Biotechnology Advances xxx (xxxx) xxxx

Contents lists available at ScienceDirect

Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Enzyme co-immobilization: Always the biocatalyst designers' choice…or

not?
Sara Arana-Peñaa,1, Diego Carballaresa,1, Roberto Morellon-Sterllinga, Ángel Berenguer-Murciab,
⁎
Andrés R. Alcántarac, Rafael C. Rodriguesd, Roberto Fernandez-Lafuentea,
a
Departamento de Biocatálisis, ICP-CSIC, Campus UAM-CSIC, 28049 Madrid, Spain
b
Departamento de Química Inorgánica e Instituto Universitario de Materiales, Universidad de Alicante, Alicante 03080, Spain
c
Facultad de Farmacia, Departamento de Química en Ciencias Farmacéuticas, Universidad Complutense de Madrid, Plaza de Ramón y Cajal, s/n, 28040 Madrid, Spain
d
Biocatalysis and Enzyme Technology Lab, Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Av. Bento Gonçalves, 9500, P.O. Box
15090, Porto Alegre, RS, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: The increasing relevance of cascade reactions in biocatalysis has sparked a great interest for enzyme co-im-
Diffusion limitations mobilization. Enzyme co-immobilization allows access to some kinetic advantages that in some instances are
Enzyme stability necessary to get the desired product, avoiding side-reactions. However, the kinetic effect is very relevant mainly
Enzyme reuse at the initial reaction rates, while it may be less relevant in the whole reaction course, depending on the kinetic
Enzyme inactivation
parameters of the involved enzymes. This review not only critically discusses the advantages but also the
Step by step co-immobilization
drawbacks of enzymes co-immobilization: immobilization on the same support and surface, under similar
Enzyme spatial distribution
conditions, discarding the whole biocatalyst when one of the co-immobilized enzymes is inactivated. We will
discuss when co-immobilization is almost compulsory, when the advantages of co-immobilization may not be
enough to compensate their problems and when it should be fully discarded. The co-immobilization of cofactors
and enzymes bears special interest, as this can open up the opportunity to the building of artificial cells and
extremely complex one-pot transformations. Finally, some recent strategies to overcome some the co-im-
mobilization problems will be presented.

1. Introduction
Enzyme immobilization was initially designed to overcome the
problems associated with enzyme recovery (Sheldon, 2007; Sheldon
and Van Pelt, 2013), as enzymes form a colloidal solution (due to their
size) hard to separate from the reaction medium (Sarangapani et al.,
2015) sometimes also formed by a protein solution. Moreover, im-
mobilization facilitates downstream processing and continuous opera-
tion (Sheldon, 2007; Sheldon and Van Pelt, 2013). Later on, im-
mobilization was used to solve many other enzyme limitations, like
enzyme activity (Mateo et al., 2007c; Rodrigues et al., 2013), stability
via multipoint or multisubunit covalent attachment (Brady and
Jordaan, 2009; Fernandez-Lafuente, 2009; Garcia-Galan et al., 2011;
Hernandez and Fernandez-Lafuente, 2011; Iyer and Ananthanarayan,
2008; Klibanov, 1979; Liu and Dong, 2020) or purity, by coupling en-
zyme immobilization to its purification (Barbosa et al., 2015). Enzyme
activity may be also improved, as in some instances enzyme
stabilization may produce a more active enzyme under harsh conditions
(Dal Magro et al., 2019b, 2020).In other cases a more active con-
formation of the enzyme may be obtained (Rodrigues et al., 2019),
while may also result in a decrease in inhibitions or some diffusional
problems with positive effects on enzyme activity in other cases (Ge
et al., 2012; Mateo et al., 2007c; Rodrigues et al., 2013). Immobiliza-
tion is also useful in tuning enzyme selectivity or specificity, and it may
reduce enzyme inhibition (Bilal et al., 2019; Garcia-Galan et al., 2011;
Mateo et al., 2007c). Enzyme immobilization allows tailoring enzyme
microenvironments by co-immobilizing enzymes and ionic polymers,
generating partition effects that can also have positive effects on en-
zyme activity or stability (Tacias-Pascacio et al., 2019a; Virgen-Ortíz
et al., 2017a). That way, immobilization objectives are nowadays far
away from the initial mere enzyme recovery, that is losing relevance
due to the decrease in the price of commercial enzymes. Thus, a proper
immobilization method that optimizes the different enzyme features
may become a critical step in the design of an industrial biocatalyst (Di

⁎
Corresponding author at: ICP-CSIC, C/ Marie Curie 2, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain.
E-mail address: rfl@icp.csic.es (R. Fernandez-Lafuente).
1
Both authors have evenly contributed to this paper.

https://doi.org/10.1016/j.biotechadv.2020.107584
Received 24 March 2020; Received in revised form 24 June 2020; Accepted 24 June 2020
0734-9750/ © 2020 Elsevier Inc. All rights reserved.

