Chem Soc Rev Dynamic Article Links Citethis: DOI: …ciencias.uis.edu.co/~rtorres/wp-content/uploads/2014/04/... · 2014-04-11 · obtained his Degree in Chemistry at the University

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.

Chem Soc Rev Dynamic Article Links

www.rsc.org/csr TUTORIAL REVIEW

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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.

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