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This journal is c The Royal Society of Chemistry 2012 Chem. Soc. Rev.
Cite this: DOI: 10.1039/c2cs35231a
Modifying enzyme activity and selectivity by immobilizationw
Rafael C. Rodrigues,aClaudia Ortiz,
bAngel Berenguer-Murcia,
cRodrigo Torres
d
and Roberto Fernandez-Lafuente*e
Received 29th June 2012
DOI: 10.1039/c2cs35231a
Immobilization of enzymes may produce alterations in their observed activity, specificity or
selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon
immobilization (caused by the distortion of the enzyme due to the interaction with the support) in
some instances such properties may be enhanced by this immobilization. These alterations in
enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally,
these variations will be positive. For example, they may be related to the stabilization of a
hyperactivated form of the enzyme, like in the case of lipases immobilized on hydrophobic
supports via interfacial activation. In some other instances, these improvements will be just a
consequence of random modifications in the enzyme properties that in some reactions will be
positive while in others may be negative. For this reason, the preparation of a library of
biocatalysts as broad as possible may be a key turning point to find an immobilized biocatalyst
with improved properties when compared to the free enzyme. Immobilized enzymes will be
dispersed on the support surface and aggregation will no longer be possible, while the free enzyme
may suffer aggregation, which greatly decreases enzyme activity. Moreover, enzyme rigidification
may lead to preservation of the enzyme properties under drastic conditions in which the enzyme
tends to become distorted thus decreasing its activity. Furthermore, immobilization of enzymes on
a support, mainly on a porous support, may in many cases also have a positive impact on the
observed enzyme behavior, not really related to structural changes. For example, the promotion
of diffusional problems (e.g., pH gradients, substrate or product gradients), partition (towards or
away from the enzyme environment, for substrate or products), or the blocking of some areas
(e.g., reducing inhibitions) may greatly improve enzyme performance. Thus, in this tutorial
review, we will try to list and explain some of the main reasons that may produce an
improvement in enzyme activity, specificity or selectivity, either real or apparent, due to
immobilization.
1. Introduction
Enzymes are nowadays reaching high levels of implementation
in areas as diverse as fine and pharmaceutical chemistry, food
modification or energy production (e.g., biodiesel and bioethanol).1
Immobilization of enzymes is a requisite for their use as
industrial biocatalysts in most of these instances, since
immobilization permits the simple reuse of the enzyme and
simplifies the overall design and performance control of the
bioreactors.2–4 Thus, many efforts have been devoted to
convert this requirement into a powerful tool to greatly
improve enzyme performance.5 For example, stabilization of
monomeric enzymes via multipoint covalent attachment or
generation of favorable environments surrounding the enzyme
has been reported in many instances,6 while multimeric enzymes
have been stabilized by immobilizing all enzyme subunits, thus
preventing subunit dissociation.7 In any case, immobilization is
compatible with any other strategies to yield a more stable
biocatalyst, such as chemical modification,8 use of enzymes from
the thermophile microorganisms,9 or genetic manipulation.10
Immobilization is in many instances associated with a decrease
in enzyme activity or a worsening of other catalytic features.
a Biocatalysis and Enzyme Technology Lab, Institute of Food Scienceand Technology, Federal University of Rio Grande do Sul, Av. BentoGoncalves, 9500, P.O. Box 15090, ZC 91501-970, Porto Alegre, RS,Brazil
b Escuela de Bacteriologıa y Laboratorio Clınico, UniversidadIndustrial de Santander, Bucaramanga, Colombia
c Instituto Universitario de Materiales, Departamento de QuımicaInorganica, Universidad de Alicante, Campus de San Vicente delRaspeig, Ap. 99 – 03080, Alicante, Spain
d Escuela de Quımica, Grupo de investigacion en Bioquımica yMicrobiologıa (GIBIM), Edificio Camilo Torres 210, UniversidadIndustrial de Santander, Bucaramanga, Colombia
eDepartamento de Biocatalisis, Instituto de Catalisis-CSIC, CampusUAM-CSIC, C/Marie Curie 2, Cantoblanco, 28049, Madrid, Spain.E-mail: [email protected]; Fax: +34 915854760; Tel: +34 915854941w Part of a themed issue on enzyme immobilization.
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However, some reports in the literature show how immobiliza-
tion of an enzyme may also improve its activity (the rate of the
reaction per milligram of enzyme), specificity (discrimination
between substrates) and selectivity (production of one among
several possible products).11 Immobilization, in most cases, will
produce slight distortions in the enzymes structure, and this
may alter the final properties of the enzyme.12 These changes
will be largely uncontrolled, but building a large library of
biocatalysts prepared following quite different immobilization
strategies to have a large diversity of situations may permit to
find solutions where the enzyme properties improve.11
However, the improvements in enzyme performance after
immobilization are not always really related to the production
of a more active or selective enzyme molecule, but to some
artifact which can alter the activity of the free or immobilized
enzyme, or just affect the stability of the enzyme. Thus, this
review will try to present and discuss the facts and artifacts
that can promote improvements in enzyme activity, specificity
and selectivity after immobilization. These improvements in
enzyme activity may be considered in any case more the
exception than the rule, as in most instances immobilized
enzymes will exhibit a lower catalytic performance (also cause
by real effects on enzyme structure or artifacts similar to those
described here). In this sense, this review is quite far from
other reviews on immobilization methods that may be found
in the literature that usually list immobilization methods or the
different uses of them.2–12
2. Improvements in enzyme activity
by immobilization
2.1 Aggregation of the ‘‘soluble’’ enzyme
In some instances, the researcher compares the activity of
the immobilized enzyme under conditions in which the free
enzyme is insoluble, i.e. comparison of an aggregated enzyme
(formed by enzyme precipitation in that medium) with an
enzyme immobilized and dispersed on the surface of the support
(Fig. 1). This systematically occurs when using anhydrous
media, where an enzyme is not soluble,13 but may also occur
under other reaction conditions (e.g., pH near the isoelectric
point, high protein concentration, etc.). Actually, this may be
viewed as a comparison between two immobilized forms of the
enzyme, an aggregated enzyme with severe diffusional problems
versus an enzyme immobilized on a porous support with lower
diffusional limitations.14 The result may be that, in certain
cases, an ‘‘improved’’ activity is observed using the immobilized
enzyme compared to the ‘‘aggregated’’ enzyme. However,
this improved activity should be considered an artifact and
critically scrutinized.
2.2 Prevention of enzyme inhibition
Inhibition may be another cause for enzyme activity altera-
tion. Some enzymes may be inhibited by high concentrations
of the substrate or by some of the reaction products,
Claudia Ortiz
Prof. Claudia Cristina OrtizLopez was born in 1966. In2004 she obtained her PhD inpossible uses of the complexmechanism of interfacial acti-vation of lipases as a usefultool to improve biocatalyticalprocesses with immobilizedenzymes. This work wassupervised by ProfessorsGuisan and Fernandez-Lafuente at ICP-CSIC(Spain). In 2004, she obtaineda tenure track position at theSchool of Bacteriology, wherecurrently she works as an
Associate Professor. She also directs the Research Group inBiochemistry and Microbiology at the Universidad Industrial deSantander, Colombia, from 2010. Her research interests includeIndustrial Microbiology, Bioprocess Technology, Biocatalysisand Biotransformations.
