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Enhancing the functional properties of thermophilic enzymes by chemical

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 Enzyme and Microbial Technology 49 (2011) 326–346

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

journal homepage: www.elsevier.com/locate/emt

Review

Enhancing the functional properties of thermophilic enzymes by chemical

modification and immobilization
Don A. Cowan a,∗ , Roberto Fernandez-Lafuente b,∗∗
a
Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape, Bellville 7535, Cape Town, South Africa
b
Departamento de Biocatálisis, Instituto de Catálisis-CSIC, C/Marie Curie 2, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain

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

Article history: The immobilization of proteins (mostly typically enzymes) onto solid supports is mature technology and
Received 26 April 2011 has been used successfully to enhance biocatalytic processes in a wide range of industrial applications.
Received in revised form 28 June 2011 However, continued developments in immobilization technology have led to more sophisticated and
Accepted 29 June 2011
specialized applications of the process. A combination of targeted chemistries, for both the support and
the protein, sometimes in combination with additional chemical and/or genetic engineering, has led to
Keywords:
the development of methods for the modification of protein functional properties, for enhancing protein
Thermophilic enzymes
stability and for the recovery of specific proteins from complex mixtures. In particular, the development of
Enzyme immobilization
Enzyme stabilization
effective methods for immobilizing large multi-subunit proteins with multiple covalent linkages (multi-
Chemical modification of enzymes point immobilization) has been effective in stabilizing proteins where subunit dissociation is the initial
Modulation of enzyme properties step in enzyme inactivation. In some instances, multiple benefits are achievable in a single process.
Here we comprehensively review the literature pertaining to immobilization and chemical modifica-
tion of different enzyme classes from thermophiles, with emphasis on the chemistries involved and their
implications for modification of the enzyme functional properties. We also highlight the potential for
synergies in the combined use of immobilization and other chemical modifications.
© 2011 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
1.1. Enzymes as industrial biocatalysts: ways to improve their properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
1.2. Enzymes from thermophilic microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
1.3. Chemical modification of enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
1.4. Immobilization of enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
2. Enhancing the properties of thermophilic enzymes by chemical modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332
2.1. Chemical crosslinking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332
2.2. Chemical modification of the enzyme surface without introducing crosslinkings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
3. Improvement of the thermophilic enzymes properties via immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
3.1. Use of immobilized enzymes from thermophiles as industrial biocatalysts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
3.1.1. Immobilization of thermophilic proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
3.1.2. Immobilization of thermophilic amylases and xylanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
3.1.3. Immobilization of thermophilic sugar isomerases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
3.1.4. Immobilization of thermophilic redox enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
3.1.5. Immobilization of thermophilic glycosidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336
3.1.6. Immobilization of thermophilic esterases and lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
3.1.7. Immobilization of other thermophilic enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
3.1.8. Non conventional uses of immobilized thermophilic proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
3.2. Use of immobilized thermophilic enzymes as biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339

∗ Corresponding author.
∗∗ Corresponding author. Tel.: +34 91 5854941; fax: +34 91 585 47 60.
E-mail addresses: dcowan@uwc.ac.za (D.A. Cowan), rfl@icp.csic.es, rfernandezlafuente@hotmail.com (R. Fernandez-Lafuente).

0141-0229/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2011.06.023
 D.A. Cowan, R. Fernandez-Lafuente / Enzyme and Microbial Technology 49 (2011) 326–346 327

4. Simultaneous use chemical modification and immobilization to improve the properties of enzymes from thermophiles . . . . . . . . . . . . . . . . . . . . . . . 340
4.1. Chemical modification of immobilized enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
4.2. Chemical modification of the enzyme to improve immobilization efficiency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
5. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

1. Introduction 1.2. Enzymes from thermophilic microorganisms

1.1. Enzymes as industrial biocatalysts: ways to improve their The term ‘thermophile’ is used in a generic sense for organ-
properties isms capable of growing optimally at elevated temperatures (i.e.,
above 37 ◦ C) [5,6]. This group is rather arbitrarily sub-divided on the
Enzymes catalyze complex chemical processes, mostly under basis of cardinal growth temperatures into ‘moderate’ thermophiles
mild conditions. The exploitation of enzymes in industrial, (Topt ∼ 60 ◦ C), extreme thermophiles (Topt ∼ 75 ◦ C) and hyperther-
commercial and laboratory processes is the result of their mophiles (Topt ∼ 85 ◦ C) [63]. Habitats suitable for the survival and
high activity in aqueous media and high substrate specificity growth of thermophiles are widespread. Moderate thermophiles
and product selectivity [1–4]. However, some enzymes have are commonly found all warm habitats, including hot desert soils,
been selected through natural evolution to perform their phys- natural hydrothermal waters, industrial wastewaters and domestic
iological functions under so-called extreme conditions, and water sources [63]. The distribution of extreme- and hyper-
operate optimally at temperatures, salinities, pHs, and in sol- thermophiles is more restricted, largely because their favored
vent environments which fall well outside ‘normal’ physiological habitats, terrestrial and deep-sea hydrothermal systems, are very
ranges [5,6]. localized [64].
To be successful in industry, biocatalysts need to be stable It is now widely accepted that the enzymes from these organ-
and functional under conditions that may be quite far from their isms are, in general, adapted to operate optimally at or near the
physiological environments [7–9]. Most typically, natural enzymes optimum growth temperature for the organism, be it 60 ◦ C for
do not fulfill all the requirements of an industrial biocatalyst a moderate thermophile or 95 ◦ C for a hyperthermophile. Typi-
and require improvement of one or more functional characteris- cally, intracellular enzymes follow this ‘rule’ more precisely, while
tics before implementation [10–12]. Such improvements can be extracellular enzymes may be stable to temperatures considerably
generated using different tools. For example, the development higher than the optimum growth temperature [63].
of metagenomic gene discovery has made enzymes from ther- The stability of all enzymes as an ordered polymer may be
mopilic environments readily accessible, even when the parent characterized as a function of their Tm (melting temperature) –
microorganisms are not cultivatable [13,14]. The rapid cloning the temperature at which the protein undergoes reversible (or
and over-expression of target enzyme genes in suitable hosts is irreversible) unfolding [65,66]. Such gross conformational changes
now a routine and rapid process [15,16]. Site-directed mutagene- may be visualized using spectral methods such as Circular Dichro-
sis and directed evolution methods [17–20] have made it possible ism or intrinsic fluorescence monitoring [67]. In practice, however,
to manipulate virtually any enzyme property. Advances have also protein stability is most commonly determined by loss-of-function,
been made in the design of new immobilization supports, in the most readily assessed in enzymes by loss of catalytic activity and
development of more efficient immobilization protocols and better leading to half-life and temperature-inactivation (Tinact ) data [Tinact
bioreactors [21–29]. is a rate constant of enzyme inactivation under a defined set of con-
Traditionally, the different (i.e., chemical versus genetic) tools ditions]. The correlation between Tm and Tinact is not particularly
to improve enzyme performance have been used as almost precise, as the conformational changes resulting in inactivation of
independent strategies. However, the joint use of different strate- catalytic function may be much smaller than those involved in full
gies has shown a synergistic effect [30] where, for example, protein unfolding.
the properties of genetically modified enzymes may be further One of the most active sectors of research in the field of ther-
improved via immobilization [31–34] or chemical modification mophiles has been in developing an understanding of the molecular
[35–37], while chemically modified enzymes may be further mechanisms of protein stability. This field has been very com-
stabilized via immobilization [38,39]. There are many other prehensively reviewed [65,66,68–72]. A consensus view, derived
examples of coupled use of different tools to improve enzyme from numerous studies including comparative analysis of struc-
performance [40–57]. tural analogues from different thermal groups, site directed and
The use of enzymes from thermophilic microorganisms (i.e., random mutagenesis and numerous other approaches, is that
organisms growing optimally in the range of 60–100 ◦ C) has been multiple subtle molecular mechanisms are used to establish the
frequently proposed as a solution to the problem of enzyme sta- requisite stability of a protein [73]. Most of these mechanisms
bility. While the intracellular enzymes from other extremophiles are intramolecular and not necessarily obvious or predictable [74].
(acidophiles, alkalophiles, etc.) may not be exposed to the extreme Nevertheless, most of these subtle molecular mechanisms function
elements of the external environment [58–60], thermophiles are by increasing the complement of non-covalent intracellular cross-
at complete thermal equilibrium with their microenvironments linking, thereby reducing conformational mobility. It is generally
and it is therefore an evolutionary imperative that their enzymes assumed that, for enzymes, a reduction of conformational mobility
are sufficiently stable at the ambient temperature to support the is associated with a reduction in activity [75,76], although protein
physiological processes of the organisms [6,61,62]. evolution studies have demonstrated that these two processes may
However, even using these stable enzymes, there are room for be decoupled [77,78].
some improvements in their properties. In this review, we analyze With a few high profile exceptions (specifically, the DNA
the coordinated use of naturally stabilized enzymes with physic- polymerases), thermophilic enzymes have not revolutionized the
chemical tools to improve enzyme properties. Table 1 reviews the world of biotechnology. The reasons include the following: not
enzymes and applied protocols. all enzyme technology processes require a thermostable biocat-
328 D.A. Cowan, R. Fernandez-Lafuente / Enzyme and Microbial Technology 49 (2011) 326–346

Table 1
Thermophilic enzymes described in this review, together to the physic-chemical modification performed.