Please cite this article as: Sara Arana-Peña, et al., Biotechnology Advances, https://doi.org/10.1016/j.biotechadv.2020.107584
S. Arana-Peña, et al.
Cosimo et al., 2013; Garcia-Galan et al., 2011; Liese and Hilterhaus,
2013; Mateo et al., 2007c). In fact, the number of papers related to
enzyme immobilization have increased steadily over time (Gonçalves
et al., 2019).
Considering the impact of immobilization on enzyme features, co-
immobilization is an even more complex problem. Co-immobilization
should be defined so as to confine two or several enzymes in the same
space (in the same particle, for example), and it has been the subject of
many recent reviews (Hwang and Lee, 2019; Idan and Hess, 2013; Ji
et al., 2016; Jia et al., 2014; Kazenwadel et al., 2015; Liang et al., 2016;
Memon et al., 2018; Ren et al., 2019; Wang and Zhang, 2015; Zhang
and Hess, 2017).
The interest of enzyme technology in co-immobilization grew to-
gether with the interest in cascade reactions (Grondal et al., 2010;
Guterl et al., 2012; Han et al., 2020; Lin et al., 2014; Mayer et al., 2001;
Ngo et al., 2016; Nicolaou et al., 2003, 2006; Peters et al., 2014; Ricca
et al., 2011; Song et al., 2019; Sperl and Sieber, 2018; Tietze, 1996). A
cascade reaction may be defined as that where the product of enzyme 1
is the substrate for enzyme 2, and that may involve many consecutively
acting enzymes in a more or less complex synthetic route. In these
cases, it has been shown that the initial reaction rate may be ac-
celerated when using co-immobilized enzymes, and that way many
researchers attempted the design of co-immobilized enzyme biocata-
lysts, mimicking nature (Hwang and Lee, 2019; Idan and Hess, 2013; Ji
et al., 2016; Jia et al., 2014; Kazenwadel et al., 2015; Ren et al., 2019;
Rodrigues et al., 2013; Wang and Zhang, 2015; Zhang and Hess, 2017).
The initial reaction rate is accelerated using co-immobilization because
this reduces or even eliminates the lag time that is produced using
several enzymes immobilized on different enzyme particles or even free
enzymes. This lag time is produced because the concentration of the
intermediate products will be initially very low and this will not permit
the other enzymes in the reaction chain to express their activities from
the beginning of the reaction (Fig. 1). Using co-immobilized enzymes,
the initial concentration of intermediate products, produced in a con-
fined space, may be very high and may permit the other enzymes to
express all the activity from the initial moments of the reaction (Fig. 2).
However, in the whole reaction course, this lag time may not be so
significant (Garcia-Galan et al., 2011). Catalyst confinement and
P1 concentration is high inside the
biocatalyst particle with Enzyme 1
Porous supports
[P1]
[S]
[P1]
P1 must go out of the particle of Enzyme
1 and is diluted in the reaction medium
Enzyme 1
Enzyme 2
Fig. 1. Schematic representation of the pathway that product 1 should follow from the biocatalysts with enzyme 1 to the particle with enzyme 2 (suffering a dilution
in the overall reactor). Enzyme 2 cannot function under saturating conditions in the first moments of the reaction. This produces an increase of lag time.
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Biotechnology Advances xxx (xxxx) xxxx
compartmentalization can be performed at different spatio-temporal
levels by using different formats of reactors, such as stirred tanks or
fixed-bed reactors, microreactors, etc. (Benítez-Mateos et al., 2018b;
Bolivar et al., 2016, 2019; Semenova et al., 2020; Viefhues et al., 2017).
This way, enzyme co-immobilization may be found in many recent
papers, in many instances without any previous justification or any
proof of the advantages that co-immobilization can give to the overall
process. This review will try to perform a critical evaluation of the
advantages, even the necessity in some instances, and new opportu-
nities that co-immobilization can provide in the design of cascade re-
action processes, but making a special emphasis on the problems that
co-immobilization can raise, mainly in the design of the biocatalysts. In
this context, we will discuss when the advantages of the co-im-
mobilization overcome the drawbacks and some recent solutions where
the problems are sought to be minimized. That way, this review will
aim at taking a wholly different focus to the other recent reviews on this
area.
2. Use of enzymes in cascade reactions: problems and advantages
derived from the simultaneous use of different enzymes
The simultaneous use of several enzymes in free, immobilized or co-
immobilized forms has some positive effects in reaction performance in
cascade reactions, additional to the reduction of the lag time (Guterl
et al., 2012; Lin et al., 2014; Ngo et al., 2016; Sperl and Sieber, 2018). It
can shift the reaction towards the desired direction simulating the
metabolic chains, some inhibitions may be reduced by transforming one
product that inhibits one of the initial enzymes in the chain into another
compound without that inhibitory effect, and it permits the use of a
single reactor, with the relevance from the point of view of process costs
that this may have (Guterl et al., 2012; Lin et al., 2014; Ngo et al., 2016;
Sperl and Sieber, 2018). The simultaneous use of several enzymes is per
se a complex goal, because it is mandatory to find conditions where all
the involved enzymes have high enough stability and activity (Guterl
et al., 2012; Lin et al., 2014; Ngo et al., 2016; Sperl and Sieber, 2018).
In certain cases, the failure to meet these requirements may fully pre-
vent the possibility of using a one-pot reaction using the desired en-
zymes. However, biodiversity offers such a diverse range of enzyme
[P1]
[P2]
At this low concentration, P1 must go inside
the particle of biocatalysts with Enzyme 2
S. Arana-Peña, et al.
P1 is rapidly produced by enzyme 1 in a confined space
S1 P1 P2
Enzyme 2 may be exposed to the P1 saturation concentration from
the beginning of the reaction. That way, P1 is rapidly modified
Enzyme 1
Enzyme 2
Fig. 2. Using co-immobilized enzymes in porous supports, product 1 is produced in a confined space where enzyme 2 is also immobilized, causing enzyme 2 to
perform its function from the first moments of the reaction and reducing the lag time.
activities that it is relatively likely to find enzymes whose properties
can fit each other and permit the design of the process (Fernández-
Arrojo et al., 2010; Ferrer et al., 2015, 2016; Kennedy et al., 2008;
Lorenz et al., 2002; Uchiyama and Miyazaki, 2009; Voget et al., 2003).
Another point to be considered is the possibility that one of the
products along the cascade may have enzyme inactivating effects. These
compounds may be free radicals (Kim et al., 2015; Surmeli et al., 2010;
Tang et al., 2009; Zavada et al., 2016), vinyl groups (Hanzlik and
Thompson, 1984; Krafft and Katzenellenbogen, 1981; Marcotte and
Walsh, 1976; Walsh, 1977) or hydrogen peroxide (Anjem and Imlay,
2012; Cho et al., 2010; Hernandez et al., 2012; Jiao et al., 2020). In a
one-pot system all enzymes will be exposed to these deleterious com-
pounds, making it compulsory to look for enzymes that are stable versus
these deleterious reagents. This may reduce the range of useful enzymes
to be used in the process.
When using just an enzyme in a mono step process, process opti-
mization is complex because the problems of the enzyme usually also
involve some compounds that are labile or present low solubility. From
this perspective, the difficulties in the appropriate design of one-pot
cascade reactions will be even more complex (Grondal et al., 2010;
Guterl et al., 2012; Lin et al., 2014; Mayer et al., 2001; Ngo et al., 2016;
Nicolaou et al., 2003, 2006; Peters et al., 2014; Ricca et al., 2011; Sperl
and Sieber, 2018; Tietze, 1996). In any case, the use of different en-
zymes bearing different activity/stability profiles with the reaction
conditions will reduce the range of conditions where the processes can
be designed. Thus, designing this kind of processes is a very appealing
goal in biocatalysis and success is achieved only after a great effort
(Grondal et al., 2010; Guterl et al., 2012; Lin et al., 2014; Mayer et al.,
2001; Ngo et al., 2016; Nicolaou et al., 2003, 2006; Peters et al., 2014;
Ricca et al., 2011; Sperl and Sieber, 2018; Tietze, 1996).
That way, the researcher should balance on one hand the ad-
vantages that may be obtained by the simultaneous use of several en-
zymes, such as one-step and one-pot reactions and the kinetic ad-
vantages discussed early, and on the other hand the problems derived
from this combined use, such as the necessity of using enzymes that fit
the activity/stability profiles, and the drawbacks listed above.
The potential of enzyme biotechnology in cascade reactions is
reaching new maxima, that some years ago had been unthinkable. For
example, in a very new proposal, the researchers generated a new ac-
tive center in an esterase (Santiago et al., 2018). Later on, the catalytic
activity of this artificially designed new active center was improved
(Alonso et al., 2020). After designing a serine-hydrolase inhibitor
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Biotechnology Advances xxx (xxxx) xxxx
S1
containing a metal-complex catalytic site for oxidation and Friedel–-
Crafts alkylation reactions, they were able to build a cascade reaction
using just this single enzyme, using the esterase as one of the active
centers of the plurizyme and artificially designed metal-complex as the
other active center (Alonso et al., 2020). Obviously, also in this instance
it is necessary to find conditions where the activity/stability profile is
adequate for metal catalyst and the enzyme.
3. Requirements of co-immobilization from the point of view of
the biocatalysts design
We have presented some of the requirements when using one-pot
processes involving several enzymes. Next, we will discuss some of the
requirements that can arise when trying to co-immobilize several en-
zymes on the same particle.
3.1. Different sizes of the involved proteins
One usually ignored factor in the design of combi-biocatalysts is the
difference in size of the co-immobilized enzymes. This may have re-
levance in several aspects, depending on the co-immobilization
strategy. Using crosslinked enzyme aggregates (CLEAs), the size of the
proteins is not relevant, as the immobilization method does not depend
on this protein feature (Cao et al., 2000; Chmura et al., 2013; Sheldon,
2011a, 2011b, 2019) (Fig. 3). Using nonporous materials (nano-
particles, nanotubes, etc.) to immobilize several enzymes, they may be
co-immobilized irrespective of their size, as all enzymes will be located
on the support surface as shown in the Fig. 4 (Ansari and Husain, 2012;
Cipolatti et al., 2016; Garcia-Galan et al., 2011; Liu and Dong, 2020;
Munir et al., 2020; Sharifi et al., 2020). However, using preexisting
porous supports, the situation becomes very different. The pore dia-
meter in the support will be determined by the size of the larger en-
zyme. If the pore diameter increases, the specific area of the support
will decrease and the enzyme loading capacity and mechanical re-
sistance of the support will decrease (Santos et al., 2015), as re-
presented in Fig. 5. This means that a lower volumetric activity of the
immobilized biocatalysts will be reached, with the economic drawbacks
that this may raise.
3.2. Importance of the enzyme immobilization rate
The advantages of enzyme co-immobilization, as it will be discussed
S. Arana-Peña, et al.
Precipitant
Aggregates of large
and small proteins
Large protein
Small protein
Fig. 3. Co-immobilization of large and small proteins using crosslinked enzyme aggregate (CLEA) technology has no setbacks.
later in this review, may be altered by the distribution of enzymes on
the particle (Gooding et al., 2000; López-Gallego et al., 2017; Mathesh
et al., 2017; Rocha-Martín et al., 2012; Schmid-Dannert and López-
Gallego, 2019; Velasco-Lozano and López-Gallego, 2018; You and
Zhang, 2013). Using CLEAs, it is not possible to control the ordering of
the different enzymes on the final particles in a precise way (Garcia-
Galan et al., 2011), although we can speculate that it may be possible to
alter the enzyme ordering by altering the aggregation conditions, even
just using an empirical method to find the best aggregation conditions.
Using porous supports, if the immobilization is very rapid and the
diffusion pathway of the enzymes inside the pores is short before en-
zyme immobilization, it is possible to obtain crowns of the different
enzymes. The first immobilized one will be at the outermost area of the
pores, and the other enzymes will be successively immobilized after
that, forming concentric crowns in an ideal case as shown in Fig. 6
(Bolivar et al., 2011; Do et al., 1982; Do and Hossain, 1987; Gutenwik
et al., 2004; Hossain and Do, 1992; Ladero et al., 2001; Scharer et al.,
1992; Schmid-Dannert and López-Gallego, 2019; Van Roon et al., 2002,
2003). If the immobilization rate of the enzymes is not high enough to
prevent enzyme diffusion inside the particle before immobilization, a
Mixture oflarge
and small proteins
Non-porous
nanoparticle
Large protein
Small protein
Fig. 4. Co-immobilization of large and small proteins using non-porous nanoparticles has no problems.
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Crosslinking
reagent
Combi-CLEA
of large-small proteins
CONTROLLED ORDERING
IS NOT POSSIBLE
perfectly ordered co-immobilized enzyme system may be very hard to
get, as the second set of enzyme molecules may become immobilized on
the holes between the first immobilized enzyme molecules, thus being
easier to get a co-localized combi-enzyme, but not ordered crowns, as
schematized in Fig. 7. However, the “perfect” enzymes co-localization
may be also hard to reach, and it may be determined by the im-
mobilization rate of the different enzymes (Bolivar et al., 2011; Do
et al., 1982; Do and Hossain, 1987; Gutenwik et al., 2004; Ladero et al.,
2001; Scharer et al., 1992; Schmid-Dannert and López-Gallego, 2019;
Van Roon et al., 2002, 2003). If all enzymes immobilize very rapidly,
but at different rates, using the mixture of enzyme, very likely the most
rapidly immobilized enzymes will locate mainly towards the outermost
surface of the particle pores, while the most slowly immobilized en-
zymes will be preferentially located in the inner surface of the particle
pores, as shown in Fig. 8. This needs to be more deeply studied to give
clearer answers, but at first glance, not always all possible distributions
of native enzymes will be accomplished if a careful design of the combi-
biocatalysts is not performed. In this sense, the new strategies to get an
optimal spatial organization of enzymes based in scaffolds and enzyme
mutations may be very suitable solutions, as the reactivity of the groups
Large and small proteins may
be immobilized on the external
surface of the nanomaterials
S. Arana-Peña, et al. Biotechnology Advances xxx (xxxx) xxxx

INDIVIDUAL ENZYMES IMMOBILIZATION

Large protein Small protein

Small diameter Good immobilization of Small diameter pores

pores closed by the large protein in large support high enzyme
large proteins pore diameter supports loading

ENZYMES CO-IMMOBILIZATION

Large protein
Small protein
Larger pores
Lower volumetric activity
Fig. 5. Individual immobilization and co-immobilization of large and small proteins using porous supports: the pore diameter is determined by the largest enzyme.

Immobilization Immobilization Immobilization

of the first of the second of the third
enzyme enzyme enzyme

SEQUENTIAL IMMOBILIZATION OF ENZYMES

THAT IMMOBILIZE MORE RAPIDLY THAN
THEY DIFFUSE INTO THE SUPPORT PORES

First enzyme
Second enzyme
Third enzyme

Fig. 6. Step by step co-immobilization of enzymes. A hypothetic example where the enzymes immobilize more rapidly than they can diffuse in porous supports.

5
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SEQUENTIAL IMMOBILIZATION:
LOW IMMOBILIZATION RATE
OF ONE ENZYME

Rapid immobilization Slow immobilization of

of the first enzyme the second enzyme

Slow immobilization
of the first enzyme
Rapid immobilization rate
CO-LOCALIZATION
IS NOT SIMPLE
Rapid immobilization
of the second enzyme

Slow immobilization rate

Fig. 7. The location of sequentially co-immobilized enzymes on the support depends on the immobilization rate of the first enzyme. Ordered co-immobilization is not
always guaranteed by sequential co-immobilization.

IMMOBILIZATION ON POROUS
SUPPORTS OF MIXTURES OF
ENZYMES THAT IMMOBILIZE AT
DIFFERENT RATES:
Immobilization rate: > >
IMPROPER CO-LOCALIZATION

IMMOBILIZATION ON POROUS
SUPPORTS OF MIXTURES OF
ENZYMES THAT IMMOBILIZE AT
SIMILAR RATES:
Immobilization rate: = =
PROPER CO-LOCALIZATION
Fig. 8. Co-immobilization of all enzymes at the same time. There is no guarantee of reaching a co-localization, if the immobilization rates are different enough.

added to the enzymes will be, at first glance, similar (Gooding et al.,
2000; López-Gallego et al., 2017; Mathesh et al., 2017; Rocha-Martín
et al., 2012; Schmid-Dannert and López-Gallego, 2019; Velasco-Lozano
and López-Gallego, 2018; You and Zhang, 2013).
3.3. The immobilization strategy must be similar for all involved enzymes
If all enzymes are immobilized on the same particle, all enzymes
should be under similar conditions for some time and all enzyme mo-
lecules will be on the same support surface, bearing the same active