Angel Berenguer-Murcia
Dr Angel Berenguer-Murciaobtained his Degree inChemistry at the Universityof Alicante in 2000, and in2005 he obtained his PhD. In2006 he moved to the Univer-sity of Cambridge (UK) towork under the supervision ofProf. Brian F. G. Johnsonon the design of ‘‘smartmaterials’’. In 2009 he movedback to the Materials Instituteof the University of Alicantewhere he is a Research Fellow.His research interests includethe development of membranes,nanoparticle synthesis, and thedesign of porous materials.
Rafael C. Rodrigues
Prof. Rafael Costa Rodrigueswas born in Rio Grande, RS,Brazil, in 1980. He obtainedhis PhD in enzymatic syn-thesis of biodiesel under thesupervision of Prof. Ayub atthe UFRGS (Brazil). Heperformed a part of hisresearch in the group of Prof.Guisan (ICP-CSIC, Spain)studying new immobiliza-tion–stabilization methods forlipases to apply in biodieselproduction reactions. In 2010he obtained a lectureship atthe Food Science and Techno-
logy Institute, UFRGS (Brazil). His research interests areimmobilization-stabilization of enzymes and reaction engineer-ing. He has coauthored 32 papers and 1 patent, presenting anH number of 11.
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decreasing the observed activity.15,16 Immobilization has been
reported to prevent or at least diminish enzyme inhibition in
certain instances; therefore, in this specific case an increase in
enzyme activity after immobilization may be expected
(Fig. 2).11 Caldolysin, a metal chelator-sensitive extracellular
protease from Thermus aquaticus strain T351 is the first
reported example of how after its immobilization, enzyme
substrate inhibition may be avoided.17 The proposed mechanism
involved steric exclusion of the substrate from an ‘‘inhibition
site’’ without significant interference with the active site. Lactases
from different origins are inhibited by the substrates (lactose)
and reaction products (glucose and galactose); it has been
shown that this inhibition may be reduced by partial blocking
or just by inducing a certain distortion in the inhibition site by
immobilization18 (Fig. 2).
In other examples, rigidification of the enzyme structure
by multipoint covalent immobilization has reduced some
allosteric inhibitions (e.g., as shown in the synthesis of antibiotics
catalyzed by penicillin G acylase).19 Thus, a higher activity of a
given enzyme after immobilization may be in some instances
derived from a decrease in enzyme inhibition, and not from the
production of a more active conformation of the enzyme.
From an applied point of view, this inhibition reduction
problem may have the same, or even more merit than an actual
increase in activity (e.g., yields may progress until conversion
reaches 100%), but the researcher needs to check on the existence
of this kind of problem before assessing the real cause for the
improvement in enzyme activity.
2.3 Activity determination under harsh conditions
It should be considered that enzymes are quite unstable
biocatalysts (i.e. their optimum operating range is considerably
narrow) whose activity strongly depends on the experimental
conditions.20,21 The immobilization of an enzyme inside a porous
support may have several protective effects on the enzyme
structure under different situations. For example, many deter-
gents may produce a decrease in enzyme activity by inhibition
or by enzyme distortion,22 and if the enzyme is inside the pores
of a support, it may be partially protected from this cause of
activity loss (e.g., micelles may have more problems in pene-
trating inside the pores) (Fig. 3). The final results may be an
apparent increase in activity, if measured in the presence of
detergent. Similar effects may be produced if the enzyme is
subjected to strong stirring which is able to inactivate the
enzyme (e.g. to disperse the substrate, introduce oxygen into
the system, etc.). An enzyme inside the pores of a support will
also be protected from this negative effect14 (Fig. 3).
Another variable that strongly determines enzyme activity
is the reaction pH. This is important considering that in
many instances the support may be an ionic exchanger. These
ionic exchangers may behave as a ‘‘solid’’ buffer, generating a
Fig. 1 Prevention of enzyme precipitation by immobilization.
Rodrigo Torres
Prof. Rodrigo Torres was bornin Valparaıso, Chile, in 1966.He obtained his PhD in 2005working under the supervisionof Profs Jose Manuel Guisanand Roberto Fernandez-Lafuente. After a stay as avisiting scholar at the Depart-ment of Microbiology ofCornell University, he movedback to the School of Chemis-try of Universidad Industrialde Santander, Bucaramanga,Colombia, where he is currentlyan associate professor. Hisresearch interests include
enzyme immobilization, biocatalysis and biotransformation,proteomics, peptide synthesis and nanobiotechnology for thedevelopment of new applications of nanocompounds in environ-mental and pharmaceutical applications. He has coauthored 42papers and 2 patents.
Roberto Fernandez-Lafuente
Prof. Roberto Fernandez-Lafuente was born in 1964.He obtained his PhD underthe supervision of Prof. Guisanin ICP-CSIC (Spain). After apostdoctoral period in UCL(UK) under the supervisionof Prof. Cowan, he returnedto ICP-CSIC, where heobtained a permanent positionin 2001. Since 2008, he is aResearch Professor. Hisresearch interests are thedevelopment of strategies forthe preparation of improvedbiocatalysts and biosensors:
enzyme purification, immobilization, stabilization and alsoreaction design. He has coauthored over 270 papers and20 patents, and has supervised 15 doctoral theses, presentinga h number of 45.
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pH inside the biocatalyst bead that may greatly differ from the
pH value in the reaction medium (Fig. 4).
If the immobilized enzyme is stored at a pH near its optimal
value, while the activity measurement is far from this pH and it
is performed in short times, the immobilized enzyme will
remain in the optimal pH value even though the pH in the
bulk may be far from it. Thus, the immobilized enzyme may
be ‘‘apparently’’ more active at a pH value far from the
optimal value. Thus, some precautions should be considered
before stating that enzyme activity increases after immobiliza-
tion, and that it is not a protective effect caused by the
immobilization step.
2.4 Enzyme rigidification
When considering enzyme performance, there are several
factors that should be weighed in apart from purely chemical
ones. Another point to be considered is that enzyme activity is
Fig. 2 Prevention of enzyme inhibition by immobilization.
Fig. 3 Prevention of interaction of immobilized enzymes with external surfaces.
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linked to the stability of its structure. Changes in this structure
tend to decrease enzyme catalytic activity.20 A proper enzyme
immobilization may produce a strong rigidification of the
enzyme structure, mainly if a very intense multipoint covalent
attachment is achieved.23 In these cases, if the support matrix
is rigid and the spacer arms are short, all enzyme groups
involved in the enzyme immobilization process should main-
tain their relative positions under any circumstance, because
one group cannot move regardless of the others. This multi-
point covalent attachment may not be simple to achieve but
using a proper support and suitable enzyme–support reaction
conditions, it has revealed itself as one of the most powerful
strategies to stabilize enzymes.11,14 Thus, a stabilized-immobilized
enzyme should be expected to retain its structure under much
more drastic conditions than the free enzyme (for example
presenting a higher optimal temperature).11 If a very high
stabilization has been achieved, this optimal temperature for
the immobilized form may yield a free enzyme activity almost
null due to thermal enzyme distortion (Fig. 5).
Thus, activity measurements of an immobilized enzyme at
temperatures above the optimal for the free enzyme may result
in a significant increase in enzyme activity after immobilization.
Considering that multipoint covalent attachment should prevent
enzyme conformational changes induced by any reagent, it is
expected that enzyme activity may be retained under any distorting
conditions. Thus, similar improvements in enzyme activity upon
immobilization may be caused by the presence of organic solvents,
urea, guanidine and any other distorting agents. These will decrease
the activity of the free enzyme while having a lower effect on the
activity of the stabilized-immobilized enzyme.
A special case lies in multimeric enzymes, formed by different
subunits that may be in association–dissociation equilibrium.24
Multisubunit immobilization is able to fully prevent this pheno-
menon, thus avoiding this effect on enzyme activity and stability
when assayed under dissociation conditions7 (Fig. 6).