Enzyme Source Treatment

Alcohol dehydrogenase Thermoanaerobacter brockii Physical adsorption

Alcohol dehydrogenase Thermus thermophilus HB27 Covalent immobilization
Physical adsorption
Alcohol oxidoreductase Sulfolobus solfataricus Covalent immobilization
Amidase Bacillus pallidus RAPc Covalent immobilization
Ionic exchange
Entrapment
Aminoacylase Thermococcus litoralis Entrapment
Physical adsorption
␣-amylase Bacillus licheniformis Chemical modification (no
crosslinking)
Covalent immobilization
l-arabinose isomerase Thermoanaerobacter mathranii CLEAs
Aspartate transcarbamoylase Aquifex aeolicus Chemical crosslinking
ATP synthase Bacillus stearothermophilus Liposome encapsulation
BPL:BCCP complex Aquifex aeolicus Chemical crosslinking
Caldolysin Thermus aquaticus T351 Chemical modification (no
crosslinking)
Covalent immobilization
Physical adsorption
Catechol 2,3-dioxygenase Bacillus stearothermophilus Covalent immobilization
Catalase Thermus thermophilus Covalent immobilization plus
crosslinking
CLEAs
Cytochrome P450 Sulfolobus tokodaii 7 Chemical modification (no crosslinking)
Cyclodextrin glycosyltransferase Thermoanaerobacter Chemical modification (no crosslinking)
Covalent immobilization
Dehalogenase Sulfolobus tokodaii Physical adsorption
Diaphorase Bacillus stearothermophilus Physical adsorption
Diguanylate cyclase Thermotoga maritime Entrapment
DNA-binding protein Methanopyrus kandleri Chemical crosslinking
DNA ligase Thermus scotoductus Chemical modification (no crosslinking)
Esterase Bacillus stearothermophilus Covalent immobilization
Ferredoxins Synechococcus vulcanus Covalent immobilization
␣-galactosidase Thermus sp. strain T2 Covalent immobilization Covalent immobilization plus crosslinking Physical
adsorption
␤-glucosidase Bacillus stearothermophilus Entrapment
␤-galactosidase Thermophilic anaerobe NA10 Covalent immobilization
␤-galactosidase Talaromyces thermophilus Covalent immobilization CBS 236.58
␤-galactosidase Sulfolobus solfataricus Covalent immobilization
␤-galactosidase Pyrococcus furiosus Covalent immobilization
␤-galactosidase Thermus sp. strain T2 Covalent immobilization Covalent immobilization plus crosslinking Physical
adsorption
Glucokinase Bacillus stearothermophilus Entrapment
Glucose dehydrogenase Thermophilic bacterium SM4 Chemical crosslinking
␣-Glucosidase Thermococcus AN1 Chemical modification (no crosslinking) Chemical crosslinking
␤-glucosidase Pyrococcus furiosus Covalent immobilization
Glutamate dehydrogenase Thermus thermophilus Chemical crosslinking
Covalent immobilization
Covalent immobilization plus
crosslinking
Gutamate dehydrogenase Bacillus stearothermophilus Entrapment
Hydrogenase Pyrococcus furiosus Physical adsorption
d-hydantoinase Bacillus stearothermophilus Chemical crosslinking
Physical adsorption
Laccase Bacillus sp. Covalent immobilization
(+)-␥-lactamase Sulfolobus solfataricus MT4 CLEA
l-lactate dehydrogenase Thermotoga maritima Chemical crosslinking
l-lactate dehydrogenase Thermus caldophilus GK24 Chemical modification (no crosslinking)
l-lactate dehydrogenases Clostridium “bilayer encagement”
Thermohydrosulfuricum
l-lactate dehydrogenase Bacillus stearothermophilus Ionic exchange
Lipase Thermomyces lanuginosus Chemical modification
Immobilization (covalent or
physical adsorption)
Combined
immobilization/chemical
modification
Combined chemical
modification/immobilization
Lipase Bacillus thermoleovorans Physical adsorption
Lipase Bacillus sp. Physical adsorption
Lipase Bacillus stearothermophilus Ionic exchange
MC 7
 D.A. Cowan, R. Fernandez-Lafuente / Enzyme and Microbial Technology 49 (2011) 326–346 329

Table 1 (Continued)

Enzyme Source Treatment

Lipase Bacillus thermocatenulatus Covalent immobilization

Ionic exchange
Physical adsorption
Modification plus covalent
immobilization
Lipase Rhizopus oryzae Physical adsorption
Lipase Thermus thermophilus Physical adsorption
Lipase Thermus aquaticus Physical adsorption
l-lysine 6-dehydrogenase Bacillus stearothermophilus Physical adsorption
Photosystem II Synechococcus bigranulatus Physical adsorption
Photosystem II Synechococcus elongates Entrapment
Proteinase Thermus Rt41A Covalent immobilization
Quinol oxidase Bacillus PS3 Chemical crosslinking
Pyrophosphatase Thermus thermophilus Affinity immobilization
Thermitase Thermoactinomyces vulgaris Covalent immobilization
Trehalose synthase Thermus thermophilus HJ6 Covalent immobilization
Xylanase Thermotoga FjSS3-B.1 Covalent immobilization
Xylanase Thermomyces lanuginosus SSBP Covalent immobilization
Entrapment
Ionic exchange
Xylanase Talaromyces thermophilus Covalent immobilization
d-xylose isomerase Thermus aquaticus Covalent immobilization
CLEA
d-xylose isomerase Thermotoga maritima Covalent immobilization
d-xylose isomerase Thermotoga neapolitana 5068 Covalent immobilization

alyst; a thermostable homologue may not be readily available;
existing thermostable homologues may lack other suitable prop-
erties (such as selectivity and turnover) or existing or new
thermophilic/thermostable enzymes may not fulfill commercial
requirements (production volume/cost).
1.3. Chemical modification of enzymes
Although genetic modification has generally displaced the
chemical modification of enzymes, the latter remains an effective
method for stabilizing proteins [79–81] (Fig. 1). Chemical mod-
ification will generally not be site-directed, except when using
specific chemistries directed to unique groups in the protein struc-
ture (e.g., Diels-Alder cycloaddition [82], thiol exchange, reaction
with terminal amino groups, etc.). For example, site-directed chem-
ical modification has been achieved using thiol reactive compounds
and proteins containing only a single surface Cys (natural or intro-
duced via site-directed mutagenesis) [49,50,83] (Fig. 2). Despite the
enormous power of genetic manipulation, chemical modification
has some distinct advantages [30,84]. For example, chemical modi-
fication allows a wider range of chemical groups to be introduced to
the enzyme structure [81] (Fig. 2), does not require foreknowledge
of the protein structure, is rapid (compared with genetic modifica-
tion) and is performed on the correctly folded enzyme.
If the modification is performed in solid phase (using immo-
bilized enzymes), there are added advantages [30]. There is a
greater control of the chemical modification, minimizing undersir-
able protein–protein interactions (Fig. 3). The use of an immobilized
enzyme facilitates the use of a non-compatible medium (e.g., an
anhydrous organic medium) and may simplify the step by step
chemical modifications of enzymes [30,43,85]. The use of enzymes
stabilized via multipoint or multi-subunit immobilization also lim-
its the extent of enzyme inactivation that may result from chemical
modification processes [30,85].
Chemical modification of an enzyme may be used to change
the overall properties of the enzyme surface or for the modifica-
tion of key residues [30,80,81]. For example, the modification of
seventeen Lys residues of B. amyloliquefaciens ␣-amylase with cit-
raconic anhydride significantly stabilized the enzyme at very high
temperatures (an effect not found at lower temperatures) [86]. The
modification of enzymes with polyethylenglycol or detergents is
known to improve their solubility and stability in organic media
[87]. Alternatively, chemical modification may be used to modu-
late catalytic properties [88,89]. However, the effects of chemical
modification on enzyme properties are generally difficult to predict
[30,90].
Chemical modification is also used to generate covalent
crosslinking bonds between different groups on the enzyme sur-
face [30,91]. Where such crosslinking occurs between different
structural elements of a protein, it will typically enhance struc-
tural rigidity and therefore increase protein stability with respect
to agents that induce conformational changes (such as chaotropic
agents or heat). Intramolecular crosslinking is not necessarily
straightforward; the crosslinking agent must be of an appropri-
ate length [92], and when using homo-bifunctional reagents there
may be strong competition between intramolecular crosslinking,
single-point modification and inter-molecular cross-linking (Fig. 4)
[30]. The use of multifunctional polymers simplifies the genera-
tion of multiple crosslinking bonds and is less limited by distance
constraints or competition for single-point-modification (Fig. 5).
However, the flexible structures and length of the polymers limits
the degree of protein rigidification that can be obtained [93]. Con-
versely, these polymeric multifunctional reagents have effectively
prevented dissociation of multimeric proteins or multi-protein
complexes [30,43,44] (Fig. 6).
1.4. Immobilization of enzymes
The immobilization of enzymes is used to facilitate the control
of bioreactors and, where the enzyme is sufficiently stable, the
recovery/re-use of the biocatalyst [94–98]. Interestingly, immo-
bilization has also been used to concurrently enhance enzyme
properties [99,100].
Where the enzyme is immobilized within a porous solid (such
as a protein aggregate or crystal, an inert porous support) some
sources of enzyme inactivation, such as aggregation, adsorption
onto hydrophobic surface and auto-proteolysis [100,101] (Fig. 7)
are minimized. However, this class of support may also induce dif-
fusion problems (substrate, pH gradients, etc.) [96,97]. To take full
advantage of enzyme immobilization, multipoint or multisubunit
immobilization is necessary (Fig. 8) [100].
330 D.A. Cowan, R. Fernandez-Lafuente / Enzyme and Microbial Technology 49 (2011) 326–346

Fig. 1. Chemical modification versus genetic modification.

