneous
distributions depending on carrier
particle size
Verification of uniform distribution
Homogenous incorporation in
amorphous sol–gel
Heterogeneous immobilization of
enzyme with preferential binding
to outer shells
Homogeneous distribution of the
enzyme on the support surface
avoiding the attachment of
enzyme aggregates
Homogeneous distribution was
obtained
Enzymes accumulated preferably
in the shell of the particles
Localization of enzymes in an outer
rim of 50 – 85 and 10 – 20 ␮m
thickness for the PMM and PS
catalysts
Homogeneous enzyme
incorporation
Enzymes uniformly dispersed
throughout the particles because of
the successful incorporation of the
two enzymes
Application Ref.
Biocatalysis application. Fluorescence [60]
labeling needed
Biocatalysis application. [61]
Micro-fluorometry enabled the
visualization without previous processing
of the carrier
Chromatography application. [84]
Fluorescent-labeled Protein A was used as
an indirect measure of the distribution of
IgG
Chromatography application. [85]
Fluorescent-labeled Protein A was used
Chromatography application. [86]
Fluorescent-labeled proteins were used.
Agreement between model based on
indirect measurements and direct
measurements was found
Chromatography application. How the [64]
diffusional control affects the adsorption of
multi-component systems has been
studied
Chromatography application. Light [63,65]
scattering and absorption has an effect in
the interpretation of protein profiles
Chromatography application. [67]
Methodological considerations to assess
the influence of light scattering and
absorption
Chromatography application. The radial [35]
distribution of the protein concentrations
was predicted using transport models
Chromatography application. Decrease in [68]
diffusivity and increase of hindered
adsorption is studied depending on carrier
geometrical features
Chromatography application [69]
Chromatography application. [70]
Heterogeneous or homogeneous
adsorption dependent on pore size
Biocatalysis application. Contrast matching [71]
was necessary to overcome opaque
particles
Biocatalysis application. Increased catalytic [72]
effectiveness was observed with
heterogeneous distributions
Biocatalyst characterization [39]
Biocatalyst characterization [73]
Biocatalyst characterization [74]
Biocatalyst characterization. Biocatalyst [76]
optimization
Biocatalyst characterization [77]
Biocatalyst characterization [78]
Biocatalyst characterization [79]
Biocatalyst characterization [82]
Biocatalyst characterization [80]
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Table 2 (Continued)

Method Protein and carrier system Information gained Application Ref.

Glutaminase encapsulated in Homogeneous encapsulation of the Biocatalyst characterization [81]

mesoporous silica
Cross-linked enzyme aggregates
(CLEAs and CLEAs entrapped in
polyvinyl alcohol lenses (lentikats)
Lipase immobilized in mesoporous
silica
Fluorescent proteins (His-GFP and
His-mCherryFP) immobilized on
different 4% crosslinked
agarose-type by different methods
IgG immobilized into
heterofunctional metal
chelate-glyoxyl supports
(Ag–Me2+ /G)
Different redox enzymes onto
agarose beads
Lipase immobilized in agarose
beads
enzyme
Localization and quantification of
CLEAs entrapped in lentikats
Pore size dependent. PPL diffusion and
protein distribution
Modulation of the distribution within
porous matrices by smart-control of
the immobilization rate
Modulation of the distribution within
porous matrixes by control of
immobilization rate
Modulation of the colocalization
within porous matrices by control of
immobilization rate
Dynamic protein distribution inside
the porous beads that evolves from
heterogeneous to homogeneous along
the postimmobilization time
Biocatalyst characterization [75]
Biocatalyst optimization. Small pores lead [83]
to a hindered protein penetration, an
optimal higher pore size is found given a
uniform distribution and highest loading
capacity
Biocatalysis optimization. Different [87]
co-immobilization patterns were
generated
Chromatography optimization. The [88]
binding activity of this bioconjugate was
optimized by controlling the antibody
distribution throughout the bead’s surface
in order to avoid high antibody densities
and therefore a low binding activity
Biocatalyst optimization. A spatial [89]
distribution was obtained, thereby
resulting in different biocatalysts with
different properties. Homogeneous
distribution of both enzymes over the
porous surface of the same carrier seemed
to be optimal for the redox cofactor
regeneration during the biotransformation
Biocatalyst characterization. Migration of [90]
reversibly immobilized protein was proved

stability enhancement might be related to detectable and ideally of protein conformations in solution are suitable for analy-
also quantifiable conformational distortions in the immobilized sis of proteins on surface, especially that of a porous solid
enzyme [19,21,91]. Problem is that only few of the vari- support. A summary of methods and their limitations can be
ous spectroscopic techniques applied routinely to the study found in Table 1. Common difficulties are: complicated sample

Fig. 3. Structural studies of immobilized enzymes as heterogeneous biocatalysts are shown. Protein visualization in solid materials and structural elucidation of surface-bound
enzymes provides critical information to succeed in enzyme immobilization in high quantity and quality.