To use the term ‘‘artifact’’ when the researcher finds
an increased activity under these drastic conditions due to
immobilization perhaps is not exact. The most accurate way to
express it would be that the observed enzyme activity
under these conditions increases due to immobilization-
induced rigidification. The increased enzymatic activity under
those conditions, however, is not caused by the generation of a
more active enzyme form, but by avoiding distortion of the
enzyme structure.
2.5 Effect of medium partition
In some instances, immobilization may greatly alter the physico-
chemical properties of the enzyme surroundings, generating
a much more hydrophobic or hydrophilic environment that
can produce some partition of different compounds away or
towards the enzyme.14 This is in fact a strategy to stabilize
enzymes versus some inactivation agents, such as oxygen,
hydrogen peroxide, dissolved gases or organic solvents.25–27
If the immobilized enzyme is further modified with polymers,8
the stabilizing effect becomes impressive (Fig. 7).
In an aqueous–organic co-solvent system, the organic
solvent may in many instances greatly reduce enzyme activity
(by inhibition or enzyme distortion).28 If the enzyme is
exposed to lower organic co-solvent concentration by parti-
tion, the observed result is an increase in enzyme activity
(Fig. 7). In this case, we are reducing the cause for decrease
in enzyme activity. However, as in the aforementioned cases,
the researcher will observe an increase in activity after
Fig. 4 Immobilization support as a solid ‘‘buffer’’: effects on enzyme activity.
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immobilization, which once again will be fully unrelated to the
production of a hyperactivated form of the enzyme.
2.6 Effect of substrate or product partition
As explained above, immobilizationmay in some instances produce
a partition of different compounds.14 If a partition of the substrates
or products is achieved after immobilization, this may affect enzyme
activity depending on the different possibilities of the enzymatic
kinetics. And in some cases, the effect may be positive.
If the used substrate concentration is below that required to
saturate the enzyme, and the enzyme environment permits parti-
tioning of the substrate towards the enzyme, an apparent increase
in activity will be observed. The actual situation will be a decrease
in the apparent KM, while Kcat will remain unaltered (Fig. 8).
If a high concentration of substrate is used and the partition
effect reduces the concentration of substrate, this can promote
a positive effect on the observed activity, provided that the
substrate is able to inhibit the enzyme. Once again, Kcat will
remain unaltered, but an increase in KM and Ki will be
observed (Fig. 8). A similar positive effect could be detected
if the products were excluded from the enzyme environment
and were able to inhibit the enzyme. These effects will be
similar to that found in any biphasic system.29
In all these cases, a complete kinetic study of the reaction
will clarify the actual causes for the observed increases in
Fig. 6 Multisubunit enzyme immobilization prevents subunit dissociation.
Fig. 5 Enzyme rigidification by immobilization decreases enzyme distortion.
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activity after immobilization. As in the other cases, this
operational increase in enzyme activity upon immobilization
is unrelated to the production of an enzyme structure with
better properties induced by its fixation to the support.
2.7 Diffusional limitations
Diffusion limitations have been usually considered to be a
problem that reduces enzyme activity.14 If substrate diffusion
inside the support particle is slower than its catalytic modifica-
tion, enzymes in the core of the catalyst particle will not
receive the same substrate concentration as the enzyme near
the surface of the particle. However, in some instances these
diffusional problems may turn out for the best.
The decrease of substrate in the enzyme environment can
only produce an improved activity if the substrate may
produce a strong inhibition on the enzyme and we are using
substrate concentrations high enough to produce this negative
Fig. 7 Effect of medium partition on properties of immobilized enzymes.
Fig. 8 Effect of substrate partition towards or away the enzyme environment on the enzyme properties.
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effect on enzyme activity, as it has occurred in many industrial
processes.19 This way, the decrease in substrate concentration
in the enzyme environment far from producing a decrease in
enzyme activity may actually increase it (Fig. 8).
Internal pH gradients may be formed in enzymes immobilized
on porous supports if the activity of the biocatalyst is high
enough30,31 (Fig. 9). This is usually considered a disadvantage
since the particle internal pH becomes different from that of the
bulk. These different pH values have been used to increase
enzyme operational stability.32 In a similar way, if the external
pH value does not correspond to the optimal pH for the enzyme
activity, it is possible that the situation in the presence of pH
gradients may result in enhanced activity, e.g. if the pH value
inside the biocatalyst particle is nearer to the optimal pH value
than the one in the bulk. This may occur in the laboratory when
using standard measurements protocols, and also in industry if
we must work under conditions far from the optimal ones of
the enzyme, some times due to substrate solubility or stability,
or process thermodynamics.
Another possibility where diffusion may increase enzyme
activity is when two coupled enzymes are used, co-immobilized on
the same porous particle, mainly when determining the final
product to state the ‘‘global’’ activity14,33 (Fig. 10). If the produc-
tion of product 1 is fast enough, this compound will accumulate
inside the pore and can cause the second enzyme to act under a
higher substrate concentration. In these cascade reactions, the
second enzyme will be frequently working at concentrations of
substrate under the saturation conditions, and this increase in its
substrate concentration may yield an improved activity. This has
been recently exemplified using two coupled redox enzymes.34
The activity using low cofactor concentrations was higher
using the co-immobilized enzymes, not only compared to the
enzymes immobilized in different particles, but also when
using similar amounts of soluble enzymes.
However, measuring each enzyme individually and using the
adequate concentrations of their respective substrates, enzyme
activity did not improve for any of the enzymes after immobiliza-
tion, because the enzyme structure was unaltered. This effect
was only observed when measuring the whole biocatalyst using
substrate 1 and the whole cascade (the actual industrial target),
where substrate availability for the second enzyme is improved.
2.8 ‘‘Freezing’’ of a more active conformation
It is important to remark that some enzymes exist in different
conformational states with different activities and/or stabilities.35
Moreover, many multimeric enzymes may exist in different
degrees of aggregation, having different catalytic properties.36
Perhaps the best known case is that of lipases.35 Lipases
exist in two main forms, open and closed ‘‘forms’’.37,38 In
aqueous medium, the equilibrium between these two forms
is displaced to the closed form, where the active center is
Fig. 9 Effect of pH gradients inside the particle of biocatalyst on enzyme properties.
Fig. 10 Improving enzyme activity of co-immobilized enzymes due to
partition of substrates and products inside the pores of the biocatalysts.
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secluded from the reaction media by a polypeptide chain called lid
or flap, which in many cases is almost fully inactive. On the other
hand, in the presence of a hydrophobic interface, like drops of
their natural substrate (oils), the lid is displaced and the active
center becomes exposed to the medium, displacing the equilibrium
to the open and active form. The open form of the lipases becomes
adsorbed via the large hydrophobic pocket exposed (formed by
the internal face of the lid and the area surrounding the active
center) to the hydrophobic surface (Fig. 11).
This is the so-called interfacial activation of lipases.35
Immobilization may become a tool to fix this open form of
the lipase. It has been shown that this may be easily achieved
by immobilization of the enzyme at low ionic strengths
on hydrophobic supports,39 and also by cross linking40 or
lyophilization in the presence of detergents.41
The effect of immobilization on hydrophobic supports using
lipases against fully soluble substrates may be even more
beneficial. Lipases have a trend to form bimolecular aggre-
gates, interfacing the active centers of two open forms of
lipases, and that enzyme conformation tends to be less active
than the monomeric form, because the lipase active center is
partially blocked.42 Adsorption on hydrophobic supports
(presenting large surfaces) will give a dispersed open form,
cleaving these dimers (Fig. 12).