6
S. Arana-Peña, et al.
groups and being exposed to the same end-point support-enzyme re-
action whenever required (Garcia-Galan et al., 2011). This way, co-
immobilization can make taking full advantage of the immobilization
improvement of the properties of all enzymes impossible, unless by
coincidence, the optimal immobilization protocol was identical for all
involved enzymes. As stated above, it cannot be forgotten that im-
mobilization is a potent tool, in some instances critical, to improve
enzyme features (Barbosa et al., 2015; Brady and Jordaan, 2009;
Fernandez-Lafuente, 2009; Garcia-Galan et al., 2011; Hernandez et al.,
2011; Iyer and Ananthanarayan, 2008; Klibanov, 1979; Liu and Dong,
2020; Mateo et al., 2007c; Rodrigues et al., 2013).
Heterofunctional supports are supports bearing different groups
able to immobilize the enzymes by different mechanisms (Barbosa
et al., 2013). They may open up the opportunity of solving this situa-
tion, as the enzymes may be immobilized using different strategies.
However, it is still a requirement that all enzymes are under the same
conditions, at least while the last enzyme is immobilized. The idea is
that this last enzyme is immobilized under the milder experimental
conditions. This strategy has been utilized in at least one case, using
heterofunctional glyoxyl-IMAC supports (Rocha-Martín et al., 2012),
and enabled immobilizing two enzymes on the same support, stabilizing
the one that stood stable and active at pH 10 by multipoint covalent
immobilization on the glyoxyl groups (Mateo et al., 2005, 2006b) and
immobilizing the other enzyme via a poly-His tag on the IMAC groups
(Franken et al., 2000; Hochuli et al., 1988; Porath et al., 1975; Xie et al.,
2010) under mild conditions. However, this means that the intensity of
the multipoint covalent attachment of the enzyme immobilized on the
glyoxyl supports is not the maximum, as some groups in the support are
used to introduce the IMAC groups (and not glyoxyl groups), and also
these IMAC groups generate some steric hindrances to the enzyme-
support reaction (Barbosa et al., 2013; Mateo et al., 2003, 2007a,
2007b) (Fig. 9). The coupled effects of the steric hindrances and the
decrease in the number of groups will originate a lower stabilization of
the enzyme immobilized on the glyoxyl groups compared to the results
obtained using nonfunctional glyoxyl supports. This very smart solu-
tion, however, has been utilized in just one instance (Rocha-Martín
MONOFUNCIONAL SUPPORTS:
easy multipoint covalent
enzyme support reaction
Chemically reactive groups
Adsorption groups
Fig. 9. Steric problems for the enzyme-support reaction to get an intense multipoint covalent attachment generated by the use of heterofunctional supports, where
the adsorbing groups are over the chemically reactive groups.
7
Biotechnology Advances xxx (xxxx) xxxx
et al., 2012). Some solutions for these co-immobilization problems have
been recently presented and will be discussed in Section 5 of this re-
view.
3.4. The loading capacity of the support for each specific enzyme is reduced:
multipurpose biocatalysts
This problem is quite evident: the specific area of a support is lim-
ited. That way, if several enzymes are immobilized on the particle; only
a reduced amount of each of the enzymes can be co-immobilized
compared to the immobilization of the enzymes on individual particles.
For this reason, co-immobilizing two enzymes that are not used in a
simultaneous way has no sense. Co-immobilization should involve en-
zymes that simultaneously perform their catalytic activity (Garcia-
Galan et al., 2011) (Fig. 10). However, it is not unusual that some pa-
pers do not show that a new support or immobilization method is useful
to immobilize different enzymes and that perhaps the optimal condi-
tions for each enzyme may be quite different. Instead, the papers show
the preparation of combi-biocatalysts of non-related enzymes without
considering all the problems of enzymes co-immobilization. This kind
of co-immobilized biocatalysts involving not related enzymes have even
received a specific name, “multipurpose biocatalysts” (Bilal and Iqbal,
2019; Hepziba Suganthi et al., 2019; Liang et al., 2016; Mahmod et al.,
2015; Nouaimi-Bachmann et al., 2007). In this sense, the best multi-
purpose biocatalyst is a whole cell, where all enzymes will be present
and the cofactor recycling system will be ready to work (Jeandet et al.,
2020; Rudroff, 2019; Wu and Li, 2018).
This lower loading of each specific enzyme may become critical if
the pursued catalytic effect using one of the enzymes is only achieved
using high loadings of the involved enzyme. For example, it has been
recently published that immobilized ficin utilized in milk clotting has
optimal activity when used at maximum ficin loading, while poorly
loaded ficin biocatalysts were unable to produce milk coagulation (Siar
et al., 2020).
This competition in the loading between co-immobilized enzymes
may be solved if using multilayer enzyme biocatalysts, where one
HETEROFUNCTIONAL SUPPORTS:
steric hindrances for the covalent
enzyme support reaction generated
by the protein adsorption groups
S. Arana-Peña, et al. Biotechnology Advances xxx (xxxx) xxxx

BOTH ENZYMES ARE

IMMOBILIZED SEPARATELY

MAXIMUM LOADING
CAPACITY FOR
EACH ENZYME

OPTIMAL
IMMOBILIZATION
PROTOCOL FOR
EACH ENZYME

BOTH ENZYMES ARE

SUB-MAXIMUM
COIMMOBILIZED
LOADING CAPACITY
FOR EACH ENZYME

COMPROMISE
IMMOBILIZATION
PROTOCOL GOOD
Small protein
ENOUGH FOR BOTH
ENZYMES
Large protein

Fig. 10. Reduction of the loading capacity of a support for a specific enzyme by co-immobilization with another enzyme.

enzyme is immobilized on an enzyme layer previously immobilized on
the support as shown in Fig. 11 (Deng et al., 2010; Forrest et al., 2005,
2007; Güleç et al., 2010; Gutiérrez et al., 2011; Ondul et al., 2012;
Virgen-Ortíz et al., 2017a; Wong et al., 2013). Using non-porous sup-
ports, there are no limits to the number of enzyme layers. That way,
many different enzymes may be co-immobilized using this strategy
(Fig. 11). Using porous supports, the limit will be the remaining pore
size in the particle, that must be large enough to allow immobilizing the
new enzyme layer (Virgen-Ortíz et al., 2017a). It can be used to co-
immobilize different enzymes, and also, if desired, the same enzyme,
but altering the immobilization mechanism (Arana-Peña et al., 2019b,
2020a; Rios et al., 2019a) (Fig. 12). The different immobilization

MULTILAYER BIOCATALYSTS IN
NON-POROUS SUPPORTS

NO LIMITS TO THE
NUMBER OF
ENZYME LAYERS
First enzyme
Second enzyme
Third enzyme

MULTILAYER BIOCATALYSTS
IN POROUS SUPPORTS

THE PORE DIAMETER

DEFINES THE
NUMBER OF LAYERS

Fig. 11. Preparations of multilayer biocatalysts on non-porous and porous supports; problems generated by the diameter of the pore as a limiting factor to im-
mobilization.

8
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FIRST ENZYME LAYER

IMMOBILIZED ON
THE SUPPORT SURFACE

FIRST ENZYME LAYER

COATING WITH PEI
PEI

SECOND ENZYME LAYER

IMMOBILIZED ON THE PEI
LAYER

COATING OF THE
PEI SECOND ENZYME LAYER
WITH PEI

THIRD ENZYME LAYER

IMMOBILIZATION ON THE
PEI LAYER

CONTINUE THE SAME PROTOCOL UNTIL REACHING THE DESIRED NUMBER

OF ENZYME LAYERS
Fig. 12. Schematic representation of the preparation of an enzyme multilayer biocatalyst using PEI as glue.

Substrate
, , ,
, , , , One layer enzyme biocatalyst:
S , , , , Moderate diffusional problems
, NO TORTUOSITY

Multilayer biocatalyst:
, Smaller pore diameter
, ,
INCREASE SUBSTRATE
S , ,
, DIFFUSIONAL LIMITATIONS

S
Multilayer biocatalyst:
INCREASE IN THE DIFFUSIONAL
PATHWAY OF THE SUBSTRATE
TO REACH THE INNER ENZYME
LAYERS

Fig. 13. Increase in tortuosity in the substrate path and substrate diffusional limitations using a multilayer biocatalyst on a porous support.

protocols may alter enzyme selectivity or specificity, and that way
when immobilizing a single enzyme some diversity on enzyme prop-
erties may be expected (Fernández-Lorente et al., 2001, 2007; Palomo
et al., 2003; Rodrigues et al., 2013; Terreni et al., 2001). Although this
permits to greatly increase the enzyme mass loading in the biocatalyst,
the increase in catalytic volumetric activity is not guaranteed, as the
number of enzymes layers increases the substrate diffusional limita-
tions. These diffusional limitations are increased because each new
enzyme layer reduces the pore size, and this produces a reduction of the
rate of substrate entry in the pores of the biocatalyst particle. Fur-
thermore, it also generates an increased tortuosity in the substrate
pathway to reach the inner layers of enzyme as represented in Fig. 13,

9
S. Arana-Peña, et al.
ACCESSIBILITY OF LARGE SUBSTRATES TO
THE INNER LAYERS OF MULTILAYER BIOCATALYSTS
STANDARD BIOCATALYST:
The large substrate can
reach all enzyme molecules
Substrate Enzyme
Fig. 14. Steric hindrance to large substrates for multilayers biocatalysts generated by the presence of polymer and proteins in the way of the substrate towards the
enzymes in the bottom.
the substrate cannot reach the active center following the most direct
pathway but must avoid the immobilized polymer molecules and en-
zyme molecules (Bolivar et al., 2013; Boniello et al., 2010; Pronk et al.,
2014; Regan et al., 1974; Shen and Chen, 2007). In some instances,
positive or negative substrate partition may be promoted, increasing or
reducing the concentration of the substrate in the enzyme environment
(Tacias-Pascacio et al., 2019a; Virgen-Ortíz et al., 2017a). That way,
using multilayers composed of a single enzyme, if the problems raised
by the new enzyme layer are higher than the advantages, it is possible
that even a lower enzyme activity may be observed under specific
conditions if the immobilization mechanism produces an enzyme with a
lower activity than the lower layers of enzymes and the diffusional
limitations are high and this more active enzyme molecules does not
receive substrate at the appropriate rate (Arana-Peña et al., 2019b,
2020a; Rios et al., 2019a). Thus, it may be expected that the activity of
the enzymes in the inner layers of the combi-biocatalysts may be un-
derutilized. The problem is maximized if the substrate is large and may
suffer steric problems to penetrate the net formed by the different en-
zyme layers (e.g., proteases (Tavano et al., 2018)), as shown in Fig. 14.
3.5. Significant differences on the stabilities of the co-immobilized enzymes
Another problem in the use of co-immobilized enzymes is the pos-
sibility of very large differences among the stabilities of the involved
enzymes (Garcia-Galan et al., 2011). This problem, that can be con-
sidered mainly technical, can greatly reduce the feasibility of the in-
dustrial implementation of the cascade process, as it may be possible
that while the least stable enzyme is almost fully inactivated, the other
enzymes can maintain full activity. However, this fact is usually ignored
in the papers related to co-immobilized enzyme biocatalysts. One of the
consequences is the necessity of using an excess of the least stable en-
zyme to maintain a reasonable level of the overall combi-biocatalysts
activity. This means a decrease in the volumetric activities of the other
enzymes as it is necessary to use more support surface to load more of
the least stable one. This also causes that the careful optimization re-
commended in the biocatalysts design (Abu and Woodley, 2015; Bayer
et al., 2015; Fessner, 2015; France et al., 2017; Ladkau et al., 2014; Lin
and Tao, 2017; Muschiol et al., 2015; Oroz-Guinea and García-Junceda,
2013; Ricca et al., 2011; Riva and Fessner, 2014; Schrittwieser et al.,
2011, 2018; Wachtmeister and Rother, 2016; Wu et al., 2016; Yue et al.,
2017) must consider that one of the enzyme activities will be changing
throughout the operational lifetime of the biocatalysts, making its
10
Biotechnology Advances xxx (xxxx) xxxx
MULTILAYER BIOCATALYST:
The large substrate cannot
reach the inner layers of enzyme for
steric reasons
design and modelling more difficult (Fig. 15). The importance of this
enzyme inactivation will depend on the position of this enzyme in the
reaction chain.
Another serious problem is the necessity of discarding not a support,
but a much more expensive biocatalyst containing one or several fully
active immobilized enzymes because one of the enzymes has been in-
activated (Garcia-Galan et al., 2011). Using independently immobilized
enzymes, the solution will be to add more of this unstable enzyme when
necessary. Using co-immobilized enzymes, it is not possible to just add
the inactivated enzyme. The maintenance of the process features will
require a deep kinetic modelling study to ensure that the global bio-
catalyst activity is properly utilized. This problem will be treated in a
deeper way later on in this review.
4. Cases where co-immobilization is convenient or even necessary
Before deciding to use a co-immobilized enzyme preparation, the
pros and cons of the different possibilities should be considered (Garcia-
Galan et al., 2011). Using independently immobilized enzymes, each
enzyme may be immobilized following a different protocol, a different
support and a different active group in the support that may be opti-
mized (Santos et al., 2015). When one enzyme is partially inactivated, it
is possible to add fresh biocatalyst of just that enzyme, being un-
necessary to discard or treat the other enzyme biocatalysts. Using co-
immobilized enzymes, the situation is not so simple, as we will discuss
in this point.
4.1. Use of lytic enzymes to protect enzymatic biocatalysts from microbial
contamination
There are many papers showing that immobilized lytic enzymes
may prevent contamination of surfaces or even of the medium (Datta
et al., 1973; Crapisi et al., 1993; Guadarrama-Zempoalteca et al., 2016;
Lee et al., 1994; Mascheroni el at., 2010; Wu et al., 2018; Xiao et al.,
2019).
Based on this idea, one likely application of enzyme co-im-
mobilization is when the objective is to protect the target immobilized
enzyme from the attack of microorganisms that contaminate the sub-
strate used under non-sterile conditions using an immobilized lytic
enzyme. This is not a real cascade reaction but it is a likely application
of enzyme co-immobilization, as there are two different enzymes in-
volved. We have been able to find only two papers showing the
S. Arana-Peña, et al. Biotechnology Advances xxx (xxxx) xxxx

(A)
100

80
Residual activity, (%) Variation of the activities of the co-
60 immobilized enzymes over time in
40 a combi-biocatalyst when all
20
enzymes have similar stabilities
0
0 60 120 180 240 300 THIS IS NOT A PROBLEM
Time, (min)

(B)
100
Variation of the activities of the co-
80
Residual activity, (%)

immobilized enzymes over time in

60
a combi-biocatalyst when all
40 enzymes have different stabilities
20
IT IS A PROBLEM AT DIFFERENT
0
0 60 120 180 240 300 LEVELS
Time, (min)

Fig. 15. Theoretical variation of the enzyme activities in a combi-biocatalyst when all enzymes have similar stabilities (A) or when the enzyme stabilities are very
different (B).