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preparation (e.g. drying) involving a high risk of protein
denaturation, strong interference from solid material and insuf-
ficient sensitivity and resolution to detect small conformational
changes.
The most widely used method is Fourier transformed infrared
spectroscopy (FTIR) [92,93]. Changes in the protein’s relative
composition in secondary structure and also in the tertiary fold
on immobilization can be monitored using FTIR, as shown in
Table 3. Protein surface interactions, hydrogen bond formations
and other bond parameters are detectable by FTIR too [21,93–95].
FTIR studies of different enzymes revealed retention of secondary
structural composition in the immobilizate. Several lipases, the
protease pepsin, glucose oxidase, horseradish peroxidase and
RNase were examined covalently or noncovalently bound to
surfaces of metals (e.g. gold), organic and inorganic polymeric
materials [31,41,96–102]. The overall protein conformation and
the orientation of the protein on the surface were also elucidated
by FTIR, however, only for nonporous supports [22,38,40,102,103].
Use of gold surface is highly beneficial for optimizing the spectral
resolution due to low background signals of the gold. However,
unless the real immobilization can be mimicked on gold, the
problem is that gold is a highly uncommon and usually by far
too expensive support material for practical use of immobilized
enzymes in biocatalysis [96,102]. With other words, more con-
venient materials (e.g. mesoporous silica, porous polymer beads)
using FTIR, the interference with the measurement is unfortunately
quite significant [22,38,41,94,104–107].
Circular dichroism (CD) and Raman spectroscopies serve as
common alternatives or complements of FTIR spectroscopy in the
characterization of enzyme immobilizates [21,22,108]. Both meth-
ods are capable of revealing protein secondary structural content
and are sensitive to spectral contributions of amino acid side
chains, hence to the protein conformation overall. Being a non-
invasive and conveniently applied technique, CD was often used
to analyze enzyme immobilizates, however, with the severe limi-
tation that colloidal particles or transparent material surfaces were
required [21,22,94,108–110] (Table 3). A relatively large num-
ber of enzyme immobilizates obtained with different methods
have been analyzed using CD [21,22,94,108,111–114]. Alterations
in protein secondary structure after immobilization were noted
[21,22,94,108,112,115–118], and efforts were made with limited
success to relate these to changes in enzyme activity [108] and
stability [94,116].
Like FTIR, Raman spectroscopy is in principle well suitable for
the analysis of proteins in solid samples, be it that dried pro-
teins or frozen protein solution are investigated [21,108,119–121]
(Table 3). Background signals in solid-supported enzyme prepara-
tions can be unexpectedly high however [122]. Raman spectra are
well resolved and signal quality is high when metal nanoparticles
of 10–100 nm size fabricated from colloidal silver or gold are used
[123–125]. The application of Raman spectroscopy to the charac-
terization of immobilized proteins is increasing [37,120,125–129].
Loss of secondary protein structure on immobilization was ana-
lyzed [120,129]. Orientation of enzymes on solid surface was
determined and attachment via covalent bonds was also elucidated
[130]. Method compatibility with a variety of solid supports (e.g.
organic polymers) and the requirement to use high protein loading
are severe drawbacks.
New techniques like TOF-secondary ion mass spectrometry
[20,101,108,131], solid-state NMR [21,73,132–135], Foster reso-
nance energy transfer (FRET) measurements [136] and Trp/Tyr
fluorescence measurements [137,138] are gaining momentum, and
exciting developments are expected in upcoming research. The
direct characterization of the conformational properties of immo-
bilized enzymes needs to be advanced through further method
development.