Fig. 11 Interfacial activation of lipases on hydrophobic supports: congealing a hyperactivated enzyme form.
Fig. 12 Hyperactivation of lipases by breaking the dimers by immobilization on hydrophobic supports.
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Thus, in this case, the observed enzyme activity is improved
by displacing the equilibrium towards the monomeric open
form of the lipase without the need for adding an external
hydrophobic interface. Moreover, by changing the support
morphology and hydrophobicity, it has been shown that it is
likely to have an optimal open form of the lipase, with an
activity even higher than that of the enzyme adsorbed on
drops of insoluble substrates.14
Some other enzymes may be hyperactivated by a conformational
change induced by an effector (an activator, some specific
medium).43 If the enzyme is attached to the support via many
points, or if lyophilization is performed in the presence of this
effector, enzyme molecules with this hyperactivated form may
be produced, which will remain hyperactivated in the absence
of the effector (Fig. 13).
In this case, immobilization is stabilizing a hyperactivated
enzyme form induced by a molecule or reaction medium, in a
way that this hyperactive form will be retained even in the
absence of the effector.
2.9 Production of a new more active conformation
Immobilization of enzymes produces conformational changes
and/or chemical modifications when incorporated to the
support. It is very likely that enzyme activity versus its natural
substrate may suffer a certain decrease. However, in many
instances the target substrate is quite far from the physio-
logical one. Although we will discuss this point more extensively
at a later stage, if we prepare a large library of immobilization
methods, for example involving different enzyme regions in the
immobilization and giving different degrees of enzyme–support
interaction or generating different enzyme microenvironments,
it is not unlikely to find some biocatalysts with a higher specific
activity versus a particular substrate.11 This random hyper-
activation produced by a particular immobilization method
against a particular substrate will be based on the casual
generation of a more active enzyme form, and it would be
more likely to occur with enzymes having a flexible active
center (e.g., lipases, multimeric enzymes), and if a sufficiently
large biocatalyst library is prepared.
3. Changes in enzyme specificity or selectivity by
immobilization
In this section we will discus how immobilization may greatly
affect enzyme specificity and/or selectivity.11 These changes
may deeply alter enzyme performance in several reactions of
industrial relevance:
- Resolution of racemic mixtures:44 enzymes are in many
instances used as catalysts in the dynamic resolution of
racemic mixtures of substrates different from the natural
ones (where a complete specificity should be expected), via
hydrolysis, esterification, amination, transesterification, etc.
Using these unnatural substrates, specificity may not be
complete. If immobilization alters KM or Kcat towards one
or both enantiomers, the enantioselectivity of the enzyme and
the obtained enantiomeric excess may be greatly affected.
- Enantioselective modification of prochiral compounds
(e.g., reduction of prochiral ketones, asymmetric hydrolysis
of prochiral dicarboxylic esters or asymmetric acylation of
prochiral carboxylic acids, etc.):44 again, the use of substrates
far from those natural to the enzyme may produce moderate
enantioselectivity values. Immobilization may alter the preferred
produced isomer, by favoring one or the other transition state.
- Regioselective modifications of poly-functional compounds:45
for instance, regioselective hydrolysis of peracylated poly-
hydroxy compounds, regioselective synthesis using polyhydroxy
(e.g., sugars, glycerin) or poly carboxylic acids, oxidations of
poly-alcohols, etc. In this case, immobilization may affect
the adsorption of the substrate on the enzyme active site,
confronting different groups with the catalytic residue thus
modifying the selectivity of the process. The final percentage
of the target molecule will also depend on the rate of the
Fig. 13 Effect of effectors during enzyme immobilization on enzyme properties.
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successive modifications of the other groups in the substrate.
Therefore it should also be related to the specificity of the
enzyme between the different possible compounds.
- Kinetically controlled synthesis: this process is based on
the use of an activated acyl donor46 such as an ester or an
amide (or vinyl adduct) to reach maximum transient yields
that depend on the balance of three different reactions cata-
lyzed simultaneously by the enzyme: the formation of the
target product, the hydrolysis of the activated acyl donor,
and the hydrolysis of the target product. Examples of this
reaction are transesterifications, transformations of esters by
amides, transamidations, transglycosylations, etc. The kinetically
controlled synthesis of antibiotics47 or the synthesis of biodiesel48
may be some of the most relevant examples. Enzyme perfor-
mance on kinetically controlled synthesis depends on the
adsorption of the nucleophile on the active center of the
enzyme, the specificity of the enzyme versus the active acyl
donor and the product, the possible inhibitions of one or the
other reaction, etc.46 (Fig. 14). Obviously, all these processes
depend on the enzyme and will be deeply modulated by the
enzyme structure after immobilization.47
- Interesterification and acidolysis (e.g., to produce struc-
tured triglycerides):49 the mechanism of these reactions is quite
complex. Interesterification may be carried out using a blend
composed of several oils of different sources, employing just
one oil which presents different fatty acids or mixing one oil
together with esters of the desired fatty acid. In the case of
glycerides, if we want to introduce a new fatty acid into the
glycerol moiety, the ester bond between the native fatty acid
residue (the original substituent group) and the glycerol
moiety must first be hydrolyzed. This reaction liberates the
native fatty acid and produces a lower (less substituted)
glyceride containing at least one hydroxyl group. The hydrolysis
step is followed by the formation of a new ester bond by
reaction of the newly created hydroxyl group with the incoming
replacing fatty acid (that also needs to be released from the
ester)49 (Fig. 15).
The acidolysis mechanism in this reaction is similar to an
interesterification. After hydrolysis of an ester bond between
the native fatty acid residue (the original substituent group)
and the glycerol moiety of the triglyceride, the native fatty acid
is released and a glyceride containing at least one hydroxyl
group is produced. The hydrolysis step is followed by the
formation of a new ester bond by reaction of the newly created
hydroxyl group with the incoming new free fatty acid49
(Fig. 16). Thus, the specificity of the enzyme by the different
fatty acids and triglyceride positions becomes a key point
towards the final yields of the structured triglyceride.
Like in the case of the activity, the enzyme properties in all
these processes may be strongly modulated by immobilization
via different ways. Next, we will explain some of the most
relevant.
3.1 Effect of diffusion limitations
At first glance, diffusional problems will always have either a
null or a negative effect on the observed results in these
processes. In any reaction where enzyme specificity may play
an important role, the concentration of the best substrate will
decrease more rapidly than that of the less suitable substrate.
Fig. 14 General scheme of kinetically controlled synthesis of ampicillin.
Fig. 15 General scheme of interesterification.
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Thus, for example, in the resolution of racemic mixtures,
substrate diffusional problems can only produce a decrease
in the apparent enzyme enantiospecificity. In kinetically
controlled syntheses, the consumption of the nucleophile and
the accumulation of the product along the pore of the support
can only produce a decrease in the maximum yields, by
decreasing the saturation of the enzyme by the nucleophile
and favoring the hydrolysis of the formed product.14 In
selective reactions, enzyme selectivity did not appear to be
influenced by the substrate concentration, and only a decrease
in the reaction rate should be observed. Thus, at first sight, low
enzyme loadings and enzyme distributions along the particle
pores in a way that overcome these diffusional limitations
(e.g., forming a crown on the external part of the bead) should
be always advantageous.34
However, as discussed above, diffusional problems may
alter not only substrate concentration, but also the pH inside
the particle.30 The reaction pH may exert a critical influence
on any enzyme property, and this effect of the pH on such
properties may also be altered by immobilization (as will be
discussed later). Thus, while substrate diffusional problems
can hardly have any positive effect on the enzyme performance
in this kind of processes, pH gradients may be used as a tool to
improve the results after enzyme immobilization, and can
bring forth improvements in the enantiomeric excesses
obtained (in the dynamic resolution of racemic mixtures and
in enantioselective processes) and also in the maximum yields
in kinetically controlled processes.45,46 Thus, even without directly
affecting the enzyme structure, the promotion of internal pH
gradients inside the particles of porous biocatalysts may
produce a significant improvement in enzyme performance
upon immobilization in this kind of reaction.