INDIVIDUAL IMMOBILIZED MAIN AND LYTIC CO-IMMOBILIZED MAIN AND LYTIC

ENZYMES: PROTECTION IS LIMITED ENZYMES: PROTECTION IS HIGH

Microorganism

X Main enzyme

Lytic enzyme

Fig. 16. Co-immobilization of lytic enzymes and target enzyme to prevent contamination using porous supports: individual co-immobilization versus co-im-
mobilization.

protection of lactase by co-immobilizing with lysozyme (Li et al., 2017, biocatalysts by microorganisms when using porous supports (Fig. 16).
2019b). Only of the lytic enzyme is inside all the biocatalysts particles, it can
Although this is not shown in the paper, we can guess that enzymes destroy all the problematic cells and that way maintains the target
co-immobilization is the only way to fully prevent contamination of the enzyme intact. Moreover, this will prevent the biocatalysts from acting

11
S. Arana-Peña, et al. Biotechnology Advances xxx (xxxx) xxxx

MULTILAYER CO-IMMOBILIZED MAIN ENZYME AND LYTIC

ENZYME OVER IT: THE PROTECTION IS PERFECT Microorganism

Main enzyme

Lytic enzyme

Fig. 17. Advantages of the immobilization of the lytic enzyme over the target enzyme to protect the target enzyme from microbial contamination.

MULTILAYER CO-IMMOBILIZED LYTIC ENZYME AND MAIN ENZYME

OVER IT: NO PROTECTION AT ALL FOR STERIC REASONS Microorganism

Main enzyme

Lytic enzyme

Fig. 18. Steric hindrances using different lytic/main enzymes space distribution in a multilayer configuration.

as reservoirs of the microorganism and contaminate future substrate
batches. That way, for this specific use, even considering that the co-
immobilization may have great complication, it is the only way to en-
sure the desired effect. The order in which the enzymes are immobilized
will be very important. The authors use the lytic enzyme immobilized
over β-galactosidase, as represented in Fig. 17. The immobilization of
the β-galactosidase over the immobilized lytic enzyme will block the
lysozyme and will avoid the desired protective effects for steric reasons
(Rodrigues et al., 2013) (Fig. 18).
On the other hand, using individually immobilized lytic enzymes
and the target enzyme on non-porous nanoparticles, co-immobilization
may not be a strict requirement. In this case, all the surface of the target
enzyme biocatalysts particles will be susceptible to be attacked by the
immobilized lytic enzyme as shown in Fig. 19. This way, if the stabi-
lities or the immobilization protocols do not fit for both enzymes, the
same effect may be achieved using independently immobilized en-
zymes, saving the co-immobilization problems (Garcia-Galan et al.,
2011).
4.2. Problems and solution of the use of commercial enzyme cocktails
The tendency of some companies to commercialize cocktails of en-
zymes containing all the enzymes required to perform the reaction can
be highlighted in this context. This is especially popular in poly-
saccharide degradation, as discussed below (Kornecki et al., 2020). In
other instances, natural enzyme cocktails are used, using one or several
enzyme sources to get a mixture of enzymes. In this instance, the en-
zyme mixture is not optimized, since it is produced by the micro-
organism (Buaban et al., 2010; Passos et al., 2009; Sørensen et al.,
2007; Verma et al., 2010; Yamada et al., 2010).
Even using an enzyme cocktail prepared for a company where the
relation between amounts of the different enzymes is properly de-
signed, the use of co-immobilized enzymes may be problematic. The
relation between the enzymes has been designed to be used in free form
for one cycle under some specific conditions. That way, they will only
exhibit suitable properties under the conditions recommended by the
supplier as stability of the different enzymes under different conditions
to those proposed by the supplier may be quite dissimilar (Dal Magro
et al., 2019c).
If the researcher wants to use an immobilized form of the enzymes
forming the enzyme cocktail, it is necessary to purify each of the
components, although this may be in some instances an unnecessarily
long and tedious work, as in many instances the cocktails are prepared
mixing the different enzymes. Then, each purified enzyme may be
immobilized under each enzyme optimal conditions or all of the en-
zymes may be co-immobilized following a good enough immobilization