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6. Advanced direct characterization of the performance of
immobilized biocatalysts
Assuming that both substrate conversion and space–time yield
will constitute fixed target parameters of a biocatalytic process
operated in apparatus of given size, the relevant question in reac-
tion engineering therefore is how much enzyme must be supplied
to the reactor in order to fulfill the processing objectives [4,20,23].
Activity and also stability of the immobilized enzyme under opera-
tional conditions will thus determine the loading of heterogeneous
biocatalyst required. Theory of the coupled reaction and diffusion in
solid-supported immobilized enzymes is well developed. The cat-
alytic effectiveness factor is the activity ratio of the immobilized
enzyme compared to the corresponding soluble enzyme [4,20,23].
Lowering of the experimental effectiveness factor below a value of
unity as result of effects of external film diffusion and internal pore
diffusion is usually explained by considering the dimensionless
Damköhler number and the also dimensionless Thiele modulus,
respectively [4,139] (Scheme 1b). Problem of the approach is that
model validation and parameter estimation are usually done exclu-
sively on the basis of indirect evidence, that is, measurements from
the liquid phase. Moreover, stability of the enzyme or the bio-
catalyst as a whole presents another issue that may be related
to but can also be completely distinct from reaction and diffu-
sion. Again, stability parameters are usually inferred indirectly
from measurement of the progress of the reaction in the liquid
phase, more specifically, from the lack of agreement between the
expected (modeled) reaction time course and the time course mea-
sured. However, as was already mentioned, stability is only one
of many possible effects that influence the progress of the reac-
tion. Therefore, direct evidence from advanced characterization of
heterogeneous biocatalysts in in-operando studies (Figs. 2 and 4)
would be extremely helpful to enable clear assignment of cause
to effect in biocatalytic process development with immobilized
enzymes (Scheme 1b).
Two levels of spatial resolution are considered here in the
course of process characterization (Tables 4 and 5). First of all,
macroscopic stability studies look at mechanical disintegration
of the porous support, for example, under agitation and stirring.
Detachment of enzymes from the solid surface either as a con-
sequence of the disintegration or occurring separately from it is
also monitored. Secondly, knowledge about the heterogeneous
environment in which enzymes work is the key. Concentration
gradients may develop inside the porous supports and thus result
in conditions very much different from those in the liquid phase.
Activity and stability parameters of the enzyme but also thermo-
dynamic parameters of the reaction may be affected. Local sensing
within the solid support provides relevant evidence. Finally, local
inhomogeneities at the reactor-scale level must also be consid-
ered and recent developments in in situ sensing methods are
described.
6.1. In-operando studies of macroscopic stability
Mechanical stability of porous supports was traditionally deter-
mined through visual or microscopic analysis of samples taken
from the reactor. To perform the same analysis in operando and
also continuously, fluorescence and microscopy methods were
developed for application directly in the reactor with minimum
perturbation of the ongoing reaction. Table 4 summarizes these
developments [82,129,140]. Interfacing the analytical devices with
the reactor presents a challenge. Immersion probes in combination
with fiber-optic technology are used to record fluorescence data.
Microscopic evidence is collected through observation windows
implemented in the reactor.
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Table 3
Enzyme carriers and carrier used in the elucidation of immobilized protein structure in immobilized enzymes.