3.2 Generation of micro-environments around the enzyme
Enzyme properties and performance on the processes described
above are strongly influenced by the concentration of both
substrates and products. If the enzyme is immobilized in a very
hydrophobic (e.g., supports made of divinylbenzene)50 or
hydrophilic environment (e.g., polymeric beds anchored to
the support surface, formed by polyethylenimine or dextran-
sulfate), some partition of the substrates may be expected.8,14
A positive partition of the substrates may have some beneficial
effects on the enzyme performance, e.g., the enzyme may be
saturated for longer periods of time by the preferred substrate
in dynamic resolutions or by the nucleophile in kinetically
controlled processes. A partition of the product that will
reduce the product concentration around the enzyme may be
positive in a kinetically controlled process, by reducing the
hydrolysis of the product and increasing the maximum yields.46
In some cases, inhibitions caused by the products may be also
relevant for the final results (e.g., after hydrolysis of the
preferred isomer, the product may be an effective inhibitor
of the hydrolysis of the undesired isomer). Thus, some partition
of substrates and/or products from the immobilized enzyme
environment in the right direction may greatly improve enzyme
performance, and this may be achieved without really affecting
enzyme conformation but just altering the availability of the
different compounds involved in the reaction.
Enzyme properties are also governed by the experimental
conditions, and the nature of the support may promote some
partition on the components of the medium. This is very clear
in the presence of organic solvents. If the enzyme is in a highly
hydrophilic polymeric bed,8,14 the concentration of solvent
around the enzyme will be lower than that in the reaction
medium. If this lower concentration of organic solvent improves
enzyme performance (e.g., producing a higher selectivity or
specificity), after immobilization we can detect an improvement
in the enzyme performance in the reaction.45 This will not be a
consequence of changes in the enzyme structure, but will be due
to changes in the reaction conditions under which the enzyme
operates. In any case, the final effect will be an improvement in
enzyme performance in this kind of processes.
3.3 Immobilization of mixtures of enzymes
In some instances, commercial preparations of enzymes or the
crude extract obtained in a laboratory contain several enzymes
that are able to catalyze a similar reaction.51–53 Although this
is not an ideal situation, many researches have been carried
out using this mixture of enzymes. In some instances, it is
difficult to even detect the presence of the contaminant enzyme
that may be in trace amounts but being very active versus some
specific substrates (e.g., chymotrypsinogen B in preparations
of porcine pancreatic lipase).51 In others the microorganism
produces a collection of isoforms (e.g., the isoforms of lipases
in Candida rugosa).54 The properties observed using these
crude preparations will be the average of the whole mixture
of enzymes able to catalyze the target reactions (and will
depend on the exact batch).
Upon immobilization, several factors may decrease the
relevance in the reaction for some of the enzymes. First, not
all enzymes will become immobilized on all supports, and
either by chance or on purpose (e.g., when immobilization is
designed to associate immobilization and purification of the
target enzyme) the contaminant enzyme may not become
immobilized on the support.51 If the enzyme that did not
become immobilized on the support is the one having the
poorest performance in the process, we can observe an
improvement in the results obtained using the immobilized
Fig. 16 General scheme of acidolysis.
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preparation when compared to the free ‘‘enzyme’’. This
is produced by the purification of the enzyme during immo-
bilization, not by an actual alteration of the enzyme properties
(Fig. 17).
Another possible effect derived from immobilization is that
some enzymes become significantly more inactivated versus the
target substrate than others. The practical effect will be similar
to the one described above, if the most inactivated enzyme
is the one having the worst performance, the immobilized
biocatalyst will exhibit a better behavior than the collection
of free enzymes. In this case, this improvement will be a
consequence of the selective inactivation of one of the enzymes
during immobilization.
Different stabilizations due to the immobilization of the
different components of the crude extract which are able to
perform the reaction may also produce an effect on the
performance of the immobilized biocatalyst. This mainly
produces different behaviors when using conditions where
the free enzyme suffers conformational changes reducing its
activity. If one enzyme is much more stabilized than another
after immobilization, it may retain more activity under these
drastic conditions.11 If it is the one having the best perfor-
mance, an improved behavior of the immobilized biocatalyst
compared to the free enzymes preparation under these condi-
tions will be observed. Now, this will be a consequence of the
preferential stabilization of one enzyme during insolubiliza-
tion of the enzymes.
Obviously, all these positive effects derived from immobili-
zation of an enzyme mixture will depend on the substrate used
(e.g., using a substrate where only one enzyme has activity, this
effect is not possible). Thus, a deep characterization of the
crude preparation may be necessary to fully understand the
changes in enantio- or regioselectivity or specificity, or in
kinetically controlled synthesis after immobilization.
3.4 Enzyme rigidification
As commented in Section 2.4, a strong rigidification of the
enzyme structure via multipoint covalent attachment may help
keeping this conformation when the conditions are altered.11
If the enzyme is utilized under conditions (e.g., using solvents
to solubilize the substrate) under which the free enzyme suffers
some distortion that decreases its specificity or selectivity,
and the immobilization permits to keep the enzyme features,
the observed result after immobilization may be an ‘‘improve-
ment’’ in the results (Fig. 5).
3.5 Changes in enzyme structure due to immobilization
As it was previously mentioned in Section 2.9, immobilization
of an enzyme will most probably alters its structure to some
degree, due to unspecific enzyme support interactions or the
interactions that cause the immobilization.10,11,14 In many
cases, immobilization produces a change in enzyme activity,
but these changes may also be correlated to changes in the
behavior of the enzyme in any of the aforementioned processes.
If the enzyme has a rigid active center, it may be very hard to
find an immobilized preparation with improved properties.
However, the situation may be different if the enzyme has a
flexible active center.
3.5.1 ‘‘Conformational engineering’’ of enzymes suffering
structural changes during catalysis. Some enzymes suffer drastic
conformational changes during the catalytic process. As stated
above, lipases are perhaps the best known enzymes in this
aspect. All lipases have the capability of acting at the interface
of oil drops by interfacial activation, their adsorption on
these drops takes place via the large hydrophobic pocket
formed by the internal face of the lid and the hydrophobic
areas surrounding the active center that interact with it.37,38
Fig. 17 Effect of the selective immobilization of a determined enzyme when using mixtures of enzymes.
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The lid may be very small, like the one of the lipase B
from Candida antarctica55 not fully secluding the active center
from the medium, or quite complex, like the double lid
recently described for the thermoalkaline lipase from Bacillus
thermocatenulatus.56 These movements do not affect only the
lid, but alter the overall structure of the lipases (Fig. 18).
In fact, although the crystal structure of a lipase shows only
one open form, it is not hard to imagine that depending on the
conditions under which the lipase moves its lid, different
conformations of the active center may be found. That will mean
that lipases will have a very flexible active center that may
tolerate some distortions without losing its catalytic activity.