12
S. Arana-Peña, et al.
X NON-POROUS NANOPARTICLES: INDIVIDUALLY
X
X Lytic enzyme
X MAIN ENZYME VERY LIKELY MORE EFFICIENTLY
Fig. 19. Co-immobilization of lytic enzymes and target enzyme to prevent contamination using non-porous nanoparticles: individual immobilization versus co-
immobilization.
method for all the enzyme components. This co-immobilization pro-
tocol will be hardly optimal for all the enzymes in the cocktail.
Moreover, not all enzyme components will retain the same activity after
immobilization under similar conditions and this can change again the
optimal relation between the different enzymes in the cocktail (Dal
Magro et al., 2019b, 2019c, 2020).
Furthermore, if one enzyme activity decreases much more than
other enzyme activity during immobilization, it is not possible to add
more of this enzyme, as the researcher only has the enzyme cocktail,
producing a biased or non-compensated combi-biocatalyst (Deng et al.,
2010; Forrest et al., 2005, 2007; Güleç et al., 2010; Gutiérrez et al.,
2011; Ondul et al., 2012; Virgen-Ortíz et al., 2017a; Wong et al., 2013).
Even with the enlargement of the operational window of immobilized
enzymes when stability is increased, (Amaral-Fonseca et al., 2020; Dal
Magro et al., 2019b, 2019c, 2020) it is very likely that the ratio between
the different enzymes may be only valid under a specific condition and
a specific reaction, also reducing the interest of the direct im-
mobilization of these enzyme cocktails (Chen et al., 2017; Dutta and
Wu, 2014).
4.3. Parameters to be considered in the design of co-immobilized enzymes
The co-immobilized biocatalysts, taking into consideration the
previous problems, will require a careful design of the ratio between the
enzymes involved to maximize the overall biocatalyst activities (Deng
et al., 2010; Forrest et al., 2005, 2007; Güleç et al., 2010; Gutiérrez
et al., 2011; Ondul et al., 2012; Virgen-Ortíz et al., 2017a; Wong et al.,
2013). The use of too much of one enzyme means the use of too few of
another enzyme, and that way the advantages of enzyme co-im-
mobilization will decrease. As showed above (see Section 3), there are
some technical issues that demand a careful analysis of the advantages
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Biotechnology Advances xxx (xxxx) xxxx
IMMOBILIZED LYTIC ENZYME CAN PROTECT THE
MAIN ENZYME FROM MIROORGANIMS
CONTAMINATION
Microorganism
Main enzyme
NON-POROUS NANOPARTICLES: CO-IMMOBILIZED
LYTIC ENZYME CAN PROTECT THE
FROM MICROORGANISM CONTAMINATION
and drawbacks of enzyme co-immobilization before taking on the de-
cision of the suitability of the combi-biocatalyst. If, by chance, optimal
immobilization support and immobilization conditions are similar and
stabilities of the involved immobilized enzymes are comparable, and
only the advantages of co-immobilization remain, co-immobilization
may be considered a good solution in any cascade reaction. This occurs
in some instances, although this has been scarcely studied (Mafra et al.,
2019).
However, this is quite unlikely, as it has been shown that small
differences on the support surface affect different enzymes in a very
different way, and even the same enzyme is affected in a different
manner when changing the substrate or the conditions (Barbosa et al.,
2013; Garcia-Galan et al., 2011; Shen and Chen, 2007). In this more
general case, even in cases where some advantages of the co-im-
mobilization are observed, co-immobilization should be considered
only after a proper evaluation of all parameters.
If the enzymes are not acting in a synergistic way versus the sub-
strate and there is no clear necessity in the use of co-immobilized en-
zymes, co-immobilization should be discarded. If individually im-
mobilized enzymes may offer similar results, better optimized
individually immobilized enzyme biocatalysts should be designed and
utilized. This will be better than co-immobilizing several enzymes just
to produce a multi-catalyst. Multipurpose catalysts production (Bilal
and Iqbal, 2019; Hepziba Suganthi et al., 2019; Liang et al., 2016;
Mahmod et al., 2015; Nouaimi-Bachmann et al., 2007), just “in case”
the different activities are required, should never be recommended.
4.4. Cascade reactions
Cascade reactions, presented in Section 2, include many different
processes with a great interest and involve a wide diversity of applied
S. Arana-Peña, et al.
areas (Grondal et al., 2010; Guterl et al., 2012; Lin et al., 2014; Mayer
et al., 2001; Ngo et al., 2016; Nicolaou et al., 2003, 2006; Peters et al.,
2014; Ricca et al., 2011; Sperl and Sieber, 2018; Tietze, 1996). Some of
these reactions are not usually considered a cascade reaction, but they
may be considered as belonging to this kind of processes. For example,
the degradation of large polymers, like polysaccharides and lingo-ma-
terials (Jeoh et al., 2017; Kim et al., 2014; Kornecki et al., submitted.;
Kostylev and Wilson, 2012; Meng and Ragauskas, 2014; Zhang and
Lynd, 2004) or proteins (Pedroche et al., 2004, 2006, 2007), involves
many steps, and some of them are catalyzed by different specific en-
zymes. Although in many instances each individual activity is possible
in the absence of the other enzymes, in some cases a synergetic effect
among the different enzymes has been described (Kim et al., 2014;
Kostylev and Wilson, 2012), other inhibitions caused by some of the
intermediate products may be eliminated (Grondal et al., 2010; Guterl
et al., 2012; Lin et al., 2014; Mayer et al., 2001; Ngo et al., 2016;
Nicolaou et al., 2003, 2006; Peters et al., 2014; Ricca et al., 2011; Sperl
and Sieber, 2018; Tietze, 1996). It can be expected that if some sy-
nergetic effect (e.g., an effect that requires the simultaneous action of
several enzymes) (Kim et al., 2014; Kostylev and Wilson, 2012) of the
different enzymes occurs, enzyme co-immobilization may be required
to get the desired effect even using non-porous nanomaterials.
4.4.1. Hydrolysis of polysaccharides
Starch, cellulose and pectin are some of the polysaccharides which
are usually enzymatically hydrolyzed (Kornecki et al., 2020). Starch is
enzymatically hydrolyzed to produce High Fructose Syrup (HFS), a
nutritive sweetener, which is a Generally Recognized As Safe (GRAS)
food ingredient and has a great demand in the global food market
(White, 2014).
Conventionally, HFS is produced from starch by a multi-enzymatic
process, involving a mix of enzymes such as α-amylase, amyloglucosi-
dase and glucose isomerase (Ahmad et al., 2015). In some cases, a
debranching enzyme, pullulanase, is added. The starch is first treated at
high temperature for gelatinization. Then, the gelatinized starch is
saccharified using α-amylase at high temperature. The second enzy-
matic step is the hydrolysis of saccharified starch into dextrose by
amyloglucosidase. The dextrose hydrolysate contains 94–96% of dex-
trose. The third stage of the process is isomerization, where dextrose is
converted into fructose using glucose isomerase (Singh et al., 2018).
Some examples used enzyme co-immobilization for starch hydrolysis.
Most of them reported the co-immobilization of two enzymes: amy-
loglucosidase and pullulanase (Chakrabarti and Storey, 1990; Talekar
et al., 2013a), β-amylase and pullulanase (Atia et al., 2003), α-amylase
and glucoamylase (Salgaonkar et al., 2018). However, it has been
shown that the tri-enzyme biocatalysts were more effective for starch
hydrolysis (Talekar et al., 2013b, 2017). Thus, a combi-CLEA of alpha
amylase, glucoamylase and pullulanase was prepared. The one pot
starch hydrolysis reaction yielded 100%, 60% and 40% conversions
with combi-CLEAs, separate CLEAs mixture and free enzyme mixture,
respectively, after 2 h of reaction (Talekar et al., 2013b). This paper
clearly shows the kinetic advantages of co-immobilization along the
whole process, suggesting some synergetic effects. Later, the same
group prepared a magnetic nano-biocatalyst of α-amylase, glucoamy-
lase and pullulanase, this permitted to reach a 100% starch conversion
in only 90 min, very likely by eliminating the diffusion limitations
barriers as they use non-porous nanoparticles.
Another example of synergetic effect of enzymes for polysaccharide
hydrolysis is pectin hydrolysis. Pectic substances are high molecular
weight, acidic and complex glycosidic macromolecules that are present
in plants (Jayani et al., 2005). Pectin is hydrolyzed by a group of en-
zymes named pectinases that can be divided in two main categories:
esterases, responsible for the de-esterification of pectin removing the
methoxy esters; and depolymerases, responsible for the cleavage of the
α-(1 → 4)-glycosidic bonds in the D-galacturonic acid moieties of the
pectic substances (Kashyap et al., 2001; Ward et al., 1989). The
14
Biotechnology Advances xxx (xxxx) xxxx
commercial cocktails for this use are composed of 14 different enzy-
matic activities, but the main enzymes are polygalacturonase, which
catalyzes the hydrolytic cleavage of the polygalacturonic acid (Van Den
Brink and de Vries, 2011), pectin methyl esterase that catalyzes the de-
esterification of methyl ester linkages of the backbone of pectic sub-
stances releasing acidic pectin and methanol (Whitaker, 1984), and
pectin lyase that performs the non-hydrolytic breakdown of pectates or
pectinates, characterized by a trans-eliminative split of the pectic
polymer (Sakai et al., 1993). Thus, considering that for pectin hydro-
lysis, especially in juice industry, the participation of all these enzymes
is necessary; enzyme co-immobilization is an attractive solution.
Usually, pectinases are produced by fungal microorganisms and several
of these enzymes are expressed. They are commercialized as mix pre-
parations of pectinases, generally with a main representative activity.
The immobilization of these preparations in several supports may be
found in diverse publications (Benucci et al., 2019; Dal Magro et al.,
2016, 2018, 2019a; Fang et al., 2016; Goetze et al., 2017; Hosseini
et al., 2020; Martín et al., 2019; Mohammadi et al., 2019a; Muley et al.,
2018; Nadar and Rathod, 2019; Rajdeo et al., 2016; Soozanipour et al.,
2019; Wu et al., 2019a, 2019b; Yang et al., 2019). Most of them did not
consider the activity of each of the involved enzymes but only the total
pectinase activity. However, it was reported that each of these enzymes
has different optimal immobilization protocols (Dal Magro et al.,
2019b, 2020) and additionally they present different activities and
stabilities, range of pH where may be used, etc. (Dal Magro et al.,
2019c).
As well as for starch and pectin, the hydrolysis of cellulose and
hemicellulose depends on the activity of several enzymes (Houfani
et al., 2020). Cellulases and hemicellulases are complex mixtures of
enzymes similar to pectinases. Cellulases hydrolyze the β-1,4-D-glucan
bonds of cellulose yielding glucose, cellobiose and cellooligosacchar-
ides as primary products (Sethi and Scharf, 2013). The different com-
ponents of the enzyme cocktail act synergistically and simultaneously
hydrolyze internal bonds of cellulose (endoglucanase), chain ends
(cellobiohydrolases) and cellobiose to glucose (β-glucosidase) (Haldar
et al., 2016). The mechanism of cellulose hydrolysis can be via its ad-
sorption on the cellulase via a carbohydrate binding module, the com-
plexation of cellulose with the catalytic domain, the hydrolysis of the
glyosidic bonds, and finally the release of cellulose fragments (Jeoh
et al., 2017). Thus the immobilization of cellulases complexes is a
challenge for researchers. First, because it is necessary to co-immobilize
the three enzyme families listed above, and second, due to the me-
chanism of action of cellulases, and the insoluble nature of cellulose,
the initial activity of the immobilized biocatalyst is generally low.
Several attempts were made for co-immobilization of cellulase com-
ponents using a broad range of protocols and supports such CLEAs
(Bhattacharya and Pletschke, 2014; Dal Magro et al., 2016; Jafari
Khorshidi et al., 2016; Li et al., 2012; Periyasamy et al., 2016; Perwez
et al., 2019), chitosan (Hamzah et al., 2019; Mandal, 2019;
Mohammadi et al., 2019b), or non-porous materials. The latter have the
advantage that as all enzyme molecules are on the surface of the sup-
port, and thus they should be able to attack even insoluble substrates.
Examples of these materials are magnetic nanoparticles (Ariaeenejad
et al., 2020; Asar et al., 2019; Dal Magro et al., 2018; Giannakopoulou
et al., 2019; Muley et al., 2018, 2020; Raza et al., 2019) and carbon
nanotubes (Abraham and Puri, 2020; Li et al., 2019a; Papadopoulou
et al., 2019; Raza et al., 2019; Sillu and Agnihotri, 2020). In some cases,
some exogenous β-glucosidase is added to the cellulase components
mixture to avoid the inhibition caused by cellobiose in some of the
cocktails components. An interesting strategy was the layered co-im-
mobilization of β-glucosidase and cellulase on a polymeric film (Wang
et al., 2019). The authors prepared a layered structure with a thin PEG
hydrogel as the inner layer and sodium polyacrylate brush as the outer
layer on low density polyethylene film. The β-glucosidase and the cel-
lulase were separately co-immobilized onto this film. The β-glucosidase
was entrapped into the inner PEG hydrogel layer during the graft
S. Arana-Peña, et al.
polymerization from the polyethylene surface. After graft polymeriza-
tion of sodium acrylate on the PEG hydrogel layer was reinitiated,
cellulase was covalently attached on the outer polyacrylate layer. The
biocatalyst maintained 93% of its initial activity after 10 cycles using
carboxy methyl cellulose as substrate, and 89% after 6 cycles using
filter paper as substrate. Compared to the single cellulase and isolated
β-glucosidase/cellulase immobilization, the layered system showed
82% and 20% increase in catalytic activity using filter paper as sub-
strate, respectively (Wang et al., 2019). Possibly, the layered co-im-
mobilization of cellulases and β-glucosidase could be the ideal system
for the challenge of cellulase immobilization. β-glucosidase is the last
enzyme in cellulose hydrolysis, and the one that uses the smallest
substrate, soluble cellobiose. Thus, it can be immobilized on the inner
core of the biocatalyst, while endoglucanase and cellobiohydrolases,
that should attack the largest substrates, should be located in the outer
part of the biocatalyst, because they are responsible for the hydrolysis
of the larger chains of the cellulose. This enzyme ordering may be not
the most efficient in a general term, but in this instance there is no other
option.
4.4.2. Full modifications of fats and oils
In other instances, it is not so obvious that the process is a cascade
reaction. This occurs when having a substrate composed of many dif-
ferent multifunctional compounds and that is degraded step by step.
One outstanding example of this kind of processes is the full mod-
ification (hydrolysis or alcoholysis) of the glycerides contained in an oil
or fat. It has been discussed that an oil is in fact a mixture of many
different compounds, as it is composed of many different triglycerides,
as well as some amount of di and monoglycerides, formed by different
fatty acids (Arana-Peña et al., 2020b.). Because there are so many
substrates, it is unlikely that just one lipase may be the best for all of
them. Moreover, some of the components may act as an inhibitor of the
lipase that is the optimal one for the main components of the oil. In
hydrolysis reactions, the medium during the process is changing; for
example the pH may decrease in full hydrolysis of an oil (Arana-Peña
et al., 2020b.). Moreover, the best lipase at the initial reaction condi-
tions may not be the optimal one at the end of the reaction under a
more acid pH value. Even starting from a pure triglyceride, di and
monoglycerides will be produced along the reaction and for some time
they will be the main substrates (Arana-Peña et al., 2020b). It is again
unlikely that a lipase may be the best one in the modification of all this
diversity of substrates. That way, the use of several lipases has been
recommended to get full modification of oils and fats (Alves et al.,
2014; Amoah et al., 2016; Araki et al., 2018; Karmee, 2016; Lee et al.,
2006, 2008, 2010, 2011; Pedro et al., 2017; Poppe et al., 2015, 2018a,
2018b; Qiao et al., 2017; Rodrigues and Ayub, 2011). There are reports
showing that the mixture of immobilized biocatalysts of just one lipase
obtained by immobilization of the enzyme under different conditions or
on different supports may be enough to get some positive effects (Toro
et al., 2019). Lipase immobilization using different protocols or im-
mobilization conditions may affect enzyme selectivity or specificity
(Lokha et al., 2020; Pinheiro et al., 2019; Rios et al., 2019c; Tacias-
Pascacio et al., 2019b; Verdasco-Martín et al., 2019) There are some
reports using co-immobilized lipases for this purpose (Ibrahim et al.,
2008; Lee et al., 2013a, 2013b, 2019; Nouaimi-Bachmann et al., 2007;
Sánchez-Bayo et al., 2019, Shahedi, et al., 2019).
4.4.3. Classical cascade reactions
Moreover, there are many classical cascade reactions; the product of
one enzyme is the substrate of the other enzyme, whose product is the
substrate of another enzyme, and so on depending on the length of the
chain. This includes the sequential modification of an initial substrate
to produce a product quite different from the initial substrate (Grondal
et al., 2010; Guterl et al., 2012; Lin et al., 2014; Mayer et al., 2001; Ngo
et al., 2016; Nicolaou et al., 2003, 2006; Peters et al., 2014; Ricca et al.,
2011; Sperl and Sieber, 2018; Tietze, 1996). Another example is the
15
Biotechnology Advances xxx (xxxx) xxxx
production of hydrogen peroxide by oxidases that permits the stepwise
addition of this dangerous compound, used by peroxidases, lipases, etc.
to perform a second reaction that is the target one (Hernandez et al.,
2012). Involving also hydrogen peroxide, another example of cascade
reaction is the destruction by catalase of the hydrogen peroxide pro-
duced by an oxidase, in this instance the target reaction is the oxidation
catalyzed by the oxidase (Hernandez et al., 2012). As a last example, we
can mention the reactions requiring a cofactor, as they involve the use
of the target enzyme and usually a cofactor regenerating enzyme
(Berenguer-Murcia and Fernandez-Lafuente, 2010; Han and Liang,
2018; Kara et al., 2014; Marpani et al., 2017; Ding and Ou, 2017;
Rodríguez et al., 2012; Uppada et al., 2014; Wang et al., 2017). In all
these reactions, the use of co-immobilized enzymes has a kinetic ad-
vantage (Rocha-Martín et al., 2012) that is mainly relevant at the start
of the reaction (see Figs. 1 and 2).
Focusing on porous preexisting supports, the problem is clear. Using
individually immobilized enzymes, the product of the first enzyme must
go outside the biocatalyst particle, becoming diluted in the medium,
and then, under these diluted conditions, go inside the particle where
the second enzyme is located (Rodrigues et al., 2013). This means that a
very significant lag time is expected until the second enzyme can per-
form its function under its substrate, saturation concentration, as its
substrate is the product of the first enzyme catalyzed reactions which is
not present before it is formed (Fig. 1). Using co-immobilized enzymes,
the first product is produced at high rate in a confined space, and the
second enzyme is exposed from the first moments to a high con-
centration of this compound, perhaps a high enough concentration of
product of the first enzyme is available to the second enzyme to act
under substrate saturation conditions since the beginning of the reac-
tion (Rodrigues et al., 2013) (Fig. 2). That way, lag time may disappear
and the co-immobilized enzymes may be even more active than the free
enzymes, product diffusion limitations is a positive fact in this case
(Rocha-Martín et al., 2012). This means that co-immobilization has a
great effect on initial reaction rates.
However, lag time may account for just between less than 1% to
10% of the total reaction time (Garcia-Galan et al., 2011). The re-
searcher must consider if the technological complication that must be
assumed to develop a co-immobilized enzyme preparation may justify
saving the lag time in each whole reaction cycle.
Using non-porous materials, the advantages of enzyme co-im-
mobilization in the support surface is not so evident, as there is not a
confined space where the product 1 concentration may accumulate and
its increase in the second enzyme environment will be minimal.
However, using a multilayer system, where the enzyme that is located
nearest to the support surface is the first enzyme in the reaction chain,
and the last in the chain is the one immobilized is the last immobilized
enzyme, some positive effects may be found, as shown in Fig. 20. Fol-
lowing this strategy, it might be possible to find some positive effects of
enzymes co-immobilization following this strategy, as the second en-
zyme will be exposed to a high concentration of its substrate, produced
by the enzyme located under it (Deng et al., 2010; Forrest et al., 2005,
2007; Güleç et al., 2010; Gutiérrez et al., 2011; Ondul et al., 2012;
Virgen-Ortíz et al., 2017a; Wong et al., 2013).
4.4.3.1. Co-immobilization to prevent the destruction of one of the
intermediate products. However, there are some cases where co-
immobilization is not just a way to save precious time, but rather it is
a requirement to get the desired final product. This scenario occurs in
those cases where one of the intermediate products is unstable and can
suffer a transformation into another undesirable product, reducing the
yields and complicating the final purification steps. Typically, this is the
situation occurring when oxidases are employed; as these enzymes
generate hydrogen peroxide as sub-product of the oxidation of the
target substrate, the deleterious effects of H2O2 are generally limited by
using catalase (Hernandez et al., 2012).
One of the most classical examples of this situation can be found in
S. Arana-Peña, et al. Biotechnology Advances xxx (xxxx) xxxx