Reversible (non-covalent) immobilization protocols are appro-
priate where enzyme stabilization is not required, and the support
can be reused after enzyme inactivation [102]. However, even
non-covalent adsorption can result in stabilization of multimeric
enzymes if all enzyme subunits are involved in the immobilization
[103]. Typically, where structural rigidification (and conformation
stabilization) is a requisite, multipoint covalent attachment is the
preferred immobilization strategy [100]. Where the matrix is rigid
and the spacer arms are short, all groups involved in the enzyme
immobilization process should maintain their relative positions
under conditions which would otherwise result in conformational
change [99,100].

Fig. 2. Site directed modification of proteins.

 D.A. Cowan, R. Fernandez-Lafuente / Enzyme and Microbial Technology 49 (2011) 326–346 331

Fig. 3. Advantages of chemical modification of proteins on solid phases.

The generation of high density multipoint covalent linkages is
not necessarily straightforward. For optimum results, the immo-
bilization support should offer a good geometrical ‘congruence’
with the enzyme [104,105]. For example, internal cylindrical pores
are more appropriate than extended widely spaced thin poly-
mer chains. Reactive groups should demonstrate good stability
under reaction conditions, have low steric hindrance, and should
preferably be reactive with amino acid side chain moieties fre-
quently located in the enzyme surface (e.g., the ␧-amino group of
Lysine). Glyoxyl [106], epoxy [107] and in some cases, glutaralde-
hyde [108,109] activated supports have all been successfully used
for this purpose. Multipoint covalent attachment typically requires
long reaction times, moderately high temperatures and alkaline pH
values [110].
Alternative immobilization strategies, such as crosslinked
enzyme aggregates (CLEAs) or crystals (CLECs), have also been

Fig. 4. Chemical crosslinking using bi-functional reagents.

332 D.A. Cowan, R. Fernandez-Lafuente / Enzyme and Microbial Technology 49 (2011) 326–346

Fig. 5. Chemical crosslinking using poly-functional reagents.

used to enhance enzyme properties [94,95]. CLECs are prepared
by covalent crosslinking of crystallized (i.e., homogeneous) pro-
teins, generating mechanically rigid biocatalyst particles which are
applicable to column reactors [111]. CLEAs are obtained by precip-
itation of the enzyme followed by crosslinking and do not require
pure enzymes. The co-aggregation of several enzymes, or enzymes
and polymers, is possible [112]. However, the resulting biocat-
alyst does not possess high mechanical resistance. While both
immobilization strategies have a common feature in not requiring
independent support elements, both may enhance enzyme stabil-
ity by reducing enzyme mobility or preventing subunit dissociation
[113]. However, only CLEAs allow engineering of the microen-
vironment (e.g., to reduce solvent interactions or reduce oxygen
dissolution) through the co-aggregation of enzymes and polymers
[40,114].
The benefits of immobilization are not restricted to the
enhancement of enzyme stability. The deformation and reduced
conformational mobility of protein structures induced by immo-
bilization can greatly alter (and in certain cases improve) enzyme
specificity and enantioselectivity, and has been shown to reduce
substrate inhibition [100].
2. Enhancing the properties of thermophilic enzymes by
chemical modification
2.1. Chemical crosslinking
Chemical crosslinking of the subunits of multimeric enzymes
is commonly used to prevent dissociation [43] (Fig. 6). Chemical
crosslinking of thermophilic enzymes has been used to confirm

Fig. 6. Subunit crosslinking with poly-functional reagents.

 D.A. Cowan, R. Fernandez-Lafuente / Enzyme and Microbial Technology 49 (2011) 326–346 333

Fig. 7. Stabilization by immobilization on a porous support.

quaternary structure, for example in L(+)-lactate dehydrogenase
from Thermotoga maritima aspartate transcarbamoylase, from
Aquifex aeolicus and DNA-binding protein from the hyperther-
mophilic methanogen Methanopyrus kandleri [115–117] and to
stabilize protein complexes (e.g., BPL:BCCP complex. from Aquifex
aeolicus) [118]. Using intra- and inter-complex crosslinking with
disuccinimidyl tartrate, 3,3 -dithiobis(succinimidylpropionate),
and ethylene glycolbis(sulfosuccinimidylsuccinate), Tanaka and
coworkers [119] showed that the quinol oxidase super complex
from thermophilic Bacillus PS3 was formed by quinol-cytochrome
c reductase (3 subunits) and cytochrome c oxidase (4 subunits).
The use of chemical crosslinking to further increase the stability
of thermophilic enzymes is the most common objective. For exam-
ple, a hetero-oligomeric glucose dehydrogenase from a moderately
thermophilic bacterium, SM4, was crosslinked with glutaraldehyde
[120]. The crosslinked enzyme showed a single optimum tem-

One-point support-enzyme attachment

No enzyme rigidification
Prevents some causes of inactivation
The presence of the surface may
alter some enzyme properties

Muti-point support-enzyme attachment

Enzyme rigidification, the support acts as a

rigid multifunctional crosslinker

The rigidification will depend on the number of

attachments, the area of the protein involved,
the inertness of the support and the length
of the spacer arm.

Fig. 8. Stabilization by multisubunit or multipoint covalent attachment.

334 D.A. Cowan, R. Fernandez-Lafuente / Enzyme and Microbial Technology 49 (2011) 326–346
perature for activity at around 65 ◦ C, whereas the native enzyme
exhibited dual optima at 45 ◦ C and 75 ◦ C.
␣-Glucosidase from Thermococcus strain AN1 was crosslinked
using glutaraldehyde, both in homogeneous preparation and with
bovine serum albumin [121]. While homogeneous cross-linking did
not offer significant stabilization under standard conditions, the
co-cross-linking strategy increased the activity half life from 5 min
to 8 min at 110 ◦ C. The enzyme was substantially stabilized in the
presence of 50% trehalose, an effect that was further enhanced for
the co-crosslinked enzyme (t1/2 of 90 min at 110 ◦ C compared with
30 min for the unmodified enzyme) [121].
The coating of the surfaces of multimeric proteins with a poly-
ionic polymer (e.g., polyethyleneimine) prevented the subunit
dissociation of oligomeric glutamate dehydrogenase from Thermus
thermophilus [122]. However, the protective effect was susceptible
to the reversible nature of the polymer adsorption. This prob-
lem was solved by crosslinking with glutaraldehyde, generating a
enzyme-polymer composite that was fully stable under conditions
favoring enzyme dissociation [122].
Intersubunit cross-linking of the tetrameric d-hydantoinase
from Bacillus stearothermophilus SD1 was achieved by using 1-
ethyl-3-(3-di-methylaminopropyl)carbodiimide [123]. The cross-
linked d-hydantoinase showed a fourfold stabilization under
operational conditions (c.f., the wild type enzyme) and was sta-
ble at temperatures where the unmodified enzyme was almost
fully inactivated. The cross-linked enzyme was more stable than
the wild-type in the presence of 20% methanol and at pH values
that favored subunit dissociation [123].
2.2. Chemical modification of the enzyme surface without
introducing crosslinkings
In vivo post translational modification of thermophilic enzymes
has been linked to thermostabilization. For example, in vivo lysine
methylation of thermophilic enzymes, catalyzed by lysine methyl-
transferase, has been proposed to play an important role in the
stability of the enzyme. Nineteen Lys groups of the RNA poly-
merase from the crenarchaeon Sulfolobus solfataricus were found
to be methylated [124] and 6 Lys groups were modified in the
glutamate dehydrogenase from the same organism [125]. The
post-translational modification of N-terminal cysteine residue of
cytochrome c-551 with diacyl-glycerol has been also been shown
to be implicated in enzyme stabilization [126]. Chemical modifi-
cation of proteins has been also used to determine the catalytic
residues of many thermophilic enzymes [127–129]. The lipase from
the thermophilic Thermomyces lanuginosus has been also modified
with different goals (e.g., change of substrate specificity or deter-
mination of target groups) [130–132].
In vitro chemical modification of thermophilic enzymes has
also been employed to enhance enzyme stability. Chemical mod-
ification using citraconic anhydride of approx. 10 lysine residues
in a thermophilic ␣-amylase from B. licheniformis was performed
[86]. This modification replaces a high pKa amino group with an
acidic group, greatly altering the net charge of the enzyme sur-
face. Although stability was slightly reduced at 80 ◦ C (G# of
protein unfolding was reduced by 3.6 kJ mole−1 ), the Lys modifica-
tion resulted in a 15-fold enhancement of catalytic activity at 25 ◦ C.
Stability was improved by addition of Ca2+ ions, possibly by estab-
lishing Ca2+ -dependent ionic bridges between proximal negatively
charged groups [133].
Cytochrome P450 from the thermoacidophilic crenarchaeon
Sulfolobus tokodaii strain 7 was modified with poly(ethylene oxide)
(PEO) [134]. The authors stated that the electrochemical responses
of PEO-modified cytochrome in PEO solvent were well-defined and
stable compared with those of wild-type enzyme: after 300 uses,
the peak current for wild-type P450st decreased to around 5%,
whereas the peak for PEO2000 –P450st was above 70% of the initial
response.
In the chemically adenylation of NAD+ -dependent DNA ligase
from Thermus scotoductus [135,136], the covalent ‘anchoring’ of
the cofactor induced a conformational rearrangement within the
active site of the enzyme, accompanied by partial compaction of
the protein structure. This chemical modification increased enzyme
resistance to thermal- and guanidine-induced denaturation, estab-
lishing a thermodynamic link between cofactor binding and the
enhancement of conformational stability.
The amidation of cyclodextrin glycosyltransferases (CDGs) from
Thermoanaerobacter by acetic anhydride has been reported to
affect the reaction selectivity of the enzyme [137]. This amidation
removes the positive charge of the amino groups and was per-
formed under conditions where most of the 24 free amino groups
of the protein were modified. These enzymes catalyze four differ-
ent reactions: cyclization, coupling (opening of the cyclodextrin
rings and transfer to acceptors), disproportionation (transfer of lin-
ear malto-oligosaccharides to acceptors) and hydrolysis of starch.
The acetylated CDG showed a significant reduction of its cycliza-
tion, coupling and disproportionation activities, while exhibiting
an improved saccharifying activity [137].
␣-Glucosidase from Thermococcus strain AN1 was modified
with polyethylenglycol and aminoglucose [121]. While the former
modification had little effect on enzyme stability, the aminoglu-
cose modification afforded a 4-fold improvement in enzyme
activity half life at 110 ◦ C. In the presence of stabilizing addi-
tives (sorbitol, ammonium sulfate), the chemical modifications
showed a synergistic effect. In the presence of 90% sorbitol, the
aminoglucose-modified enzyme exhibited an activity half life of
5 min, compared to less than 2 min for the native enzyme.
Caldolysin, an extracellular protease from Thermus aquaticus
strain T351, was modified with S-methylisothiourea (the degree of
modification was not quantified) to generate a stabilized enzyme
[138]. It was proposed that the modified side-chains guanidinium
groups might be expected to enhance intramolecular hydrogen
bonding.
l-lactate dehydrogenase from Thermus caldophilus GK24 is
not only extremely thermostable but also exhibits fructose 1,6-
bisphosphate (FBP)-dependent allostery [139]. Modification of 6 or
7 of the 23 arginines per enzyme subunit by using 2,3-butanedione
in the presence of borate generated an enzyme adduct that was
desensitized with respect to fructose 1,6-bisphosphate [139]. The
authors suggested that the enzyme might possess two classes of
fructose-1,6-bisphosphate binding sites, one being an activating
site with a high binding constant for fructose 1,6-bisphosphate
and containing a highly reactive arginine residue, while the other
would be an inhibitory site with a low binding constant for
fructose-1,6-bisphosphate, possibly located in the active centre
(and responsible for competitive inhibition). They suggested that
enzyme would be inactive when the arginine residue in the
fructose-l,6-bisphosphate-binding site was free, and would be acti-
vated when the residue interacted with fructose 1,6-bisphosphate
or was chemically modified [139].
3. Improvement of the thermophilic enzymes properties
via immobilization
3.1. Use of immobilized enzymes from thermophiles as industrial
biocatalysts
3.1.1. Immobilization of thermophilic proteases
Proteases are among the most industrially important enzymes,
for both hydrolysis of proteins [140] and for the production of pep-
tides [141], and numerous examples of immobilized thermophilic
 D.A. Cowan, R. Fernandez-Lafuente / Enzyme and Microbial Technology 49 (2011) 326–346 335