Method Enzyme Carrier used Comments Ref.

CD Cytochrome C and myoglobin Mesoporous silicas Secondary structure elucidation [113]

Hen egg white lysozyme Fused silica glass, high-density Secondary structure conservation [108]
polyethylene and
poly(methyl-methacrylate)
Hemoglobin Mesoporous silica (folded-sheet Secondary structure conservation [114]
mesoporous material)
Organophosphorus hydrolase Functional mesoporous silica supports Secondary structure alteration [22,115]
Beta-lactoglobulin Amine-functionalized mesoporous Secondary structure alteration [116]
nanoparticle
Subtilisin Silica gel (230–400 mesh) Secondary structure conservation [94]
Lipase Lewatit VP.OC 1600 (Novozyme435) No secondary structure info; tertiary [94]
structure elucidation
BSA Polystyrene particles Secondary structure elucidation [21]
Subtilisin Mesoporous silica Secondary structure alteration [21,117]
␣-Chymotrypsin Methyl methacrylate polymer Secondary structure elucidation [21]
Alcohol dehydrogenase Silica particles of different types and Secondary structure alteration [21,118]
size
Lipase Amino functionalized multi-walled Secondary structure conservation [112]
carbon nanotubes
Penicillin G acylase Silica nanoparticles functionalized Secondary structure characterization
with glutardialdehyde
Raman spectroscopy Monoclonal antibody Fractogel EMD SO3 (M) cation Secondary structure elucidation [120,129]
exchanger
Laccase Self-assembled monolayers of thiols on Secondary structure elucidation [122]
Ag and Au surfaces
CytC Gold and silver nanostructures Secondary structure elucidation [124]
Myoglobin, CytC and P450 Ag electrodes Secondary structure elucidation [125,127]
Tyrosinase Poly(indole-5-carboxylic acid) Secondary structure elucidation [128]
FTIR Lipase Carbon nanotubes Secondary structure conservation [112]
Lysozyme Nanoporous gels Secondary structure elucidation [103]
Pepsin Colloidal gold particles Secondary structure conservation [96]
BSA, bovine ␤-lactoglobulin and Spherical polyelectrolyte brushes Secondary structure elucidation [98]
bovine pancreatic ribonuclease A (SPB) = narrowly distributed
poly(styrene) core particles onto which
linear chains of anionic
polyelectrolytes are grafted
DRIFT Subtilisin Silica gel Secondary structure conservation [94]
Lipase Lewatit VP.OC 1600 (Novozyme 435) Secondary structure conservation [94]
ATR-FTIR Lysozyme Mesoporous silica SBA-15 Secondary structure elucidation [22,104]
Lipase Chitosan-based carriers Secondary structure conservation [41]
Lipase Mesoporous organosilicas (PMOs) Structure alteration [105]
Lysozyme, myoglobin Mesoporous silica SBA-15 Secondary structure alteration [106]
Channelrhodopsin Gold surface Secondary structure elucidation [40]
Peptide cecropin P1 Polystyrene (PS) surface, Secondary structure elucidation [38]
polystyrene-maleimide(PS-MA)
surface
Glucose oxidase Functionalized mesoporous silica Secondary structure conservation [107]
Glucose oxidase Sol–gel Secondary structure elucidation [99,100]
PM-IRRAS Peroxidase Activated gold surfaces Alteration or conservation of [101]
secondary structure
Nickel–iron hydrogenase Graphite electrodes Secondary structure elucidation [31]
Synchrotron infrared Lipase Macroporous polymer matrix Secondary structure elucidation [97]
microspectroscopy (SIRMS) (poly(methyl methacrylate));
Novozyme 435
Trp/Tyr fluorescence Nitrate reductase Different agarose based carriers Structural rearrangement after [137]
immobilization
Lipase Cyanogen Bromide 4B Structural rearrangement via [138]
Sepharose immobilization and chemical
modification

6.2. In-operando reaction monitoring and enzyme activity
determination
Just like in other fields of biotechnology (and chemical tech-
nology), observing the reaction progress through direct in situ
measurements of substrate, product or both inside the reactor
offers the general advantage of improved process monitoring and
control [141–148]. In-operando applications require the use of
transparent windows, optical fibers or immersed probes. Specific
problem of immobilized enzymes is that the relatively large par-
ticles used as porous support are not always compatible with the
analytical probes used and often disturb the measurements. Porous
particles also result in greater spatial inhomogeneities than is nor-
mal in a bioreactor containing suspended cells. Implementation of
bypass streams facilitates measurements but requires certain adap-
tations to the enzyme reactor [145]. Table 4 summarizes recent
developments in the field and provides an overview of the methods
used [149–154]. IR spectroscopy is often used [155,156] and Raman
spectroscopy has also received growing attention recently [157]. In
both cases, a fiber-optic probe is placed directly into the reactor, for
example, a stirred tank. Enzyme activity can sometimes be inferred
indirectly from in situ measurements of substrate consumption or
product formation. Indeed, most of the applications are still limited
to kinetic analysis rather than true in-operando characterization.

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Fig. 4. In-operando studies of immobilized biocatalysts are shown. Internal sensing is usually performed to reveal local catalytic environment determining catalytic activity.
Ideally, structural studies would provide information about catalyst stability in real time.