Based on this idea, it has been proposed that the immobilization
of this kind of enzymes on a battery of different supports, under
different immobilization conditions, involving different regions of
the enzyme surface in the immobilization, giving different degrees
of rigidification, establishing different interactions between the
enzyme and the support, or generating different microenviron-
ments (Fig. 19), may generate a library of biocatalysts based on
a single enzyme exhibiting very different enantioselectivity,
enantiospecificity, regioselectivity, and even alter the yields in
kinetically controlled synthesis.10,11,14,45
This modulation of the enzyme properties is currently
uncontrolled, because the ‘‘in silico’’ techniques can point the areas
involved in the immobilization step, but still are far from predicting
small changes caused by the enzyme–support interactions.
Moreover, the changes in enzyme properties by immobilization
will be produced even if we already have a suitable biocatalyst
and we do not want to alter these properties. In this case a very
mild immobilization (e.g., using a lowly activated aldehyde
dextran as a spacer arm)11 is preferable.
The effects of the immobilization strongly depend on the
substrate. For a particular substrate the best biocatalyst may
be one, while for other substrates the best biocatalyst may be
completely different. This is usually observed using different
lipases against different substrates.45
Furthermore, it has been established that the reaction
medium conditions exert very different effects on the enzyme
catalytic features when changing the immobilization protocol.11
Thus, in many instances a change in the reaction conditions
improves enzyme performance when it is immobilized on a
support, while it decreases the enzyme activity that was immobilized
by using another protocol.45 This may be explained by the
influence of the experimental conditions on the interaction
between the enzyme and the support (e.g., if the support is not
fully inert), by a different effect on the same change in the
medium when the enzyme structure is different (e.g. caused by
different immobilization protocols), or if some particular
region of the enzyme is stabilized and cannot move.
The suitability of this strategy to tune the enzyme perfor-
mance depends on the size of the biocatalyst library (Fig. 19).
Immobilization affects the enzyme features, but in some cases
has a deleterious effect on enzyme performance and only in
some others will improve it. Thus, the wider and more
different the biocatalysts that are included in said library,
the higher the possibility of finally finding a catalyst able to
exhibit adequate properties in a particular reaction. Therefore,
while in order to have a stabilized biocatalyst there are some
preferred strategies, such as to give an intense multipoint
covalent attachment,11 to modulate the enzyme catalytic
features almost any immobilization protocol that yields a
stable enough preparation may be interesting.14 In this sense,
some immobilization protocols that may be used to immobilize
enzymes via different orientation but using the same chemical
groups in the support have substantial interest. One of these
examples is the use of epoxide-activated supports.57 Epoxide
activated supports, despite being reactive with many different
groups of proteins, react very slowly with free enzymes. The
enzyme requires to be first adsorbed on the support, and then it
reacts covalently with the support. It has been shown that by
adding some adsorbing groups to the support surface, these
groups adsorb the enzyme, and the orientation of the enzyme
on the support may be fully altered, and that produces a change
in the enzyme features.57 Another example is the adsorption of
enzymes on polymeric beds formed by ionic polymers coating
the support surface. The ionic strength during immobilization
may permit to control the penetration of the enzyme in the
polymeric bed,58 while orientation may also depend on the
immobilization pH value. One of the oldest methods found for
enzyme covalent immobilization is the use of glutaraldehyde
chemistry. This protocol may be used to have different forms
of the lipase, at least five.59 Using high ionic strength, ionic
Fig. 18 Structure of open and closed forms of RML. The 3D structure was obtained from the Protein Data Bank (PDB) using Pymol vs. 0.99.
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adsorption is avoided, but CALB is adsorbed on the support by
interfacial activation. Using non-ionic detergents (e.g., Triton
X-100), the enzyme becomes ionically adsorbed on the activated
support. If both detergent and salt are simultaneously present
during immobilization, a covalent attachment to the support is
first produced. In the absence of detergent or high ionic
strength, a mixture of all the previous immobilization causes
should coexist. Each of these preparations exhibited different
specificity, enantiospecificity, etc., and this could be further
amplified if the immobilization via ionic exchange is studied
under different conditions.
To increase the library even more, the immobilized lipases
may be further chemically modified increasing the prospects of
success.8 Amination, succinilation, polyethylene glycol, glutar-
aldehyde modifications, but also some other less conventional
modifications, have proved to be effective methods to alter
enzyme features, and even the effects depend on the previous
immobilization protocol applied.
Even if this strategy is a trial and error one, due to the
complexity of the interactions between an enzyme and the support,
its potential to modulate lipases has been proved. Thus, the tuning
of lipase properties by immobilization is now an accepted idea. It
has been reported using purified lipases from Candida antarctica,
Candida rugosa, Rhizomucor miehei, Bacillus thermocatenulatus,
Pseudomonas, etc. Examples mainly refer to hydrolytic reactions
in aqueous medium (resolution of racemic mixtures of esters,
regioselective hydrolysis of peracetylated sugars or glycerin, etc.),45
although some examples refer to the enantiospecific synthesis of
esters in anhydrous media using a kinetically controlled process.60
In some cases, even inversion of the enantiospecificity of the
enzyme has been achieved just by changing the way as the enzyme
is immobilized. In extreme cases, the use of just one purified lipase
immobilized on two different supports, and used under different
conditions (e.g., pH 5 or 7) has permitted to have almost full
enantiospecificity for one or the other enantiomer.
The idea has been extended to other enzymes also suffering
from some changes between an open form and a closed form,
like penicillin G acylase.11
This modulation of enzyme properties by immobilization needs
to be studied using pure enzymes, otherwise effects like the ones
described in the point above may complicate the understanding of
the occurring phenomena. Actually, it looks like although we are
using a lipase having an identical chemical sequence, all properties
of the enzyme may be altered to the point of appearing like
different enzymes. In this case, we are having an enzyme-support
adduct with a fully different structure, with different sensitivities
towards changes in the medium or in the substrate.11
3.5.2 Alteration of subunit assembly on multimeric enzymes.
The activity, stability, selectivity or specificity of multimeric
enzymes are strongly dependent on the assembly of the enzyme
subunits.7 This enzyme subunit assembly may be altered by
the reaction conditions, enhanced by some additives or experi-
mental conditions, or weakened by others.
Multisubunit immobilization of a multimeric enzyme
permits to use the enzyme under conditions where the free
enzyme tends to become dissociated, rendering new enzyme
conformations that may present better properties than the
native enzyme. An example of this is the kinetically controlled
synthesis of ampicillin catalyzed by the alpha-amino acid
esterase from Acetobacter turbidans in the absence of phosphate
ions, which stabilize the multimeric structure of the enzyme but
decrease the synthetase/hydrolase ratio.7 These conditions
could be only utilized if an enzyme preparation in which
dissociation is impossible was used. Thus, immobilization is
not generating a more favorable enzyme structure, but allowing
the use of the enzyme under dissociating conditions that result
in a more favorable structure.
Moreover, immobilization of a multimeric enzyme may
produce some distortion in the tertiary and quaternary structure,
Fig. 19 A library of biocatalyst from just one enzyme: different orientations, rigidification or microenvironments.
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generating enzyme preparations with different behavior than
that of the free enzyme (in an also completely random effect of
the immobilization). In this case, as it has been described for
lipases, the use of a large library of immobilized biocatalysts
may permit to find one that can exhibit improved properties
for a particular process. Examples of this are also quite scarce.