proteases have been reported [142]. Caldolysin, a metal-chelator- from different sources and the xylanase of T. thermophilus doubled
sensitive extracellular protease from Thermus aquaticus strain T351 the efficiency of xylan saccharification, demonstrating the syner-
is the first reported example of immobilization of a thermophilic gistic action of the two enzymes [153].
protease. The enzyme was immobilized on Sepharose 4B, CM-
cellulose and controlled pore glass (CPG), with good immobilization
3.1.3. Immobilization of thermophilic sugar isomerases
yields but moderate decreases in specific activity, depending on
The use of elevated temperatures is of interest in the isomeri-
the support used [143]. Further study of the enzyme immobilized
sation of sugars due to the increased solubility of the substrates,
on bromocyanogen-activated Sepharose 4B showed that the sub-
improved fructose yields and process compatibility with upstream
strate inhibition observed for the free enzyme disappeared [138].
enzyme digestion unit operations [154]. In the free state, d-xylose
The proposed mechanism involved steric exclusion of substrate
isomerase from Thermus aquaticus was inactivated with a half-
from an “inhibition site” without significant interference with the
life of 4 d at 70 ◦ C in the absence of divalent cations [154]. In the
active site, perhaps the first time that immobilization was shown
presence of cations, full activity was retained for at least a month
to prevent inhibition [100].
at the same temperature. Attachment to epoxy-activated agarose
Subsequently, thermitase (a thermostable protease derived
and co-aggregation with bovine serum albumin gave immobilized
from Thermoactinomyces vulgaris) was covalently immobilized in
cation-free enzyme preparations with the same stability as the
polyacrylamide gels [144]. The number of covalent bonds between
free enzyme supplemented with Mn2+ or Co2+ . These results sug-
the enzyme and gel matrix was estimated to be 4 per protein, and
gest that subunit dissociation [43], which may be prevented by
the effects observed were related to this multipoint attachment
immobilization or by cation supplementation, was the probable
[100]. While the immobilized preparation retained only 20% of the
mechanism for the observed inactivation.
native enzyme specific activity, the optimal temperature was sub-
Xylose (glucose) isomerases from the hyperthermophiles Ther-
stantially higher than that of the free enzyme. At 68 ◦ C, immobilized
motoga maritima and T. neapolitana 5068 have been used in the free
thermitase was over 10 times more active than the native enzyme
and immobilized forms to produce high fructose syrups [155]. At
at that temperature.
pH 7.0 and in the presence of Mg2+ , the temperature optima for the
An extracellular proteinase from Thermus strain Rt41A was
soluble enzymes were 95 ◦ C to 100 ◦ C for the T. neapolitana enzyme
immobilized on controlled pore glass beads [145]. Immobilization
and above 100 ◦ C for the enzyme from T. maritima. Under certain
resulted in a large reduction in pH optimum for the enzyme on both
inactivation conditions, soluble forms of the enzymes exhibited
protein and peptide substrates but increased stability at 80 ◦ C over
multiphasic inactivation profiles in which the decay rate slowed
a pH range from 5 to 11. Stabilization was principally attributed to
considerably after a rapid initial decline. However, after covalent
prevention of autolysis (Fig. 7). The immobilized enzyme has also
immobilization to glass beads the inactivation of the enzymes was
been used for dipeptide synthesis [146].
characterized by a monophasic first-order decay rate intermedi-
ate between the initial rapid and the slower phase for the soluble
3.1.2. Immobilization of thermophilic amylases and xylanases
enzymes. In the presence of both Co2+ and Mg2+ , the immobilized
The hydrolysis of sugar polymers is enhanced at high tem-
enzyme preparations showed the ability to catalyze glucose-to-
peratures, which increase substrate solubility and limit microbial
fructose conversions at 80 ◦ C with estimated lifetime productivities
contamination [147,148]. This has been particularly evident in
in the order of 2000 kg fructose per kilogram enzyme, a value com-
the widespread application of Termamyl® , the thermostable ␣-
parative with enzymes currently used at 55–65 ◦ C, but with the
amylase from Bacillus licheniformis, in the industrial saccarification
additional advantage of higher fructose product concentrations.
of starch [149]. Although not required for bulk industrial applica-
At 90 ◦ C, the estimated lifetime productivity for the enzyme from
tions, the B. licheniformis ␣-amylase has been further stabilized by
Thermotoga neapolitana was 1000 kg kg−1 [155].
covalent immobilized to ␥-aminopropyl silica using glutaraldehyde
l-arabinose isomerase from Thermoanaerobacter mathranii
[150].
was immobilized by cross-linking with glutaraldehyde and
The immobilization of several thermophilic xylanases has been
polyethylenimine and used at 65 ◦ C for production of d-tagatose
reported. Xylanase from Thermotoga sp. strain FjSS3-B.1 was immo-
from d-galactose [156]. Using a 30% g/mL solution of d-galactose,
bilized to porous glass beads, making the half-life at 95 ◦ C similar
the yield of d-tagatose was 42% and no sugars other than d-tagatose
to that of the free enzyme at 90 ◦ C [151]. Interestingly, solutes such
and d-galactose were detected. Direct conversion of lactose to d-
as sorbitol and xylan increased the thermostability of the solu-
tagatose in a single reactor was demonstrated using a thermostable
ble enzyme at ultra-high temperatures (half-life at 130 ◦ C in 90%
␤-galactosidase together with the thermostable l-arabinose iso-
sorbitol, 60 min) but not the immobilized preparation (half-life,
merase. The two enzymes were also successfully combined with a
1.3 min).
commercially available glucose isomerase for conversion of lactose
Thermomyces lanuginosus SSBP xylanase has been immobilized
into a sweetening mixture comprising lactose, glucose, galactose,
by entrapment in alginate, by covalent immobilization on chitosan
fructose and tagatose [156].
and by ionic linkage to Eudragit S-100 [152]. The activity of the
non-covalently linked enzyme was greatest on Eudragit S-100 and
showed greater thermal stability than the free enzyme at 70 ◦ C, 3.1.4. Immobilization of thermophilic redox enzymes
although no change was observed at the activity pH optimum (6.5) There are many examples of immobilization of thermophilic
of the immobilized enzyme [152]. redox enzymes. Although the use of very high temperatures is not
Several immobilization protocols were applied to xylanase from favored due to the low thermal stability of the NAD(P) cofactors, the
Talaromyces thermophilus [153], the most effective of which one low functional stability of mesophilic oxidoreductases has stimu-
(with a yield of 98.8% and a 99.2% recovery of xylanase activity) was lated the search for more thermostable homologues [157].
the use of gelatin with glutaraldehyde as the crosslinking reagent. NAD+ -dependent alcohol aldehyde/ketone oxidoreductase from
The immobilized enzyme exhibited a shift in the optimal pH from the extremely thermoacidophilic archaeon Sulfolobus solfataricus
7 to 8, showed no change in the optimal temperature of activ- was covalently immobilized on Eupergit C (an epoxy support) [158].
ity and was stable over multiple successive cycles of hydrolysis at The immobilized enzyme was used in the stereospecific reduction
50 ◦ C. The immobilized preparation was used successfully for the of 3-methyl-butan-2-one on a gram scale with in situ regeneration
large-scale continuous production of xylobiose, using wheat bran of PEG-NADH in the presence of propan-2-ol. No data on enzyme
hemicellulose as substrate. Co-immobilization of the ␤-xylosidase stability or inactivation rates were reported.
336 D.A. Cowan, R. Fernandez-Lafuente / Enzyme and Microbial Technology 49 (2011) 326–346
The use of hydrogenases for the direct reduction of cofactors
with molecular hydrogen potentially provides an attractive method
for cofactor regeneration in reductive biotransformations [157].
Hydrogenase I from Pyrococcus furiosus was adsorbed to an elec-
trode by using cyclic voltammetry. In a further experiment, the
hydrogenase was adsorbed on graphite beads and utilized for the
continuous reduction of NADP+ to NADPH with molecular hydrogen
in a continuously operated fluidized bed reactor [159].
Alcohol dehydrogenase from Thermoanaerobacter brockii was
immobilized by hydrophobic adsorption on methyl-, octyl-, and
hexadecyl-Fractosil. Compared to the free enzyme, the thermal
stability of the immobilized enzyme was enhanced and both the
pH and temperature optimum values were altered [160]. Similarly,
the alcohol dehydrogenase from Thermus thermophilus HB27 was
covalently immobilized on various activated supports (activated
with aldehyde or epoxide groups), with stabilization factors (5–10-
fold) similar to those obtained by ionic adsorption of the enzyme
onto polyethyleneimine coated supports [161]. The authors recom-
mended the preferential use of ionic adsorption, on the basis that
adsorption was very strong but that the process was reversible, the
immobilization protocol was rapid with high activity yields, and
that the immobilized enzyme was more stable than the covalent
preparations in the presence of organic solvents (possibly because
of negative partitioning of the solvent from the enzyme environ-
ment). This preparation was used in the asymmetric reduction of
acetophenone to produce (S)-(−)-1-phenylethanol, with an enan-
tiomeric excess of more than 99% [161].
The glutamate dehydrogenase from Thermus thermophilus is a
homotrimer that catalyzed the reversible oxidation of glutamate
to ␣-ketoglutarate and ammonia with either NAD+ or NADP+ as