Different forms of IR spectroscopy were used to measure product
release rates in immobilized enzyme systems. Data were used to
calculate the enzyme activity [158–165]. However, it was neces-
sary that the reaction medium containing the solid biocatalyst was
by-passed in a flow-through sensor or placed in a spectrophotome-
ter cell. As such, these measurements constituted neither in situ nor
in-operando characterization.
6.3. In-operando internal sensing in heterogeneous biocatalysts
The heterogeneous environment of an enzyme immobilized in
porous solid support is likely to be different from the environment
of the surrounding bulk phase (Scheme 1b, Fig. 4). Differences in
conditions inside and outside of the support are consequences of
coupled reaction and external/internal diffusion in heterogeneous
biocatalysts. At steady state, knowledge about the magnitude of
these differences is important to explain the observable effective-
ness factor of the immobilized enzyme which in turn affects the
overall volumetric reaction rate. Dynamic response of the het-
erogeneous environment to changes in the bulk phase is also of
interest [4,10,23,139]. External diffusion can be controlled by the
fluid dynamics at the reactor level [23,27,139]. By contrast, inter-
nal diffusion effects are often pronounced and cannot be readily
controlled by an external variable since they depend on structural,
hence internal features of the support [10,12].
Progress in methods of sensing directly within solid support
enables the biocatalyst’s internal environment to be analyzed
quantitatively and in real time. Diverse analytical principles have

Table 4
In-operando studies of immobilized biocatalysts I.

Application Method and equipment System Application and information Comments Ref.
gained

Macro-stability studies
In situ reaction rates
measurements
In situ microscopy Mesoporous carriers Mechanical stability under Influence of stirring conditions [140]
stirring conditions on the velocity of particles
degradation was studied
Raman spectroscopy Mesoporous carriers Stability of attachment in Enzyme leaching was [129]
stagnant suspension of monitored
particles
Fluorescence Mesoporous carriers Stability of attachment of Enzyme leaching was [82]
microscopy lipases in continuous flow monitored
reactors
Infrared spectroscopy Free enzyme Activity measurement Kinetic analysis in IR [158–160,162,163]
spectrophotometer
Infrared spectroscopy Free enzyme Reaction monitoring of Reaction media was placed in a [155]
synthesis and hydrolysis spectrophotometer cell
reactions catalyzed by an
amidase
Infrared spectroscopy Whole cell suspensions Acetonitrile was Mid- IR spectroscopy using an [156]
biotransformed to acetamide immersion probe
by a nitrile hydratase enzyme
and subsequently to acetic acid
(carboxylate ion) by an
amidase
Infrared spectroscopy Immobilized enzyme Kinetic analysis in a flow cell Mid-IR spectroscopy [164,165]
using a flow through sensor
Raman spectroscopy Wide range of carriers Global reaction rates of stirred Immersed probes were used [157]
suspension of immobilized
enzymes

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Table 5
In-operando studies of immobilized biocatalysts II: internal sensing in heterogeneous biocatalysts.

Method and equipment System Application and Comments Ref.

information gained

Spectroscopic techniques
Electrochemical sensing
Opto-chemical sensing
Opto-chemical sensing
IR spectroscopy Used in ATR mode
NMR imaging Porous particles in a
packed bed reactor
Raman spectroscopy Porous carriers
Multiphoton microscopy Porous carriers
Polarographic O2 High porosity carrier
microsensing
Luminescence intensity; Porous carrier containing
fiber optics connected to labeled proteins
spectrofluorimeter
Dual-wavelength Porous carrier containing
rationing; CLSM labeled proteins
Dual-wavelength Fluorescence-labeled
rationing; CLSM material
Fluorescence lifetime using Fluorescent-labeled
CSLM membrane containing
immobilized enzymes
Fluorescence lifetime Fluorescence-labeled
spectroscopy using phase material
modulation
DLR using phase Fluorescence-labeled
modulation; fiber optics porous carrier containing
immobilized enzyme
Phase modulation Phosphorescence-labeled
technique porous carriers containing
immobilized oxidases
Local concentration Increasing availability of [194,201]
resolution immersion probes
Intraparticle diffusion Time consuming, high cost, [66,132]
coefficients specialized equipment
Spatial resolved Increasing availability of [197]
concentration, diffusion immersion probes and
coefficients in hydrogels confocal technology. Only
non-fluorescent species of
significant concentration
can be quantified
Local concentration Increasing use [178,179]
gradients
Internal concentration Laborious set-up; different [174–177]
gradient depending on microsensors needed;
enzyme loading and pre-characterization of
particle radius, kinetic each sensor. Invasive
parameters, concentration method. High spatial
in liquid boundary layers resolution data acquisition
is difficult
pH monitoring in stirred FITC is coupled to [168]
tank or fixed bed reactor, immobilized enzymes. Low
modeling and reactor signal-to-noise ratio. pH
design different between bulk and
intraparticle amounted to
1.5–3 units
pH influence on selectivity Luminescence [167]
of kinetically controlled intensity-based
reactions, enzyme loading, measurements. FITC is
particle and pore size, coupled to immobilized
surface modification, enzyme Internal pH was
carrier selection measured in a fixed bed
reactor during reaction
Method development and Signal dependent was on [193]
evaluation for pH sensing measurement point/depth
within the beads
Temporal and spatial pH pH profile is influenced by [166]
profiles in a buffer, incubation time,
substrate-enzyme reaction glucose concentration,
process within a thin diffusion distance and
membrane reaction of glucose oxidase
Method development and First applied with [186]
evaluation for pH sensing immobilized pH sensitive
dye in hydrogels
pH gradient between bulk DLR is compatible with [170,171]
and particle (biocatalyst different carrier surface
design), correlation functionalities, dyes
between steady-state adsorbed directly in carrier
kinetic analysis of matrix; pH gradient
immobilized enzyme and depends on geometrical
intraparticle elucidation features of carrier,
relevance for optimization
of biocatalytic conversion
processes
Intraparticle oxygen, Compatible with different [172,173]
oxygen gradients carrier surface
modifications, dyes
adsorbed directly in carrier
matrix; oxygen gradient
depends on immobilization
approach and informs
about intrinsic
immobilization chemistry