Immobilization of lactase from E. coli on different supports
proved to greatly alter the enzyme behavior in a transglycosilation
reaction.7 The enantioselectivity of a multimeric epoxide hydrolase
from Aspergillus niger was improved by immobilization on an
amino-epoxide support. In this case, an improved enzyme
structure is generated by immobilization.7
3.6 Freezing of changes induced on the enzyme by the reaction
medium
The properties of an enzyme on some of the processes listed
above are strongly modulated by a determined experimental
condition, like the presence of some organic solvent, the
reaction pH, etc. If the immobilization is able to maintain
the enzyme conformation induced by these experimental variables,
this may permit to improve the enzyme performance.61 The
improved behavior induced by this favorable conformational
change should be observed if the enzyme is immobilized under
those conditions, even it is used in other different ones.
Immobilization of lipase B from Candida antarctica on a bed
formed by polyethylenimine at different pH values (used in
resolution of racemic mixtures) or penicillin G acylase in the
presence of methanol (used in kinetically controlled synthesis
of antibiotics) are among the few examples described.11
4. Conclusions
Immobilization of enzymes on a support may alter the
performance of an enzyme in many interesting processes, such
as selective hydrolysis or oxidations, kinetic resolutions of
racemic mixtures or kinetically controlled synthesis.11 To
precisely understand what is occurring to the enzyme, several
facts need to be analyzed. First, the enzyme will be fully
dispersed on the support surface after immobilization, which
will prevent aggregation or other inactivation phenomena.14
Moreover, multipoint covalent immobilization may produce a
more rigid structure, less sensitive to conformational changes;
thus, enzyme activity under drastic conditions may become
higher than that of the free enzyme. The surface of the support
may produce some effects that may affect enzyme performance, by
partitioning of substrates, products or components of the reaction
medium.14 If the biocatalyst is porous, after immobilizing the
enzyme some diffusional limitations may alter the concentration of
substrates, products or even bring forth pH gradients that may
also influence enzyme performance.14 All these ‘‘improvements’’
were not really produced because an enzyme structure with a
better performance has been hitherto obtained. Nevertheless,
the fact is that the enzyme behaves better in the target process
than when it was used in its free form.
Among the ways to improve enzyme activity or selectivity
via immobilization, the only ‘‘rational’’ way is when we try to
congeal an enzyme structure having better properties induced
by the medium, by an effector or by the support. If this works,
it is a way to have an improved performance of the enzyme in
the absence of the stimulus that initially produces that improve-
ment. To reach this goal, it is necessary to know the agents that
produce this improvement and a strategy to ‘‘freeze’’ the
improved enzyme. Immobilization of the open structure of
lipases is very likely the best known example of this pheno-
menon, because we are using a support that ‘‘mimics’’ the cause
for enzyme hyperactivation, a hydrophobic surface. In general,
however, a strategy to significantly increase the rigidity of the
enzyme, in such a way that when this reagent is removed
the ‘‘improved’’ enzyme structure remains, may be achieved
using strategies that may stabilize this enzyme form, such as
lyophilization (using the enzyme in the form of an aggregate) or
an intense multipoint covalent attachment.
However, the highest number of reports on the improve-
ment of the enzyme properties by immobilization came from
the random promotion of favorable enzyme structural changes
produced during immobilization using different enzyme
protocols. This strategy is mainly useful in enzymes suffering
from large conformational changes during catalysis, and
that by their own nature present a very flexible active center.
After immobilization, it is probable that enzyme specificity,
selectivity and even response to changes under the experi-
mental conditions may be strongly altered.11 The prospects
of success using this strategy are closely related to the
development of immobilization methods that may involve
different areas of the proteins, promoting some rigidification,
generating different microenvironments. Currently, site-
directed rigidification of enzymes by immobilization, by
coupling a proper combination of support design and site-
directed mutagenesis has opened the door to full control over
enzyme immobilization, even though a trial and error assay
will still be necessary, since it is not possible to predict the
effects of the rigidification on a defined protein area on the
final enzyme performance on a particular process.10 Changes
in enzyme properties not necessarily mean improvements, and
in some instances a careful and extremely mild immobilization
protocol should be used to keep the good properties of the
utilized enzyme intact.
The combination of immobilization with chemical modification,
after or before immobilization, may become a source of new
biocatalysts with even larger modifications in their properties.8
Thus, immobilization, combined with rapid developments
in areas like genetic manipulation and support design, protein
chemistry, organic chemistry, reactor and reaction design, should
become an ever growing tool to improve the different aspects of
enzymes as industrial biocatalysts in the near future.
Acknowledgements
This work has been supported by grant CTQ2009-07568 from
Spanish Ministerio de Ciencia e Innovacion, and grant No.1102-
489-25428 from COLCIENCIAS and Universidad Industrial de
Santander (VIE-UIS Research Program). A. Berenguer-Murcia
thanks the Spanish Ministerio de Ciencia e Innovacion for a
Ramon y Cajal fellowship (RyC-2009-03813). The authors would
like to thank Mr Ramiro Martınez (Novozymes, Spain S.A) for
kindly supplying the enzymes used in this research. Prof. Rafael
C. Rodrigues thanks to CNPq and FAPERGS (Brazil) for
financial support.
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This journal is c The Royal Society of Chemistry 2012 Chem. Soc. Rev.
References
1 A. Schmid, J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts andB. Witholt, Nature, 2001, 409, 258–268.
2 E. Katchalski-Katzir, Trends Biotechnol., 1993, 11, 471–478.3 W. Hartmeier, Trends Biotechnol., 1985, 3, 149–153.4 P. Torres-Salas, A. Del Monte-Martinez, B. Cutino-Avila,B. Rodriguez-Colinas, M. Alcalde, A. O. Ballesteros andF. J. Plou, Adv. Mater., 2011, 23, 5275–5282.
5 R. A. Sheldon, Adv. Synth. Catal., 2007, 349, 1289–1307.6 P. V. Iyer and L. Ananthanarayan, Process Biochem., 2008, 43,1019–1032.
7 R. Fernandez-Lafuente, EnzymeMicrob. Technol., 2009, 45, 405–418.8 R. C. Rodrigues, A. Berenguer-Murcia and R. Fernandez-Lafuente,Adv. Synth. Catal., 2011, 353, 2216–2238.
9 D. A. Cowan and R. Fernandez-Lafuente, Enzyme Microb. Technol.,2011, 49, 326–346.
10 K. Hernandez and R. Fernandez-Lafuente, EnzymeMicrob. Technol.,2011, 48, 107–122.
11 C. Mateo, J. M. Palomo, G. Fernandez-Lorente, J. M. Guisan andR. Fernandez-Lafuente, Enzyme Microb. Technol., 2007, 40,1451–1463.
12 U. Hanefeld, L. Gardossi and E. Magner, Chem. Soc. Rev., 2009,38, 453–468.
13 E. P. Hudson, R. K. Eppler and D. S. Clark, Curr. Opin.Biotechnol., 2005, 16, 637–643.
14 C. Garcia-Galan, A. Berenguer-Murcia, R. Fernandez-Lafuenteand R. C. Rodrigues, Adv. Synth. Catal., 2011, 353, 2885–2904.
15 F. Ghanbari, K. Rowland-Yeo, J. C. Bloomer, S. E. Clarke,M. S. Lennard, G. T. Tucker and A. Rostami-Hodjegan, Curr.Drug Metab., 2006, 7, 315–334.