cofactors (or NAD(P)H for the reduction), and has been proposed
for the regeneration of both reduced and oxidized cofactors [162].
Due to its trimeric nature, low-pH inactivation is triggered via
dissociation of the enzyme subunits; in the absence of subunit dis-
sociation the enzyme is extremely stable, resulting in the selection
of this enzyme as a model system for studying the processes of sub-
unit dissociation in oligomeric enzymes. Immobilization on glyoxyl
agarose under standard conditions (pH 10 [106]) enhanced enzyme
stability even under conditions where dissociation was not the first
step of inactivation [162].
This enzyme was also used as a model to couple immobilization
and purification of multimeric enzymes from thermophile microor-
ganisms cloned in mesophilic hosts. One of these strategies was
by immobilization on glyoxyl supports at neutral pH value. First
immobilization on glyoxyl support requires the establishment of
several simultaneous enzyme support attachments [106]. At neu-
tral pH values, this is not possible unless the enzyme has several
terminal amino groups (pK 7–8) [163]. It should be considered
that multimeric thermophilic enzymes cloned in mesophilic hosts
are the only multimeric proteins that keep intact its quaternary
structure after thermal shock [164], thus specific immobilization
of these protein extracts should permit the purification of the
multimeric thermophilic enzyme. Glutamate recombinant Thermus
thermophilus glutamate dehydrogenase, expressed in E. coli, was
covalently immobilized on glyoxyl supports at pH 7, demonstrating
the effectiveness of a protocol capable of a single step purification,
immobilization and stabilization by prevention of subunit dissoci-
ation [165].
Immobilization via ion exchange is a multipoint process: it has
been demonstrated that low charge density ion exchangers can
only adsorb large proteins which can interact with widely spaced
ionic groups [166]. Using heterofunctional supports containing
epoxy groups (that do not immobilize proteins at a significant
rate) [107] and the lowest amount of amino groups necessary to
produce the enzyme ionic exchange on the support, recombinant
T. thermophilus glutamate dehydrogenase was used as a model
for the one step purification (via selective ionic exchange), and
immobilization–stabilization (via multipoint covalent attachment
on the epoxide groups) using this tailor-made support [167]. It was
found that the most stable enzyme preparations were those pre-
pared by immobilization on glyoxyl agarose at pH 7 followed by
incubation at pH 10 [168].
3.1.5. Immobilization of thermophilic glycosidases
3.1.5.1. Immobilization of thermophilic ˇ-glycosidases. The removal
of lactose from milk is a requirement to the generation of milk prod-
ucts for lactose-intolerant members of the population [169,170], to
prevent the precipitation of lactose during the production of some
dairy products (e.g., ice-cream), to accelerate fermentation and,
in dairy whey, improve its use as a fermentation medium. These
processes exploit that catalytic specificity of ␤-galactosidases. This
group of enzymes is also of potential use in the synthesis of
oligosaccharides [171].The use of moderately high temperatures in
the hydrolysis of lactose in milk is effective in reducing microbial
growth, and even offers the prospect of coupling the heat steril-
ization with lactose removal. As a result, the properties of both
free and immobilized thermophilic ␤-galactosidases have received
considerable attention [172].
A thermostable ␤-galactosidase (EC 3.2.1.23) from a ther-
mophilic anaerobe, strain NA10, was covalently immobilized
to a porous ceramic support, SM-10 [173]. Among the differ-
ent immobilization protocols tested, the highest residual activity
after 3 h incubation at 70 ◦ C was obtained when the enzyme
was covalently immobilized to silanized SM-10 with 3-[2-(2-
amino-ethylaminoethylamino)propyl]trimethoxysilane activated
with glutaraldehyde. The enzymatic properties were almost iden-
tical to those of the free enzyme. The half-life of the immobilized
enzyme was estimated to be approximately 450 h at pasteuriza-
tion temperature (65 ◦ C). This enzyme was also immobilized via
glutaraldehyde crosslinking [174] but showed lower functional sta-
bility. The authors suggested that the lower stability was induced
by the presence of residual free aldehyde groups on the support
surface.
␤-Galactosidase from the fungus Talaromyces thermophilus CBS
236.58 was immobilized by covalent attachment onto Eupergit C
with a binding efficiency of 95% [175]. Immobilization increased
both enzyme activity and stability at high pH values and elevated
temperatures when compared with the free enzyme. For exam-
ple, half-life values at 50 ◦ C were determined to be 8 h and 27 h
for the free and immobilized enzymes, respectively. It was evi-
dent from bioconversion experiments with 20% lactose as substrate
that the immobilized enzyme showed a strong transgalactosylation
activity, resulting in the formation of interesting galactooligosac-
charides (34% after optimization) [176].
A ␤-glycosidase from Sulfolobus solfataricus was immobilized on
chitosan activated with glutaraldehyde, with a yield of 80% [177].
Compared to the free ␤-glycosidase, the immobilized enzyme
showed a similar pH optimum (pH 7.0) and temperature-activity
profile up to 80 ◦ C. However, the immobilized enzyme was both
more thermostable and not subject to glucose inhibition [177].
The extremely thermostable ␤-glycosidase from Pyrococcus
furiosus immobilized on Eupergit C has been used for the produc-
tion of oligosaccharides with lactose as a substrate [178]. The high
stability of the enzyme made it possible to operate at high reac-
tion temperatures and to increase the substrate concentration. The
consequent reduction in water concentration decreased the rate of
hydrolysis in favor of transgalactosylation activity, with improved
oligosaccharide yields. It was noted, however, that the use of very
high temperatures promotes the formation of brown pigments via
the Maillard reaction, and that these pigments accelerated the rate
of inactivation of the enzyme. This problem was not resolved by
enzyme immobilization [178].
 D.A. Cowan, R. Fernandez-Lafuente / Enzyme and Microbial Technology 49 (2011) 326–346
A microfluidic reactor was developed for continuous flow bio-
catalytic transformations with immobilized hyperthermophilic
␤-glycoside hydrolase (CelB) from Pyrococcus furiosus at 80 ◦ C
[179]. The covalent protein attachment was performed via cross-
linking with glutaraldehyde onto amino-silanized microstructured
surfaces. While the enzyme KM was unaltered, the free energy of
activation for lactose hydrolysis was decreased from 72 kJ/mol in
the free enzyme to 38 kJ/mol in the immobilized enzyme. Cova-
lent attachment of the enzyme onto the micro-channel walls via
a dendrimeric linker restored the original Ea value. The immobi-
lized enzyme microreactor was used for continuous conversion of
100 mM lactose at 80 ◦ C and operated at a stable substrate con-
version of 60% for five days. In further studies with this enzyme
using microstructured flow reactors, immobilization was carried
out on ␥-aluminum oxide functionalized with aminopropyl tri-
ethoxysilane followed by activation with glutaraldehyde [180]. This
microreactor was employed for the continuous hydrolysis of lac-
tose (100 mM) at 80 ◦ C, providing a space-time yield of 500 mg
glucose/(mL h) with a stable conversion of ≥70%. The immobilized
enzyme displayed a half-life of 15 days under operational condi-
tions.
The lactase from Thermus sp. strain T2 is a tetrameric enzyme
that has been used in the development of new immobilization-
stabilization strategies [181]. The enzyme was immobilized on
various heterofunctional epoxy supports, with the epoxy-borane
support yielding preparations with the highest stability [182].
The optimal derivative was highly active on lactose substrates at
70 ◦ C (over 1000 IU/g), maintaining its activity after long incuba-
tion periods under these conditions. The enzyme was used for
reversible immobilization via ionic exchange on supports coated
with ionic polymers, such as polyethylenimine [183], aspartic-
dextran [184] and sulfate-dextran [185]. In all cases, strongly bound
but non-distorted immobilization products were obtained, suit-
able for application under a wide range of conditions without
significant risk of enzyme desorption. The large size of the Ther-
mus lactase allows adsorption via multiple linkages, making this
enzyme an excellent model for the development of strategies for
reversible immobilization, purification and prevention of subunit
dissociation under very stringent conditions. For example, highly
activated IMAC supports have been used to selectively immobi-
lize the enzyme in the presence of 50 mM imidazole [186], while
it has also been bound to highly activated anion exchangers in the
presence of 1 M NaCl [187].
The Thermus T2 ␤-galactosidase, in the form of a His-tagged
chimeric protein, was selectively immobilized and purified on
epoxy-iminodiacetic-Co2+ matrices [188]. This strategy is based
on the fact that epoxides cannot directly immobilize soluble
enzymes [107], but iminodiacetic-Co2+ will selectively adsorb
poly-His tagged proteins [189]. Very high enzyme loadings were
achieved, even using crude extracts. Given the co-operative effect
of physical and covalent protein attachment to heterofunctional
supports [190], it has recently been shown that the use of new
epoxy-glyoxyl-amino supports with low amino group densities
can achieve a concerted single step immobilization-purification-
stabilization of the large Thermus T2 ␤-galactosidase [191].
Immobilized preparations of Thermus T2 ␤-galactosidase have
been the subject of numerous kinetic studies in the hydrol-