CORRECTLY ORDERED MULTILAYER INCORRECTLY ORDERED MULTILAYER

BIOCATALYST: BIOCATALYST:
HIGH EFFICIENCY LOW EFFICIENCY

Non-porous support

Enzyme C: Last in the cascade reaction Product 1: Substrate of enzyme B

Enzyme B: Second in the cascade reaction Product 2: Substrate of enzyme C

Enzyme A: First in the cascade reaction Final product

Fig. 20. Effect of multilayer combi-biocatalysts on lag time using non-porous supports. Importance of the spatial ordering of the enzymes.

the production of α-oxo-acids via the oxidative deamination of amino
acids catalyzed by amino acid oxidases that is shown in Fig. 21. In this
case, hydrogen peroxide byproduct promotes the decarboxylation of the
desired α-oxo-acids (Butò et al., 1994; Fernández-Lafuente et al., 1998;
Lopalco and Stella, 2016; Trost and Fischer, 2002; Upadhya et al.,
1999), therefore reducing the final yields. Using individually im-
mobilized oxidase and catalase, even employing extremely large cata-
lase/oxidase ratios, some keto-acid was destroyed (Fernández-Lafuente
et al., 1998). However, using co-immobilized biocatalysts of catalase
and oxidase, more than 99% of the keto-acid could be obtained. Later,
this idea was used to get 7-aminocephanosporanic acid (7-ACA) using a
D-amino acid oxidase, catalase and glutaryl acylase. The standard
synthetic route does not use catalase, and it permitted the hydrogen
peroxide generated in the oxidation to transform the α-keto-adipyl-7-
ACA, that is the product the oxidation reaction of cephalosporin C
catalyzed by D-amino acid oxidase, to glutaryl-7-ACA, that was later
hydrolyzed by a glutaryl acylase (Volpato et al., 2010) (Fig. 22). The
new strategy proposes to co-immobilize the oxidase and catalase to
produce α-keto-adipyl-7-ACA, but it is mandatory to prevent the for-
mation of glutarayl-7-ACA, that acts as inhibitor of the glutaryl acylase
in the hydrolysis of α-keto-adipyl-7ACA. This permitted a hydrogen
peroxide-free route of production of 7-ACA, saving the problems gen-
erated by this compound in terms of enzyme damage and product sta-
bility (Lopez-Gallego et al., 2005; López-Gallego et al., 2008).
Recently, Orrego et al. (Orrego et al., 2018) reported an im-
mobilized tri-enzyme system able to produce α-oxo acids in “one-pot”
starting from L- or racemic mixtures of several amino acids; these au-
thors combined a broad-spectrum amino acid racemase immobilized on
glyoxyl-activated agarose beads with D-amino acid oxidase and catalase
co-immobilized using the same support and methodology. The coupling

Fig. 21. Mechanisms illustrating the conversion of α-aminoacids to α-oxoacids via oxidation with L or D-aminoacid oxidases, and the possible H2O2-promoted
oxidative decarboxylation of α-oxoacids.

16
S. Arana-Peña, et al. Biotechnology Advances xxx (xxxx) xxxx

Fig. 22. Traditional enzymatic production of 7-ACA from cephalosporin C and the alternative hydrogen peroxide route.

Fig. 23. Top: conversion of aldehydes into mixtures of en-

antiopure (S)-α-hydroxyacids and (S)-α-hydro-
xycarboxamides via sequential action of a (S)-selective hy-
droxynitrile lyase ((S)-HnL) and a non-specific nitrilase
(Nlase), requiring an amidase to increase the production of
(S)-acids. Bottom: conversion of aldehydes into (S)-α-hydro-
xycarboxamides via sequential action of a (S)-HnL and a ni-
trile hydratase (NHase).

of the three immobilized enzymes led to conversions of approximately hydrocyanation of aldehydes to furnish enantiopure cyanohydrins (α-
99% α-oxo acids through a dynamic kinetic resolution process, while hydroxynitriles), are robust and highly effective catalysts, which can be
retaining 100% of its initial effectiveness after 8 reaction cycles. recycled several times without loss of activity. Its use in aqueous media
Hydroxynitrile lyases, catalyzing the enantioselective is somehow limited by the fact that the cyanohydrin is prone to suffer a