been applied [27,166–186] for biocatalyst screening and charac-
terization as well as for study and optimization of the reactor
operation. Table 5 presents a summary of the immobilized enzyme
system studied, with a focus on applied aspects. Notion is that evi-
dence of the internal catalytic environment of a heterogeneous
biocatalyst facilitates process optimization. Screening of suitable
immobilization procedures is also a very interesting application
of internal sensing whereby optimization of carrier geometries
is of particular relevance for biocatalyst design. Immobilization
conditions can be optimized in a target-oriented manner once
conditions of the heterogeneous environment are well known
[167,170]. Results of characterization of immobilized enzymes with
internal sensing methods support the idea that determination of
kinetic and mass-transfer parameters for heterogeneous biocata-
lysts is strongly supported when the available evidence is not just
from the external environment [178]. Internal sensing also pro-
vides a powerful tool to distinguish effects of the immobilization
on the intrinsic enzyme activity from mass transfer effects [180].

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Opto-chemical internal sensing has received prominent attention
in this context.
6.3.1. Principles of opto-chemical internal sensing
Opto-chemical sensors are already in wide use for determi-
nation of diverse analytes: pH, O2 , CO2 , NH4 , glucose, alcohols,
amines and a variety of ions [187,187–190]. Detection is usually
by luminescence whereby different measurement principles are in
use. Application of opto-chemical sensing for in-operando char-
acterization of heterogeneous biocatalysts has two requirements
in particular. First, the enzyme carriers need to be made inter-
nally responsive through incorporation of suitable luminescence
dyes. Second, a suitable analytical set-up to provide in-operando
measurements with high temporal resolution and suitable spatial
resolution must be established. One possibility for luminescence
labeling is the direct conjugation of enzymes and luminophores
[167,168]. However, alteration of enzyme activity in consequence
of the chemical conjugation is a significant problem. Labeling of
the supports used for immobilization is therefore the preferred
approach, considering that labeling must be compatible with the
immobilization. Choice of the analytical set-ups determines which
reactor configuration is suitable for the luminescence measure-
ment measurements and also determines which level of spatial
resolution is provided. Whereas the use of fiber-optics offers high
flexibility in that both stirred suspensions and packed beds of par-
ticles can be analyzed, the use of microscopy offers higher spatial
resolution but restricts the application to stagnant suspensions of
particles or to flow-cell configurations.
6.3.2. Opto-chemical internal sensing of pH and O2
Internal sensing of pH and O2 has recently seen significant
developments. Using incorporation of oxygen- or pH-sensitive
luminophores into porous supports, internally responsive enzyme
carriers were created. This together with the establishment of
suitable read-out platforms now offers useful systems in which
spaced-averaged internal concentrations of pH and O2 can be
measured in enzyme immobilizates (Table 5). Intraparticle pH mea-
surements have been used for biotransformation optimization in
a fixed-bed enzyme reactor [168]. Spieß, Kasche and their col-
leagues have studied the hydrolysis of ␤-lactam substrates (e.g.
penicillin G), which results in net proton formation, and applied
FITC-labeled immobilized amidase to quantify the extent of over-
all carrier acidification during the reaction. The internal pH was
determined from fluorescence intensity data and shown to dif-
fer from the bulk pH by up to 3 pH units. Fluorescence intensity
measurements are disturbed by the moving particles in stirred
suspension. Self-referenced measurements and fluorescence life-
time [191] determinations exhibit superior analytical performance
in agitated systems. Dual lifetime referencing (DLR), in particular
[170,171,192], offers high versatility, independent on catalyst con-
centration, reactor configuration and scale of operation [170,171].
Significant progress was made in method development for intra-
particle O2 measurement. The quantification of spaced averaged
intraparticle oxygen concentrations in porous polymethacrylate
enzyme carriers was accomplished by labeling the enzyme car-
rier with an O2 -sensitive luminophore and application of the phase