ysis of lactose. For example, the enzyme immobilized via
its primary amino groups on a support activated with tris-
hydroxymethylphosphine [192] showed reduced enzyme activity
but increased stability. Both the free and the immobilized enzymes
were competitively inhibited by galactose, which was reduced
by 50% in the immobilized preparation. Glucose inhibition was
only observed for the free enzyme. It has also been reported
that immobilization on heterofunctional-epoxy supports greatly
reduced such inhibition [193]. In the hydrolysis of 5% lactose, the
337
yield using the free enzyme was only 90% where use of the immo-
bilized enzymes produced quantitative yields [193].
3.1.5.2. Immobilization of other thermophilic glycosidases. Soymilk
is a nutritional beverage that is commonly substituted for cow’s
milk, particularly for infants. The use of ␣-galactosidases to
hydrolyze non-digestible ␣-oligosacharides, such as raffinose,
without altering any of the other nutritional components of the
soymilk provides a means of enhancing its commercial usage [194].
A stable ␣-galactosidase from Thermus sp. strain T2 has been
covalently immobilized using supports activated with glyoxyl,
epoxy or glutaraldehyde groups [195]. In all cases a very active
biocatalyst was obtained, and the highest functional stability was
obtained using glutaraldehyde activated supports. Improved stabil-
ity was higher if the enzyme was first adsorbed onto an aminated
support and then treated with glutaraldehyde. This preparation
retained 90% of initial activity after 48 h at pH 7 and 75 ◦ C while the
soluble enzyme was fully inactivated after only 8 h under similar
conditions. In parallel with the enhanced thermostability, immo-
bilization increased the temperature ‘optimum’ for activity from
65 ◦ C (soluble enzyme) to 70 ◦ C. The reversible immobilization of
the enzyme on supports coated with polyethyleneimine or sulfate-
dextran also induced some stabilization [196].
␤-glucosidases are of potential application in the hydrolysis
of cellobiose (in the degradation of cellulose) and in the produc-
tion of ␤-glucose derivatives. Examples of the immobilization of
these enzymes include the preparation of covalent derivatives of
Pyrococcus furiosus ␤-glucosidase, and their application in trans-
glucosylation, using primary and tertiary organic alcohols and
secondary artificial organosilicon alcohols as aglycones, with cel-
lobiose as the glucose donor [197].
3.1.6. Immobilization of thermophilic esterases and lipases
Esterases and lipases are among the most widely used enzymes
in biocatalysis, in part due to their wide substrate specificities
and regio- or enantioselective properties. Interest in this group of
enzymes is reflected in the large number of review papers on these
enzymes and their applications that have been published over the
past 2–3 years (e.g., [132,198–206]).
Among the thermophilic lipases, the most commonly used
(probably due to its wide commercial availability) is the lipase from
the fungus Thermomyces lanuginosus. This enzyme has been immo-
bilized on diverse materials, but as this work has been very recently
reviewed [132] it will not be covered here.
Numerous lipases and esterases from thermophilic representa-
tives of the genus Bacillus have been reported. The reader should
note that many members of these genera have been recently reclas-
sified as Geobacillus, Axoxybacillus etc. but here we use the term
Bacillus in a generic sense.
Bacillus thermoleovorans CCR11 lipase has been immobilized
by adsorption on porous polypropylene (Accurel EP-100) in the
presence and absence of 0.1% Triton X-100 [207]. Immobilization
increased enzyme thermostability (activity recovered after 1 h of
incubation at 70 ◦ C for free enzyme was lower than that for immo-
bilized enzyme at 90 ◦ C) and reduced specific activity with short
chain ester substrates (e.g., with C6, down to 25%), but did not alter
the optimum pH.
A lipase from an unidentified thermophilic Bacillus was immobi-
lized on different solid supports and used in esterification reactions
[208]. The enzyme was adsorbed on silica and HP-20 beads (a
hydrophobic support) followed by cross-linking with glutaralde-
hyde on HP-20. This protocol improved the thermostability of
enzyme: the optimum temperature was nearly 5 ◦ C higher for the
immobilized enzyme while the half-life of the immobilized lipase
was nearly 2.5 higher than that of the free enzyme at 70 ◦ C.
338 D.A. Cowan, R. Fernandez-Lafuente / Enzyme and Microbial Technology 49 (2011) 326–346
The lipase from Bacillus coagulans immobilized on silica has
been used in esterification reactions. Immobilization significantly
enhanced thermostability and the esterification of ethanol and pro-
pionic acid generated reaction yields of in excess of 95% in 12 h at
55 ◦ C [209].
There are several published examples of immobilization of
enzymes from different strains of B. stearothermophilus, a well
known moderate thermophile. An esterase from this organism
[210] was immobilized by multipoint covalent attachment to
glyoxyl agarose [211]. After optimization of the immobilization
conditions, increases in the stability of the esterase preparations by
factors of up to 600-fold were obtained (depending on the pH) when
compared to single-point covalent derivatives, with a retention of
65% of the initial activity after multipoint covalent attachment.
Multipoint covalently linked esterase derivatives retained more
than 70% of the initial activity after 1 week in 50% DMF or DMS at
30 ◦ C. This structural stabilization was also evident under various
denaturing conditions (e.g., in the presence of high concentrations
of sodium chloride and organic solvents) [212]. Medium chain (C8)
aliphatic p-nitrophenyl ester substrates, which inactivate the sol-
uble enzyme, were shown to be more readily hydrolyzed by the
immobilized enzyme.
The thermostable lipase from a thermophilic B. stearother-
mophilus MC 7 was immobilized by different techniques [213]. The
most advantageous method for immobilization was found to be
ionic exchange on DEAE Cellulose.
Possibly the most studied thermophilic Bacillus lipase is that
derived from B. thermocatenulatus, first described by Schmidt-
Dannert and co-workers in 1994 [214]. The authors found that the
lipase bound almost irreversibly on various chromatography matri-
ces, such as Amberlite and Serolite, and was very stable in this form.
The structure of the enzyme has been recently resolved, showing
an unusual double ‘lid’ structure covering the active centre. Com-
plex conformational changes are involved in the transition from
the closed to the open form [215]. Various immobilized forms of
this lipase have been generated, and demonstrate that functional
properties can be modulated by immobilization [216] but that full
functionality (i.e., an ‘open’ lid structure) is retained on immo-
bilization. The enzyme was strongly adsorbed onto hydrophobic
supports, with the immobilized preparations exhibiting both
hyperactivation and stabilization [217–219]. B. thermocatenulatus
lipase immobilized on octadecyl-Sepabeads maintained 100% of
initial activity at 65 ◦ C after several days [218]. This enzyme was
also used as a model for the immobilization of enzymes on gly-
oxyl supports at neutral pH, where thiol compounds were used to
stabilize the imino group chemistry [220].
Thermophilic lipases from other microbial sources have also
been immobilized. For example, the lipase from a thermophilic
Rhizopus oryzae strain was immobilized to various matrices (Duo-
lites, Celite, and Amberlites), with Amberlite IRC 50 being selected
as the most suitable adsorbent [221]. Repeated use of the immo-
bilized lipase in organic media to esterify equimolar amount of
oleic acid with hexanol in cyclohexane at 30 ◦ C over a period
of three weeks resulted in a decrease by 18% of synthetic activ-
ity. In contrast, the ability to hydrolyze trioctanoin decreased up
to 80% over the same period. Enzyme leakage in aqueous media
(but not in anhydrous media) may be the explanation for these
results.
The lipases from Thermus thermophilus and T. aquaticus were
immobilized on decaoctyl-Sepabeads, yielding preparations with
10-fold enhanced activities at mesophilic temperatures and
increased functional stabilities [222]. The authors speculated that
the increased activity at mesophilic temperatures might be due to
retention of the lipase in its open form, and that the requirement
for the conformational activation might account for the low activity
of the native enzyme at low temperatures.
3.1.7. Immobilization of other thermophilic enzymes
Examples of the immobilization of many other thermophilic
enzymes are reported in the literature. For example, immo-
bilized d-hydantoinase from B. stearothermophilus was used
to produce N-carbamoyl-d-p-hydroxyphenylglycine from d,l-
5-(4-hydroxyphenyl)hydantoin [223]. The d-hydantoinase was
immobilized on various support matrices by adsorption, and
DEAE-cellulose resin was found to be most effective in terms
of the activity recovery (>90%). At 55 ◦ C and pH 9.0, the pro-
duction rate was maintained constantly over nine successive
operations.
ATP synthase from B. stearothermophilus was encapsulated in
liposomes and these were then immobilized on porous glass beads
[224]. This immobilized enzyme together with 4-aminobutyrate:2-
ketoglutarate transaminase (EC 2.6.1.19) from E. coli was employed
for the production of l-phosphinothricin [l-homoalanin-4-yl-
(methyl)phosphinic acid], from its nonchiral 2-keto acid precursor
2-oxo-4- [(hydroxy)(methyl)phosphinoyl]butyric acid.
An immobilized aspartate transaminase from B stearother-
mophilus was used with 4-aminobutyrate:2-ketoglutarate
transaminase (EC 2.6.1.19) from E. coli for the production of
l-phosphinothricin [l-homoalanin-4-yl-(methyl)phosphinic acid]
[225].
Catechol 2,3-dioxygenase (EC 1.13.11.2) from B. stearother-