16 A. Ciulli and C. Abell, Curr. Opin. Biotechnol., 2007, 18, 489–496.17 D. A. Cowan, R. M. Daniel and H. W. Morgan, Int. J. Biochem.,
1987, 19, 483–486.18 B. C. C. Pessela, C. Mateo, M. Fuentes, A. Vian, J. L. Garcıa,
A. V. Carrascosa, J. M. Guisan and R. Fernandez-Lafuente,Enzyme Microb. Technol., 2003, 33, 199–205.
19 R. Fernandez-Lafuente, C. M. Rosell and J. M. Guisan, J. Mol.Catal. A: Chem., 1995, 101, 91–97.
20 K.M. Polizzi, A. S. Bommarius, J. M. Broering and J. F. Chaparro-Riggers, Curr. Opin. Chem. Biol., 2007, 11, 220–225.
21 L. Gianfreda and M. R. Scarfi, Mol. Cell. Biochem., 1991, 100,97–128.
22 J. E. Mogensen, P. Sehgal and D. E. Otzen, Biochemistry, 2005, 44,1719–1730.
23 C. Mateo, J. M. Palomo, M. Fuentes, L. Betancor, V. Grazu,F. Lopez-Gallego, B. C. C. Pessela, A. Hidalgo, G. Fernandez-Lorente, R. Fernandez-Lafuente and J. M. Guisan, Enzyme Microb.Technol., 2006, 39, 274–280.
24 M. Bechtold and S. Panke, Chem. Eng. Sci., 2012, 80, 435–450.25 C. Mateo, B. Fernandes, F. Van Rantwijk, A. Stolz and
R. A. Sheldon, J. Mol. Catal. B: Enzym., 2006, 38, 154–157.26 D. Brady and J. Jordaan, Biotechnol. Lett., 2009, 31, 1639–1650.27 K. Hernandez and R. Fernandez-Lafuente, Process Biochem.,
2011, 46, 873–878.28 O. Abian, C. Mateo, G. Fernandez-Lorente, J. M. Guisan and
R. Fernandez-Lafuente, Biotechnol. Prog., 2003, 19, 1639–1642.29 H. R. Hobbs and N. R. Thomas, Chem. Rev., 2007, 107, 2786–2820.30 A. C. Spiess and V. Kasche, Biotechnol. Prog., 2001, 17, 294–303.31 W. Tischer and V. Kasche, Trends Biotechnol., 1999, 17, 326–335.32 J. M. Guisan, G. Alvaro, C. M. Rosell and R. Fernandez-Lafuente,
Biotechnol. Appl. Biochem., 1994, 20, 357–369.
33 F. Lopez-Gallego and C. Schmidt-Dannert, Curr. Opin. Chem.Biol., 2010, 14, 174–183.
34 J. Rocha-Martın, B. d. l. Rivas, R. Munoz, J. M. Guisan andF. Lopez-Gallego, ChemCatChem, 2012, 4, 1279–1288.
35 R. Verger, Trends Biotechnol., 1997, 15, 32–38.36 F. K. Chu, W. Watorek and F. Maley, Arch. Biochem. Biophys.,
1983, 223, 543–555.37 A. M. Brzozowski, H. Savage, C. S. Verma, J. P. Turkenburg,
D. M. Lawson, A. Svendsen and S. Patkar, Biochemistry, 2000, 39,15071–15082.
38 A. M. Brzozowski, U. Derewenda, Z. S. Derewenda, G. G. Dodson,D. M. Lawson, J. P. Turkenburg, F. Bjorkling, B. Huge-Jensen,S. A. Patkar and L. Thim, Nature, 1991, 351, 491–494.
39 R. Fernandez-Lafuente, P. Armisen, P. Sabuquillo, G. Fernandez-Lorente and J. M. Guisan, Chem. Phys. Lipids, 1998, 93, 185–197.
40 G. Fernandez-Lorente, J. M. Palomo, C. Mateo, R. Munilla,C. Ortiz, Z. Cabrera, J. M. Guisan and R. Fernandez-Lafuente,Biomacromolecules, 2006, 7, 2610–2615.
41 I. Mingarro, C. Abad and L. Braco, Proc. Natl. Acad. Sci. U. S. A.,1995, 92, 3308–3312.
42 J. M. Palomo, M. Fuentes, G. Fernandez-Lorente, C. Mateo, J. M.Guisan and R. Fernandez-Lafuente, Biomacromolecules, 2003, 4, 1–6.
43 J. Tang and R. R. Breaker, Chem. Biol., 1997, 4, 453–459.44 P. McMorn and G. J. Hutchings,Chem. Soc. Rev., 2004, 33, 108–122.45 J. M. Palomo, Curr. Org. Synth., 2009, 6, 1–14.46 V. Kasche, Enzyme Microb. Technol., 1986, 8, 4–16.47 G. Volpato, R. C. Rodrigues and R. Fernandez-Lafuente, Curr.
Med. Chem., 2010, 17, 3855–3873.48 H. Fukuda, A. Kondo and H. Noda, J. Biosci. Bioeng., 2001, 92,
405–416.49 R. C. Rodrigues and R. Fernandez-Lafuente, J. Mol. Catal. B:
Enzym., 2010, 66, 15–32.50 K. Hernandez, C. Garcia-Galan and R. Fernandez-Lafuente,
Enzyme Microb. Technol., 2011, 49, 72–78.51 J. M. Palomo, R. L. Segura, C. Mateo, M. Terreni, J. M. Guisan
and R. Fernandez-Lafuente, Tetrahedron: Asymmetry, 2005, 16,869–874.
52 J. M. Palomo, G. Fernandez-Lorente, C. Mateo, M. Fuentes,J. M. Guisan and R. Fernandez-Lafuente, Tetrahedron: Asymmetry,2002, 13, 2653–2659.
53 M. Masuda, A. Sakurai and M. Sakakibara, Appl. Microbiol.Biotechnol., 2001, 57, 494–499.
54 M. Lotti, R. Grandori, F. Fusetti, S. Longhi, S. Brocca,A. Tramontano and L. Alberghina, Gene, 1993, 124, 45–55.
55 J. Uppenberg, S. Patkar, T. Bergfors and T. A. Jones, J. Mol. Biol.,1994, 235, 790–792.
56 C. Carrasco-Lopez, C. Godoy, B. de las Rivas, G. Fernandez-Lorente,J. M. Palomo, J. M. Guisan, R. Fernandez-Lafuente, M. Martınez-Ripoll and J. A. Hermoso, J. Biol. Chem., 2009, 284, 4365–4372.
57 C. Mateo, V. Grazu, B. C. C. Pessela, T. Montes, J. M. Palomo,R. Torres, F. Lopez-Gallego, R. Fernandez-Lafuente andJ. M. Guisan, Biochem. Soc. Trans., 2007, 35, 1593–1601.
58 B. C. C. Pessela, L. Betancor, F. Lopez-Gallego, R. Torres,G. M. Dellamora-Ortiz, N. Alonso-Morales, M. Fuentes,R. Fernandez-Lafuente, J. M. Guisan and C. Mateo, EnzymeMicrob. Technol., 2005, 37, 295–299.
59 O. Barbosa, R. Torres, C. Ortiz and R. Fernandez-Lafuente,Process Biochem., 2012, 47, 1220–1227.
60 O. Barbosa, C. Ortiz, R. Torres and R. Fernandez-Lafuente,J. Mol. Catal. B: Enzym., 2011, 71, 124–132.
61 R. Fernandez-Lafuente, C. M. Rosell and J. M. Guisan, EnzymeMicrob. Technol., 1998, 23, 305–310.
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at I
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napo
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n 14
Oct
ober
201
2Pu
blis
hed
on 1
1 O
ctob
er 2
012
on h
ttp://
pubs
.rsc
.org
| do
i:10.
1039
/C2C
S352
31A
View Online