modulation technique. Formation of a large gradient between O2
concentrations in bulk solvent and the internal environment of the
carrier was detected, clearly indicating limitations in the supply
of O2 co-substrate into the solid support [172,173]. In-operando
determination of oxygen gradients between homogeneous liquid
phase and internal catalytic environment was performed during
determination of catalytic activity of heterogeneous oxidases as
biocatalysts. The internally available O2 concentration was shown
to control the catalytic effectiveness of heterogeneous biocata-
lyst. Clear distinction between physical and biochemical factors of
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effectiveness of the immobilized enzyme was made possible. Bio-
catalytic process intensification through enhanced mass transport
was suggested and internal sensing-based evidence on the het-
erogeneous environment could facilitate the optimization of the
operational conditions.
6.3.3. Internal sensing with high spatial resolution
Opto-chemical sensing in combination with confocal laser scan-
ning microscopy (CLSM) has allowed for determination of internal
parameters (e.g. pH) in time- and space-resolved manner. Refer-
enced fluorescence intensity measurements were used by Spieß
and colleagues to characterize different immobilizates of penicillin
G amidase [167]. Their study was seminal in demonstrating the
importance of considering an internal parameter for immobiliza-
tion optimization. They showed that internal pH was the key in
controlling the selectivity of the immobilized amidase, implying
the need to select carriers and immobilization procedures that sup-
port development of an optimum internal environment [167]. Huang
and colleagues applied similar analytical techniques to determine
pH gradients in biocatalytic membranes containing immobilized
glucose oxidase [169]. A pH drop resulted in this case from the
oxidation of d-glucose into d-gluconic acid. Fluorescence lifetime
provides advanced measurement capabilities [178,179,186], elim-
inating signal distortion in dependence of the scanning depth
as a known critical problem of intensity-based measurements in
CLSM [193]. Internal pH changes at spatial resolution have been
monitored in hydrogels and PEG microparticles using fluorescence
lifetime microscopy techniques [166,186]. Unfortunately, analy-
sis by CLSM depends on high-cost instrumentation that cannot be
adapted to real-life reactor configurations and has limited through-
put capacity. Multiphoton microscopy has been already used to
resolve concentration gradients in hydrogel-encapsulated biocat-
alysts. High spatial and temporal resolution is obtained enabling
the simultaneous identification and monitoring of multiple events
in immobilized biocatalysts. Diffusion and enzymatic reaction are
resolved by elucidation of local concentration gradients through
the catalytic particle [178,179].
Using microelectrodes, electrochemical sensing was applied to
measurement of internal parameters (NH4 + , O2 ) in immobilized
enzymes at spatial resolution of <50 ␮m [174–177,185]. However,
because the analyte’s concentration is recorded only at single dis-
crete space point, determination of full profiles necessitates that
the sensor tip be moved with high precision around the area to be
analyzed [174,175]. Mechanical fixation of the carrier, typically in
a flow cell, is therefore required and the internal concentration is
usually obtained at steady state while flowing substrate through
the carrier [174–176]. Notable limitations of the method are that
relatively large and soft carriers (e.g. agarose) are required and only
stable profiles with slow dynamics can be monitored. The analyt-
ical method has received only little response in the community,
most probably because of the highly specialized set-up and the
complicated micro-manipulations involved. Moreover, its strongly
invasive character is a clear drawback.
A broad variety of (powerful) spectroscopic techniques have
been applied with success for characterization of the internal envi-
ronment of solid supported chemical catalysts (e.g. NMR, Raman,
IR) [66,194–201] with high time and spatial resolution and show
some application in heterogeneous biocatalysts. Nuclear magnetic
resonance imaging has been applied to the glucose isomerization
catalyzed by heterogeneous biocatalyst in a packed bed reactor
[132] and resolution of enzyme catalyzed esterification of propionic
acid and butanol in an internal hydrogel environment [66]. Even
though the technique is highly suitable for evaluating analyte trans-