mophilus has been immobilized on highly activated glyoxyl agarose
[226]. After optimization of the immobilization conditions, the
stability of immobilized catechol 2,3-dioxygenase was essentially
independent of protein concentration whereas the free enzyme
was rapidly inactivated at low protein concentrations. Immobiliza-
tion increased the enzyme temperature where maximum activity
was achieved by 20 ◦ C, retained activity at substrate concentra-
tions where the soluble enzyme was fully inactivated and enhanced
the resistance to inactivation during catalysis. These changes were
attributed to conformational rigidification and the prevention of
the enzyme subunit dissociation [226].
A cyclodextrin glucanotransferase from Thermoanaerobacter sp.
was covalently attached to Eupergit C [227]. Soluble and immo-
bilized cyclodextrin glucanotransferase showed similar optimum
temperature (80–85 ◦ C) and pH (5.5) values, but the pH profile
of the immobilized cyclodextrin glucanotransferase was broader
at higher pH values. The half-life of the immobilized enzyme at
95 ◦ C was five times higher than that of the soluble enzyme. The
selectivity of the immobilized enzyme (determined by the ratio
of cyclodextrin to oligosaccharides) was shifted towards oligosac-
charide production. The immobilized enzyme was also used in
the synthesis of maltooligosyl fructofuranosides from starch and
sucrose, with yields over 80% [228]. Glucanotransferase from T.
cyclomaltodextrin was immobilized using different supports and
immobilization methods, with glyoxyl agarose immobilization
yielding the highest levels of activity and stability [229,230]. The
immobilization yield was almost 100% and the activity recovery
was ca. 32%. At 85 ◦ C, the biocatalyst was capable of producing
cyclodextrins (CDs) from dextrin or soluble starch (both at 1% (w/v))
at a greater rate than the soluble enzyme. In addition, the biocata-
lyst maintained 90% of its initial activity after 5 h at 85 ◦ C.
Inorganic pyrophosphatase (Pyr) from Thermus thermophilus,
fused with the C terminus of the choline-binding domain of
pneumococcal LytA autolysin, was selectively immobilized and
purified via affinity adsorption on DEAE-cellulose at high ionic
strength [231]. A thermostable trehalose synthase from Thermus
thermophilus HJ6 was immobilized on chitosan activated with glu-
taraldehyde (40% activity recovery) [232]. The half-life values for
the free and immobilized enzymes were 5.7 and 6.3 days at 70 ◦ C,
respectively.
An amidase from Geobacillus pallidus RAPc was immobilized by
entrapment in polyacrylamide gels, and covalent binding on Euper-
 D.A. Cowan, R. Fernandez-Lafuente / Enzyme and Microbial Technology 49 (2011) 326–346
git C beads at 4 ◦ C and on Amberlite-XAD57. Each process yielded
low protein binding and low activity [233] However, immobiliza-
tion on Eupergit C beads at 25 ◦ C with cross-linking resulted in
high protein binding yield and high immobilized specific activity
(80%). The pH range was somewhat broadened and stability was
enhanced (a 6-fold increase at 60 ◦ C). The immobilized preparation
on Eupergit C and crosslinked with 1-ethyl-3-(dimethylamino-
propyl) carbodiimide of the enzyme was shown to have reduced
substrate inhibition (the inhibition constant increased by 2-fold)
[234].
A thermophilic diguanylate cyclase from Thermotoga maritima,
immobilized via a sol–gel technique using methyltrimethoxysilane,
was used for enzymatic production of cyclic-di-GMP [235].
A laccase from a thermophilic strain of Bacillus was immobi-
lized on glassy carbon electrodes for biofuel cell applications [236].
The glassy carbon electrodes were functionalized via electrochem-
ical reduction of in situ generated aminophenyl monodiazonium
salts. Laccase-modified electrodes showed an optimal operational
temperature of 45–50 ◦ C and stable catalytic activity for at least
7 weeks.
Catalases from Thermus thermophilus strains HB8 and HB27 have
been immobilized on several supports, the most stable prepara-
tion being that obtained using glyoxyl agarose [237]. However, the
stability of the immobilized enzyme derivatives was dependent
on enzyme concentration, suggesting that the dissociation of sub-
units could be playing a key role in the inactivation of the enzyme
preparation. The production of CLEAs of this enzyme prevented
this dissociation, gratly improving the operational stability of the
enzyme [113].
The development of miniaturized flow reactor technology
offers a cost effective method for process development [238].
An aminoacylase from Thermococcus litoralis has been success-
fully immobilized in miniaturized flow reactor channels, either by
direct binding to monoliths or by trapping of cross-linked enzymes
on frits. Similarly, a cross-linked enzyme aggregate of a (+)-␥-
lactamase from Sulfolobus solfataricus MT4 was immobilized within
a capillary column microreactor [239]. The thermophilic (+)-␥-
lactamase retained 100% of its initial activity at the 80 ◦ C assay
temperature for 6 h. In another example, micro-reactors contain-
ing a monolith-immobilized l-aminoacylase from Thermococcus
litoralis have been developed for use in biotransformation reactions
and a study has been carried out to investigate the stereospecificity
and stability of the immobilized enzyme [240].
3.1.8. Non conventional uses of immobilized thermophilic
proteins
Immobilized thermophilic proteins have a wide range of appli-
cations. For example, ferredoxins isolated from Synechococcus
vulcanus have been immobilized on CNBr-Sepharose [241] and used
to purify ferredoxin-dependent enzymes in spinach chloroplasts.
Similarly, the co-immobilization of the Thermococcus strain
KS-1 chaperonin with heterologous mesophilic and thermophilic
enzymes [242] has been used to enhance enzyme stability. A novel
self-renaturing enzyme-chaperone chimera consisting of penicillin
amidase and a thermophilic chaperonin that functions in aqueous-
organic mixtures has been designed [243]. The design of the
chimeric structure included a flexible linker between the enzyme
and chaperone and an added chitin binding domain to facilitate the
immobilization of the chimera to a chitin support. The productivity
of the immobilized chimera for amoxicillin synthesis in aqueous-
methanol mixtures was 2.8 times higher at 95 h than that of the
immobilized penicillin amidase lacking a chaperone domain.
Thermophilic protein binding domains have also been used
to immobilize proteins. Recombinant plasmids containing fusion
proteins composed of two different modules, lactase from Ther-
moanaerobacter ethanolicus and CelD, a cellulose-binding module
339
from Anaerocellum thermophilum, were expressed in E. coli [244].
The modules encoding the CelD domain resulted in strong binding
of the fusion proteins to a cellulose carrier, allowing the construct
to be used for lactose hydrolysis in a continuous-flow system at
high temperatures.
The fusion of hyperthermophilic enzymes (from Pyrococcus
furiosus) to the carbohydrate-binding domain from the mesophile
Clostridium cellulovorans provided an elegant method for gener-
ating a stable biocatalyst with temperature-sensitive reversible
immobilization [245]. The recombinant fusion enzymes were active
in the free form at 80–90 ◦ C but immobilized by affinity adsorption
on cellulose at 30–40 ◦ C. The temperature transition between both
modes was narrow, occurring within the range of 40–50 ◦ C [245].
This system may be useful in the recycling of enzymes that are used
for the biotransformation of very large or insoluble substrates.
3.2. Use of immobilized thermophilic enzymes as biosensors
Due to their high storage and operational stability, thermophilic
enzymes have been widely considered for use in biosensors
[246,247]. In sensor design, the enzyme is typically immobilized
such that the signal is generated in close proximity to the trans-
ducer.
␤-glucosidase and glucokinase from B. stearothermophilus have
been used in the design of a ion sensitive field effect transis-
tor ␤-glucosidic glucoside sensor [248,249]. An amperometric
l-glutamate sensor was prepared by the immobilization of l-
glutamate dehydrogenase in a carbon paste electrode with a
polyethylenimine Toluidine Blue O redox polymer mediator and
lactitol/DEAE dextran stabilizing system [250]. The enzyme sta-
bility was sufficiently high such that the physical stability of the
electrode (not enzyme stability) was the limiting factor in the selec-
tion of the analysis temperature.
Stabilization of lactate dehydrogenases from Clostridium ther-
mohydrosulfuricum induced by ‘bilayer encagement’ was up to
12.8-fold (at 70 ◦ C) [251]. Conversely, acetate kinase shows no
stabilization under similar conditions. Both immobilized enzymes
were used in flow injection analysis systems for the determination
of l-lactate or acetate.
An intracellular diaphorase from B. stearothermophilus, immo-
bilized on Immobilon affinity membranes, was operational over
an analytical range of 0.1 to 1000 micromol L−1 for NADH and
showed a limit of detection (LoD) of 0.05 micromol L−1 NADH
[252]. Co-immobilization the diaphorase with different dehydro-
genases made it possible to construct amperometric sensors for
glucose (LoD 0.5 micromol L−1 ), ethanol (LoD 1.0 micromol L−1 ),
lactate (LoD 1.0 micromol L−1 ) and ␤-hydroxybutyrate (LoD
0.5 micromol L−1 ) [252].
Hexahistidine tagged variants of lactate dehydrogenase
(EC 1.1.1.27) from B. stearothermophilus were immobilized
on poly(aniline)-poly(acrylate) (PANi-PAA) or poly(aniline)-
poly(vinyl sulphonate) (PANi-PVS) composite films [253]. Both the
C- and N-terminally tagged enzymes were readily immobilized on
the polymer electrodes and catalyzed the conversion of lactate and
NAD+ to pyruvate and NADH. Both the N- and C-tagged enzyme
films showed higher NADH-dependent current outputs than the