17
S. Arana-Peña, et al.
racemization via reversible dehydrocyanation, which would lead to a
low enantiopurity of the final product (Van Rantwijk et al., 2009).
Crosslinked enzyme aggregates (CLEAs) of hydroxynitrile lyases are
excellent catalysts in organic solvents and in these media the non-en-
zymatic hydrocyanation and the previously mentioned racemization
are suppressed. However, its use in aqueous media is also hampered by
the same problem, so its co-immobilization with another enzyme which
could use the cyanohydrine as substrate is an excellent strategy. For
instance, (S)-selective hydroxynitrile lyase from Manihot esculenta
and a non-specific nitrilase from Pseudomonas fluorescens EBC 191
were co-immobilized in a CLEA for transforming benzaldehyde into (S)-
mandelic acid by sequential hydrocyanation and hydrolysis in a bi-
enzymatic cascade at starting concentrations up to 0.25 M (Chmura
et al., 2013) (Fig. 23). In fact, while the use of individual CLEAs of each
enzyme led to an overall enantiomeric excess (ee) of 94%, using the
combi-CLEA of both enzymes the ee reached 98%. Noteworthy, as the
nitrilase produced approx. Equal amounts of (S)-mandelic acid and (S)-
mandelic amide from the corresponding (S)-mandelonitrile inter-
mediate, an increase (up to 0.5 M) in the concentration of HCN induced
a marked drift towards amide production, while by including another
enzyme in the combi-CLEA (the amidase from Rhodococcus erythopolis)
it was possible to obtain (S)-mandelic acid as the only product (90%
yield, > 99% enantiomeric purity). In another report Van Pelt et al.
(Van Pelt et al., 2009) described a combi-CLEA using the (S)-selective
hydroxynitrile lyase from Manihot esculenta and a nitrilehydratase
from Nitriliruptor akaliphilus for the one-pot synthesis of five (S)-hy-
droxycarboxamides from the corresponding aldehydes, with good
yields (> 95%) and enantiomeric excess values (84–90%) similar to
those obtained following the hydrocyanation in microaqueous media
with the hydroxynitrile lyase and an excess of HCN.
4.4.3.1.1. Co-immobilization of oxidases and catalases to protect the
oxidase from hydrogen peroxide inactivation. Hydrogen peroxide is a
byproduct of one of the most utilized enzyme families, oxidases
(Hernandez et al., 2012). However, these enzymes do not withstand
high concentrations of this reactive reagent (Hernandez et al., 2012).
The use of catalase has been proposed to prevent the oxidase in-
activation by oxidation (Hernandez et al., 2012). Moreover, as catalase
generates oxygen, the immobilized oxidase activity may be improved.
Based on the data above, it may be expected that the use of co-im-
mobilized oxidase and catalase should be more efficient than the use of
individually immobilized enzymes. Unfortunately, the advantages of
co-immobilization versus individual immobilization with this goal have
not been studied in any paper. However, this protective effect has been
utilized, and many papers report the co-immobilization of catalases and
oxidases. To cite some recent references, for instance, Liao et al. (Liao
et al., 2019) reported the co-immobilization of glucose oxidase and
catalase onto poly glycidyl methacrylate (PGMA) spheres, synthesized
using suspension polymerization. Similarly, Mafra et al recently de-
scribed a combi-CLEA of these same enzymes (Mafra et al., 2019), while
Zhuang et al. (Zhuang et al., 2019) have reported the co-immobilization
of both enzymes onto a tailor-made porous polymeric resin, comparing
three different operational strategies: (i) adsorption-crosslinking, ad-
sorbing a mixed solution of both enzymes onto plain resin and per-
forming a subsequent crosslinking with glutaraldehyde; (ii) cross-
linking-adsorption, activating the support with glutaraldehyde and
adding the mixed solution as the last step, and (iii) crosslinking-ad-
sorption-crosslinking, using the derivative prepared as described in (ii)
and adding an extra amount of glutaraldehyde. This last derivative
showed the best performance, in terms of both activity and stability.
Interestingly, the use of an extrusion-based bio-printing (3D printing)
technique in a one-pot preparation of the co-immobilized enzymes has
been described by Shen et al. (Shen et al., 2019), using a sodium algi-
nate/polyacrylamide/hydroxyapatite hybrid interpenetrating polymer
network hydrogel; with this system, authors reported an increase in the
catalytic efficiency with the higher porosity of the immobilized en-
zymes, observing a good operational stability, and a high conversion of
18
Biotechnology Advances xxx (xxxx) xxxx
97% after is reuse in 4 batches.
A very remarkable example has been stated by Chen et al. (Chen
et al., 2018), using zeolitic Zn2+-imidazolate cross-linked framework
nanoparticles as “smart” glucose-responsive carriers for the controlled
release of different drugs. These nanoparticles are loaded with the re-
spective drug and glucose oxidase, so that the oxidation of glucose
proceeds leading to H2O2 and gluconic acid; thus, the degradation of
the nanoparticles caused by the lowering of the micro-environmental
pH releases the loaded drugs. To avoid the potential cytotoxicity of
these metal-organic-framework nanoparticles originated from the gen-
eration of H2O2, the co-encapsulation of catalase was carried out,
showing the potentiality of this approach.
In another example, (Wu et al., 2019a, 2019b) the co-immobiliza-
tion of galactose oxidase, catalase, and horseradish peroxidase into
Cu3(PO4)2 nano-flowers has been reported, to produce furan-2,5-di-
carbaldehyde. This is the starting material for synthesizing a plethora of
very valuable products (Irshad et al., 2019). In the paper, the authors
started from 5-hydroxymethylfurfural, obtained from lignocellulosic
biomass through the dehydration of C6 sugars fraction (Van Putten
et al., 2013). For this system, an oxidative damage was observed when
using high concentrations of 5-hydroxymethylfurfural; additionally, the
accumulated hydrogen peroxide produced by galactose oxidase causes
tri-enzyme inactivation. Remarkably, the resulting co-immobilized tri-
enzymatic system showed an increased tolerance towards the oxidative
damage compared to free tri-enzymes, as well as an enhanced thermal
stability, therefore leading to slightly higher yields. These authors
stated that this behavior could be attributed to a heightened protective
effect provided by catalase to the activity of galactose oxidase, owing to
the physical proximity between them on the same support.
Also worth noting are the reports from García-García et al. (García-
García et al., 2018) describing the co-immobilization of diamine oxi-
dase (DAO) and catalase onto glyoxyl-agarose and cyanogen bromide
Sepharose, and Gao et al. (Gao et al., 2019), showing the co-im-
mobilization of glucose oxidase and chloroperoxidase on mesoporous
TiO2 thin films.
4.5. Co-immobilization of enzymes and cofactors
Some of the most interesting cascade reactions involve enzymes that
require some cofactors (NAD(P)H, NAD(P)+ H+, ATP, etc.), that are
consumed in the reaction mol to mol (Kapp and Bamforth, 2002; Kara
et al., 2014; Kragl et al., 1996; Liu and Wang, 2007; Uppada et al.,
2014; Wang et al., 2013). These compounds are too expensive to be
used mol of cofactor per mol of product, and in order to shift the re-
action equilibrium in the product direction, a great excess of the co-
factor should be necessary. That way, there is a necessity of reusing the
cofactors, usually coupling the main reaction to a second reaction
catalyzed by another enzyme that regenerates the cofactor and helps
shift the equilibrium towards the desired direction (Kapp and Bamforth,
2002; Kara et al., 2014; Kragl et al., 1996; Liu and Wang, 2007; Uppada
et al., 2014; Wang et al., 2013). This way, a much lower amount of
cofactor than of target product may be used. However, it should be even
more interesting to design strategies to reuse the cofactor, as even in a 1
to 1000 ratio the cofactor prices may have an impact in the final price
of the product. Recently, Lopez-Gallego has shown how it is possible to
immobilize polyphosphate cofactors in anion exchangers, as for ex-
ample supports coated with polyethylenimine (PEI) (Benítez-Mateos
et al., 2017, 2018a; Sánchez-De Alcázar et al., 2019; Schmid-Dannert
and López-Gallego, 2019; Velasco-Lozano et al., 2017; Velasco-Lozano
and López-Gallego, 2018) (Fig. 24). This permits a mobility of the co-
factor that can move between the main and the cofactor regeneration
enzymes. This is a very elegant solution to the problem, as it permits not
only the cofactor recycling, but also the reuse of the cofactor molecules
by several reaction courses or the use of the combi-biocatalysts in a
continuous reactor (Velasco-Lozano et al., 2017). Using porous sup-
ports, this is another example where the system can only work if both
S. Arana-Peña, et al. Biotechnology Advances xxx (xxxx) xxxx

ENZYME CO-IMMOBILIZATION USE OF THE COMBI-BIOCATALYST UNDER

VIA ION EXCHANGE ON PEI CONTINUOUS OPERATION WITHOUT
COATED POROUS SUPPORTS ADDITIONAL EXOGENOUS COFACTOR

Ionic
adsorption of
the cofactor

ENZYMES INDIVIDUALLY IMMOBILIZED USE OF THE INDIVIDUALLY IMMOBILIZED

VIA ION EXCHANGE ON PEI COATED ENZYMES UNDER CONTINUOUS
NON-POROUS SUPORTS OPERATION WITHOUT ADDITIONAL
EXOGENOUS COFACTOR

Ionic
adsorption of
the cofactor

Main enzyme Cofactor regenerating enzyme Cofactor

Fig. 24. Enzymatic reactions involving cofactors. Co-immobilization of main enzyme and cofactor regenerating enzymes with cofactors using porous and non-porous
supports. An alternative to reuse the cofactors in a continuous way.

enzymes are co-immobilized with the cofactor in the same particle
(Fig. 24). Using non-porous nanoparticles, it may be possible that the
system can work using independently immobilized enzymes, although
perhaps with a lower efficiency (but, as far as we know, this has not
been studied) (Fig. 24).
This idea of cofactor immobilization but permitting some mobility
has opened the door to the development of artificial cell-like combi-
biocatalysts, and the potential may be huge. This has just started to be
explored (López-Gallego et al., 2017).
4.6. Importance of the spatial enzyme ordering in a combi-biocatalysts
To take full advantage of the co-immobilization on porous supports,
it has been shown that the order of the enzymes is a key point (Rocha-
Martín et al., 2012). At first glance, this is “logic”. If the second enzyme
is immobilized in the core of the particle and the first enzyme in the
external surface, a large amount of product 1 will diffuse to the out-side
and will not attacked by the enzyme as shown in Fig. 25. We should
remember that the elimination of the lag time before the product 1 is
spontaneously altered is one of the examples where the co-im-
mobilization becomes fully necessary (see Section 4). At first glance, the
best configuration will be with the first enzyme in the core and the
second in the outside, that way the product 1 will be passing through
the layer of enzyme 2 when leaving the biocatalyst particle and will be
modified at high concentrations (Fig. 25). This configuration may not
be easy to achieve because it is necessary that the involved enzymes
immobilize very rapidly to form concentric crowns (Fig. 25). The other
positive alternative is the co-localization of all enzymes, that is, the
perfect enzyme mixture (Fig. 25). Again, this may be not so easy to
achieve, as exactly similar immobilization rates on a support may not
be an easy goal. It may be required to immobilize each enzyme one by
one under conditions where the enzymes that immobilize faster reduce
this rate, enabling the existence of some holes between the immobilized
enzyme molecules and that may be covered by the other enzymes
(Fig. 7). It should be remembered that also the amount, or more ade-
quately, the enzyme activity, ratio between the different enzymes of
each of the involved enzymes must be optimized to get optimal effects
(Deng et al., 2010; Forrest et al., 2005, 2007; Güleç et al., 2010;
Gutiérrez et al., 2011; Ondul et al., 2012; Virgen-Ortíz et al., 2017a;
Wong et al., 2013). Furthermore, as stated in Section 3.5, this may
change if enzyme stabilities are not similar during the operational life
of the combi-biocatalyst. In a nice paper, using some main enzymes and
some cofactor regeneration enzymes co-immobilized on a glyoxyl-IMAC
support, it was showed by controlling the immobilization rate of the
poly-His tagged enzyme on the IMAC groups that while the “bad” lo-
calization of the enzymes produced biocatalysts with a poor perfor-
mance, the co-localization of the enzymes in the support surface per-
mitted to get biocatalysts that are more active than the free enzymes
under conditions where the cofactor was in very low concentrations
(Rocha-Martín et al., 2012).
One simple strategy to get the spatial ordering of the enzymes is the
use of a strategy involving the immobilization of one layer of enzyme
over the previous layer of enzyme. If the support is fully coated with
each enzyme, we can regulate the amount of enzyme layers and the
distribution of the enzymes in a simple way, not on the surface but in
the volume, and this will be even easier using non-porous supports,
where the number of layers will have no limit, but it is also possible
using porous supports (Arana-Peña et al., 2019b, 2020a; Deng et al.,
2010; Forrest et al., 2005, 2007; Güleç et al., 2010; Gutiérrez et al.,
2011; Ondul et al., 2012; Rios et al., 2019a; Virgen-Ortíz et al., 2017a;
Wong et al., 2013). However, as explained in Section 3.4, the tortuosity
of the path of the substrate to the enzymes in the inner layers must be
considered, along with the increase in diffusional limitations, as these
may reduce the effectiveness of the biocatalyst (Bolivar et al., 2013;
Boniello et al., 2010; Pronk et al., 2014; Regan et al., 1974; Shen and
Chen, 2007).