fer into the pores, fast concentration changes due to biocatalytic
reactions could be poorly resolved. Therefore, these techniques
have not been broadly extended to a carrier containing immobilized
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enzymes and the application is focused more in biochemical
research than in real-time monitoring. High time, cost, specialized
equipment demands for acquisition are current drawbacks [196].
Raman spectroscopy has also been applied for the spatial resolved
concentration and determination of diffusion coefficients in hydro-
gels [197].
Interest has recently been high in the use of microstructured
flow reactors for chemical synthesis [202–205]. In these miniatur-
ized systems, gas or liquid passes in single- or multi-phase flow
through small channels of typically several ten to hundred micro-
meters in size. Reactions may take place in the continuous phase
or at the surface of the microchannel walls. Internal sensing along
the microchannel wall(s) therefore constitutes a highly promis-
ing element of process monitoring and control in microreactors
[203,206–209].
6.3.4. Expanding to other analytes and biocatalysts
Method diversification to other analytes remains a challeng-
ing task for the future. Self-fluorescent molecules (e.g. NADH and
NADPH) might lend themselves directly for internal sensing using
fluorescence resonance energy transfer [145,210]. Luminescence
labeling through direct incorporation into the enzyme carrier,
as demonstrated for the relatively hydrophobic polymethacrylate
material, could be applicable to a range of other organic polymers
used in the field. More hydrophilic carriers might require adap-
tation of the labeling procedure, using covalent fixation or other
forms of deposition of the luminophore(s) on the surface, for exam-
ple. However, manufacture of optical chemical sensors has been
confronted with similar challenges, and there are already use-
ful solutions to overcome them [187,211]. Hydrogels applied for
encapsulation of enzymes and cells should be generally amenable
to the fluorescence labeling [186].
7. Conclusions
An advanced “systems approach” of characterization of solid-
supported immobilized enzymes as heterogeneous biocatalysts is
suggested. The development is envisioned to proceed through a
productive cycle, as shown in Fig. 2, whereby collection of direct
evidence from the heterogeneous system is the key. It is argued
that while conventional characterization of immobilized enzymes
still relies almost exclusively on apparent parameters determined
from measurements in the liquid phase, deepened understanding
of the behavior of the biocatalytically active solid phase is essential
for targeted development. Based on detailed summary of recent
literature with a focus on direct characterization of immobilized
enzymes, limitations to progress in the task of immobilization-by-
design are identified. Practical methods for protein conformational
analysis on solid surfaces need to be developed and adapted to the
requirements of enzymes on porous supports of up to a millimeter
in diameter. Protein visualization on porous supports has become
a widely used characterization method where microscopic imag-
ing with spatiotemporal resolution capability is preferably used.
Methods of direct characterization of activity and stability of immo-
bilized enzymes as heterogeneous biocatalysts are mainly based
on internal sensing, activity measurements and reactor operation.
Opto-chemical methods offer flexibility and versatility regarding
the material and the geometry of support used. Moreover, they
do not require complicated and costly equipment. However,
preparation of internally responsive materials compatible with
immobilization is still challenging for many carriers used and
limited to a relatively small range of analytes. Development of read-
out systems and analytical set-ups compatible with all the reactor
configurations used with heterogeneous biocatalysts also remains
a challenge. However, process analytical technologies applied in
Please cite this article in press as: J.M. Bolivar, et al., Advanced characterization of immobilized enzymes as heterogeneous biocatalysts,
Catal. Today (2015), http://dx.doi.org/10.1016/j.cattod.2015.05.004
13
heterogeneous biocatalysis are expected to gain momentum,
whereby innovative process control strategies would be based on
the on-line monitoring of internal parameters. Ability to observe
structural features of immobilized enzymes in operando remains a
distant but important target in the field.
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