wild-type.
The immobilization of photosystem complexes is perhaps
among the most sophisticated applications of thermophilic
enzymes as biosensors. Such electrodes are suitable as biosen-
sors for detecting photosystem II inhibitor based herbicides. For
example, photosystem II from Synechococcus bigranulatus was
physically adsorbed on gold electrodes via a layer of poly-
mercapto-p-benzoquinone (polySBQ), providing a mediatorless
electron transport mechanism [254]. The stability of the immobi-
lized photosystem was comparable that of the free system.
340 D.A. Cowan, R. Fernandez-Lafuente / Enzyme and Microbial Technology 49 (2011) 326–346
Immobilized (dialysis membrane entrapped) photosystem II
from S. elongatus has been used for the detection of residual
triazine-, urea- and phenolic-type herbicides [255]. The herbicide
detection was based on the fact that, in the presence of artificial
electron acceptors, the light-induced electron transfer through iso-
lated photosystem II particles is accompanied by the release of
oxygen, which is inhibited by the herbicide in a concentration-
dependent manner. This system resulted in a reusable herbicide
biosensor with good stability (50% of initial activity remained after
35 h of use at 25 ◦ C) and high sensitivity (the detection limit for
diuron was 5 × 10−10 M). Photosystem II from the same source was
immobilized on the surface of a screen-printed sensor composed
of a graphite working electrode and Ag/AgCI reference electrode
deposited on a polymeric substrate [256]. The resulting herbicide
biosensor was reusable, with good stability (half-life of 24 h) and
a low limit of detection of (approximately 0.001 ␮M) for diuron,
atrazine and simazine.
Other examples include the detection of halogenated organic
compounds using immobilized dehalogenase from Sulfolobus toko-
daii [257] or l-lysine using thermostable NAD-dependent l-lysine
6-dehydrogenase from G. stearothermophilus immobilized on gold
electrodes [258].
4. Simultaneous use chemical modification and
immobilization to improve the properties of enzymes from
thermophiles
The coupled use of immobilization and chemical modification
has been used successfully to optimize the performance of ther-
mophilic biocatalysts.
4.1. Chemical modification of immobilized enzymes
Thermus thermophilus strains HB8 and HB27 catalases were
thermo-stabilized when immobilized on glyoxyl agarose, but still
subject to subunit dissociation and loss of activity [237]. Further
crosslinking of the immobilized enzyme with aldehyde-dextran
prevented this process, yielding enzyme preparations which were
highly stable under a wide range of conditions [237]. In the case
of Thermus strain T2 ␤-galactosidase immobilized on boronate-
epoxy-Sepabeads [182], enzyme activity was extremely stable, but
protein subunits were still released under some conditions. Given
the use of this enzyme in food preparation, release of protein ‘con-
taminants’ was considered to be unacceptable. While treatment
with fully oxidized aldehyde dextran induced enzyme inactiva-
tion, treatment with partially oxidized dextran yielded a stable
quaternary structure, retaining over 80% of enzymic activity after
modification [182].
Chemical modification of immobilized thermophilic enzymes
has been used to modify catalytic behavior. For example, car-
boxylic groups on the T. lanuginosus lipase, immobilized on
cyanogen bromide agarose, were partially aminated using ethyledi-
amine and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. This
treatment improved enzyme activity and enantiospecifity in the
hydrolysis of rac-methyl mandelate [259]. The active site Cys64
residue of B. thermocatenulatus lipase, immobilized on cyanogen
bromide- or glyoxyl-agarose beads, was site-specifically modified
with pyridyldisulfide poly-aminated-dextrans or pyridyldisulfide
mono-carboxylated-polyethyleneglycol [260]. While the activity of
modified derivatives was strongly dependent on the immobilized
preparation, in many instances activity was enhanced. The fact that
the modified enzymes reacted with the irreversible inhibitors much
more rapidly than unmodified enzymes suggested that the chem-
ical modification may have stabilized the open form of the lipase
[260].
4.2. Chemical modification of the enzyme to improve
immobilization efficiency
Lipases from B. thermocatenulatus and T. lanuginosus
were reversibly immobilized by interfacial activation on
octyl-agarose, fully aminated using ethylediamine and 1-ethyl-
3-(3-dimethylaminopropyl)-carbodiimide, desorbed from the
support and then covalently immobilized on glyoxyl agarose
[56,57]. This protocol increases the number of reactive amino
groups on the protein surfaces and facilitates multipoint covalent
attachment. The pK values of the introduced amino groups are
around 9, such that a lower reaction pH can be used than is typical
in the standard immobilization protocol [56,57]. These very active
and stable biocatalysts showed enhanced performance in biodiesel
production [261,262], with the option of undergoing multiple
cycles of enzyme inactivation/reactivation [263–265]. Similarly,
the physical coating of the multimeric glutamate dehydroge-
nase from T. thermophilus by treatment with polyethylenimine
facilitated immobilization via ionic exchange [122]. After ion
exchange adsorption, the polyethylenimine–enzyme composite
was crosslinked with glutaraldehyde to prevent enzyme subunit
dissociation.
5. Future prospects
This review demonstrates that it is possible to improve the
performance of stable thermophilic enzymes using Enzyme Tech-
nology tools such as immobilization and chemical modification.
Interest in the enzymology of thermophilic organisms is
expected to continue, both with continued isolation of new taxa
and the increasing availability of gene and genome sequences from
metagenomic studies [6]. It is also projected that the exploita-
tion of these gene products, with or without chemical or genetic
engineering, will remain a focal area of the growing global biotech-
nology industry. Given this position, it is pertinent to consider
where the bottlenecks and limitations remain. One issue, which
is far from complete resolution, is the effective and reliable expres-
sion (particularly hyper-expression of thermophilic enzyme genes
in heterologous hosts). While this is not an issue which can be
resolved using Enzyme Technology tools, it remains a constraint
on the commercial implementation of thermophilic enymes.
Despite being considered as mature technology with proven
capacity to improve enzyme properties [94–100], these technolo-
gies can be further enhanced, particularly with respect to the fine
control of immobilization chemistry [26].
Most immobilization processes are far from random [26] and
control of the enzyme immobilization may be achieved through
an understanding of the variables that determine protein orienta-
tion on the support and the degree of enzyme-support interaction
[26]. This could be achieved by the development of tailor-made
heterofunctional supports, which, in conjunction with site directed
mutagenesis, would facilitate site-specific immmobilization and
rigidification of industrial enzymes. Although some progress has
bee made in the design of such supports [266,267], and the tech-
niques for targeted enzyme engineering are well established, the
combination of the two for the preparation of next-generation
immobilized enzyme product is still an unrsolved matter [26].
In many published examples of the immobilization of ther-
mophilic enzymes (Table 1), enzyme stability is of low priority
and other factors, such as specific activity and catalytic specificity,
are more relevant. The studies reviewed here have demon-
strated that properly designed immobilization protocols can be
used to solve other functional problems in these stable enzymes
(such as substrate or product inhibition and enzyme selectivity)
[100,138,192,193,234]. In other cases, enzymes present already the
 D.A. Cowan, R. Fernandez-Lafuente / Enzyme and Microbial Technology 49 (2011) 326–346
desired properties. For these cases a extremely simple, mild and
cheap immobilization process should be applied (e.g., reversible
adsorption).
Chemical modification is an additional step in the design of
an immobilized biocatalyst, where the costs and additional reac-
tion step must be balanced against the process gains [30]. To take
full advantage of this technology, there is a need for more spe-
cific chemical reagents [268], really designed to get some desired
effect on enzyme (modification, crosslinking). This could further
stimulate the design and preparation of even more effective biocat-
alysts or biosensors and further increase the industrial applications
of thermophilic (and other) biocatalysts. The industrial perfor-
mance of these modifications, should be benefited by the design
of the modification of immobilized enzymes, as we have stated
in this review. When a free enzyme is desired, full advantages
of this could be only be achieved if reversible immobilization
methods compatible with the chemical modification are designed
(that is, immobilization designed to stand the chemical modifica-
tion but that may permit the release of the modified enzyme). To
date, lipases hydrophobicallya adosrbed on hydrophobic supports
[56,57] seem the only solved situation, and even this is not effective
for all type of modifications.
Thus, an integrated development of all the strategies presented
in this review is required to actually solve the problems of the
enzymes in industry, but the solutions seem to be each day nearer.
Acknowledgments
This work was supported by grant CTQ2009–07568 from Span-
ish Ministerio de Ciencia e Innovación. DAC acknowledges financial
contributions from the National Research Foundation of South
Africa.
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