19
S. Arana-Peña, et al.
Enzyme C: Last in the cascade reaction
Enzyme B: Second in the cascade reaction
Enzyme A: First in the cascade reaction
Fig. 25. Spatial enzyme ordering on porous supports immobilizing the involved enzymes on the support surface: importance of the enzyme ordering.
5. Some current solutions to the problems of co-immobilization
Considering the kinetic advantages, but mainly the cases where co-
immobilization is a requisite to get the desired products, some research
has been performed to solve some of the co-immobilization problems
previously stated (Section 3), mainly the necessity of discarding the
whole biocatalysts when only the least stable enzyme has been in-
activated. This problem was not pointed until 2011 (Garcia-Galan et al.,
2011), and recently some solutions have been offered. At the moment,
solutions involve the immobilization of one enzyme via a reversible
mechanism (the least stable enzyme), while the other enzyme is im-
mobilized using a methodology that permits to greatly increase the
enzyme properties (e.g., stability), and may be covalent or reversible,
but following another strategy from the one used for the most stable
enzyme(s).
5.1.1. Immobilization of the least stable enzymes on the most stable enzyme
previously immobilized on the support
This first strategy is mainly useful when the least stable enzyme is
also difficult to stabilize via immobilization, making it feasible to dis-
card these stabilizing effects and focusing on the reuse of the most
stable enzyme (Peirce et al., 2016) as exemplified in Fig. 26. Following
the strategy, the most stable enzyme is immobilized using a strategy
that greatly increases its performance. The case where the system was
described for the first time was the immobilization of the lipase B from
Candida antarctica via interfacial activation on octyl-agarose (Manoel
et al., 2015), a method that permits the one-step purification, im-
mobilization, stabilization, maintenance of the open form of the lipase
and prevention of lipase dimer immobilization (Rodrigues et al., 2019).
Next, the immobilized enzyme was coated with PEI. This cationic
polymer exerts many positive effects on enzyme features (Abian et al.,
2002; Bolivar et al., 2009; Dos Santos et al., 2014; Fernandez-Lopez
et al., 2017; Mateo et al., 2006a; Virgen-Ortíz et al., 2017a; Zaak et al.,
2017a) and also generates an anion exchanger surface, either in sup-
ports (Mateo et al., 2000; Pessela et al., 2003) or in proteins. That way,
this treatment “activates” the immobilized enzyme in a way that can
20
Biotechnology Advances xxx (xxxx) xxxx
INCORRECTLY ORDERED
MULTILAYER
S BIOCATALYST:
LOW EFFICIENCY
S CORRECTLY ORDERED
MULTILAYER
BIOCATALYST:
HIGH EFFICIENCY
Product 1: Substrate of enzyme B
Product 2: Substrate of enzyme C
Final product
strongly immobilize the least stable enzyme, in this case a β-galactosi-
dase from Aspergillus niger. After lactase inactivation, it can be released
from the support by using appropriate conditions, together with some
of the cationic polymer molecules, and after a new incubation with PEI
solution to fully coat the immobilized enzyme molecules with this ionic
polymer; a new batch of lactase may be immobilized on the im-
mobilized lipase that maintained full activity for many cycles (Peirce
et al., 2016) (Fig. 26). To avoid the necessity of the PEI recoating of the
immobilized enzyme, that is also released from the enzyme, the
polymer was covalently attached to the lipase, that was previously
activated with glutaraldehyde, and the support, because heterofunc-
tional glyoxyl-acyl supports were used (Zaak et al., 2017b). Both new
steps further improved the lipase stability and in some instances even
the activity (Barbosa et al., 2012; Fernandez-Lopez et al., 2017, 2018;
Palomo et al., 2007; Rueda et al., 2016; Zaak et al., 2017a).
More recently, a similar strategy was used to co-immobilize several
lipases, some very stable ones (lipases A and B from Candida antarctica,
lipase from Thermomyces lanuginosus) and some less stable ones (lipase
from Rhizomucor miehei and Lecitase) (Arana-Peña, in preparation). The
most stable lipases were immobilized on octyl-vinylsulfone hetero-
functional supports, enabling the further stabilization of the lipases
(Albuquerque et al., 2016). The coating of the enzymes with PEI per-
mitted to immobilize the least stable lipases on these more stable ones,
enabling the reuse of the most stable lipases by several cycles of the
least stable lipase inactivation, desorption, re-coating of the im-
mobilized enzyme with PEI and immobilization of a new batch of least
stable lipase immobilization.
5.1.2. Step by step immobilization of lipases on octyl-glyoxyl agarose
Lipases are a special case of enzymes presenting the so called in-
terfacial activation, that permits the enzyme to become adsorbed on the
hydrophobic drops of their natural substrates, drops of oils or fats
(Fig. 27) (Brzozowski et al., 1991; Cheng et al., 2018; Grochulski et al.,
1993; Van Tilbeurgh et al., 1993; Verger, 1997). This immobilization
method is the most utilized to immobilize lipases (Rodrigues et al.,
2019), as it permits the one step immobilization-purification-stabiliza-
tion-hyperactivation of lipases, in many instances giving higher stabi-
lities that multipoint covalent immobilization (Dos Santos et al., 2015a,
S. Arana-Peña, et al. Biotechnology Advances xxx (xxxx) xxxx

“Activation” of the first enzyme

Most stable Least stable
enzyme PEI enzyme

Immobilization of the most Reversible immobilization

stable enzyme following REUSE OF THE MOST of the least stable enzyme
its best protocol STABLE IMMOBILIZED
ENZYME
Operation

Inactivation of
the least stable
Inactivated enzyme
enzyme release
INCUBATION
UNDER PROPER
CONDITIONS

Fig. 26. Combi-biocatalysts prepared by immobilizing the most stable enzymes on the support surface and the least stable enzymes over them by a reversible
strategy: reuse of the most stable immobilized enzyme.

Drop of oil
Lipase conformational
equilibrium

INTERFACIAL
ACTIVATION
Lipase adsorbed in its open
form on the surface of the
drop of oil
Hydrophobic
support

Lipase adsorbed via its open

form on the hydrophobic
support surface

Fig. 27. Lipase interfacial activation and its immobilization via interfacial activation on hydrophobic surfaces.

21
S. Arana-Peña, et al. Biotechnology Advances xxx (xxxx) xxxx

Incubation
under proper
conditions

Lipase immobilization via interfacial Interfacially activated and covalently

activation on heterofunctional immobilized lipase:
acyl-reactive groups support ENZYME RELEASE IS NO LONGER
POSSIBLE

Acyl group

Chemically reactive group

Covalent bond
Fig. 28. Immobilization on heterofunctional acyl supports to prevent lipase release during operation.

2015b), as this stabilized open form of the lipases is more stable than reusing the immobilized most stable enzymes (Arana-Peña et al.,
the lipase in the conformational equilibrium (Cygler and Schrag, 1999; 2019a). The inactivation conditions may be at any pH or in the pre-
Jaeger et al., 1993; Kim et al., 1997). That way, immobilization of sence of solvents, as long as the most stable lipase remains almost fully
several lipases on hydrophobic supports is a very suitable method for active under conditions where the least stable enzyme is inactivated
lipase co-immobilization. However, the problem of what to do when the (Rios et al., 2019b).
least stable lipases are inactivated remains. The desorption of the en-
zymes from the support using detergents (Rodrigues et al., 2019) may
6. Future prospects
permit the reuse of the support, but the reuse of the active enzyme may
require a purification step, that increases the price and can be too ex-
We expect that this review has offered enough reasons to use co-
pensive. Moreover, this method, although presenting many advantages
immobilized enzymes only when it has clear advantages comparing all
(Rodrigues et al., 2019) has some problems: the lipase may be un-
parameters and circumstances with the use of individually immobilized
controlledly released from the support if submitted to high tempera-
enzymes. It must be stressed that the global process course should be
tures, organic solvents or substrates/products with detergent properties
considered, not just initial rates. Operational stability of the co-im-
(Hirata et al., 2016a, 2016b; Rueda et al., 2015b; Virgen-Ortíz et al.,
mobilized enzymes adds more difficulties to the co-immobilization
2017b). Recently, heterofunctional acyl supports have been reported to
design. The studies performed to ensure these advantages should in-
be a very good alternative to just hydrophobic supports for lipase im-
clude many aspects, not only the ratio between enzyme activities and
mobilization, as they prevent the lipase release during operation
enzyme distribution inside the biocatalyst particles, but also inactiva-
(Rueda et al., 2015b) (Fig. 28). Even the immobilized enzymes may be
tion of the different enzymes over the operational lifetime.
submitted to reactivation strategies through unfolding by incubation in
Improvements in modelling of the kinetic features of the co-im-
urea or guanidine and refolding by incubation in aqueous medium
mobilized enzymes over time may be critical to determine the relation
(Rueda et al., 2015a; Suescun et al., 2015).
between all involved enzyme activities that should be used to permit
These supports have been used to get to the co-immobilization of
the use of the biocatalysts under optimal conditions for a reasonable
lipases from different sources (Arana-Peña et al., 2019a) as shown in
time period. That is, the researchers should always compare the per-
Fig. 29. The most stable lipases have been first immobilized via inter-
formance of the individually immobilized enzymes, and that of the
facial activation and later via covalent bonds, reduced with sodium
combi-biocatalysts at least for one full reaction cycle. The use of in-
borohydride to transform the imino bonds into stable secondary amino
dividually immobilized enzymes allows adding new doses of the least
bonds and the aldehyde groups in inert hydroxyl groups, that way the
stable immobilized enzyme after its inactivation without discarding the
support is no longer able to covalently immobilize enzymes (Arana-
other, despite losing the kinetic advantages of co-immobilization. Now,
Peña et al., 2019a). Next, the least stable enzymes may be immobilized
most publications show just the combi-catalysts performance and do
via interfacial activation on the chemically inert but hydrophobic sur-
not consider all the problems generated. Multipurpose biocatalysts
face. After inactivation of the least stable lipases, the combi-biocatalysts
should be substituted by individual biocatalysts, which can be used in
may be incubated in detergents to release the inactivated lipases, ex-
an individual way or combining different biocatalysts if it is necessary.
haustively washed with water to eliminate the detergent from the
However, enzyme co-immobilization may be an impressive oppor-
biocatalyst and the new least stable enzyme may be immobilized via
tunity to get new synthetic routes. The design of artificial cells bearing
interfacial activation. This may be used for many reaction cycles,
only the desired enzymes and cofactors is one of the most appealing

22
S. Arana-Peña, et al.
Most stable
lipase
Incubation
at pH 10
Heterofunctional Immobilization of the
acyl-glyoxyl support most stable lipase via
interfacial activation
Acyl group
Glyoxyl group
Imino bond
Secondary amine
bond
Reduced glyoxyl
group
DETERGENT
INCUBATION
Inactivated lipase
release
Fig. 29. Use of heterofunctional acyl supports to co-immobilize lipases enabling the reuse of the most stable immobilized lipases by a step by step lipase im-
mobilization.
possibilities, the opportunity of simultaneously co-immobilizing several
polyphosphate cofactors and enzymes to make complex “pseudo” me-
tabolic chains open the door to new prospects to produce very complex
and expensive products in a much cheaper and controlled way than
using whole resting or living cells (Benítez-Mateos et al., 2017, 2018a;
Sánchez-De Alcázar et al., 2019; Schmid-Dannert and López-Gallego,
2019; Velasco-Lozano et al., 2017; Velasco-Lozano and López-Gallego,
2018). We are pretty sure that this will be a main topic in the future
development of biocatalysis and cascade reactions. Here the price of the
biocatalysts may not be a key as these products may be expensive en-
ough to use non-optimized biocatalysts. For example, the sialyl-Lewis X
glycal, a tetrasaccharide, was one of the first great successes of cascade
biocatalytic reactions, and it used a dozen of enzymes and many co-
factors in a free form (Gijsen and Wong, 1995; Lin et al., 1995).
On the other hand, for processes where the added value of the
products is not so high and the price of the biocatalysts is a critical
factor for the economical sustainability of the process, it is not only
necessary to develop new useful strategies for any situation where it
may be possible to get the best results in the immobilization for all
involved enzymes, but also permitting the reuse of the most stable
enzymes when the least stable enzyme has been inactivated (Garcia-
Galan et al., 2011). Exploring new heterofunctional supports and na-
nomaterials to reach this goal should be a main topic in the future
trends.
Thus, the near future in this area will have two likely objectives. The
first one will be to perform extremely complex processes involving
many co-immobilized enzymes and cofactors, where the price of the
biocatalyst is not a problem and the problems of enzyme co-im-
mobilization may be diminished (although they will exist). The second
one will be to develop strategies to solve the limitations of the current
23
Biotechnology Advances xxx (xxxx) xxxx
Interfacially activated and covalently
Immobilized lipase
Reduction:
Support without
chemical reactivity
Least stable
lipase
REUSE OF THE MOST
STABLE IMMOBILIZED
LIPASE Immobilization of the
least stable lipase via
interfacial activation
Least stable
lipase
inactivation
during
operation
co-immobilization systems for process where the biocatalysts expenses
may be excessive. These new co-immobilization strategies should
permit to take full advantages of the immobilization to improve enzyme
properties, permitting to use different supports, active groups and im-
mobilization protocols to get the best possible biocatalysts from each
involved enzyme and at the same time to take advantages of the co-
immobilization. Moreover, it is necessary to avoid the problem of the
different stabilities of the enzymes, even after stabilization via im-
mobilization. This way, it will no longer be compulsory to discard fully
active immobilized enzymes when one of the enzymes has been in-
activated.
Acknowledgments
We gratefully recognize the financial support from Ministerio de
Ciencia e Innovación-Spanish Government (project number CTQ2017-
86170-R) and Generalitat Valenciana (PROMETEO/2018/076). RMS
thank to Ministerio de Educacion -Spanish Government for a FPU fel-
lowship, DC thank to Ministerio de Ciencia e Innovacion-Spanish
Government by a FPI.
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