Enzyme-catalyzed protein crosslinking

Abstract

The process of protein crosslinking comprises the chemical, enzymatic, or chemoenzymatic formation of new covalent bonds between polypeptides. This allows (1) the site-directed coupling of proteins with distinct properties and (2) the de novo assembly of polymeric protein networks. Transferases, hydrolases, and oxidoreductases can be employed as catalysts for the synthesis of crosslinked proteins, thereby complementing chemical crosslinking strategies. Here, we review enzymatic approaches that are used for protein crosslinking at the industrial level or have shown promising potential in investigations on the lab-scale. We illustrate the underlying mechanisms of crosslink formation and point out the roles of the enzymes in their natural environments. Additionally, we discuss advantages and drawbacks of the enzyme-based crosslinking strategies and their potential for different applications.

Full text

MINI-REVIEW
Enzyme-catalyzed protein crosslinking
Tobias Heck &Greta Faccio &Michael Richter &
Linda Thöny-Meyer
Received: 25 September 2012 /Revised: 1 November 2012 / Accepted: 2 November 2012
#The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract The process of protein crosslinking comprises the
chemical, enzymatic, or chemoenzymatic formation of new
covalent bonds between polypeptides. This allows (1) the
site-directed coupling of proteins with distinct properties
and (2) the de novo assembly of polymeric protein net-
works. Transferases, hydrolases, and oxidoreductases can
be employed as catalysts for the synthesis of crosslinked
proteins, thereby complementing chemical crosslinking
strategies. Here, we review enzymatic approaches that are
used for protein crosslinking at the industrial level or have
shown promising potential in investigations on the lab-scale.
We illustrate the underlying mechanisms of crosslink for-
mation and point out the roles of the enzymes in their natural
environments. Additionally, we discuss advantages and
drawbacks of the enzyme-based crosslinking strategies and
their potential for different applications.
Keywords Cross-linking .Conjugation .Ligation .Fusion
proteins .Transglutaminase .Sortase A
Introduction
The process of joining protein molecules through intermo-
lecular covalent bonds is commonly referred to as protein
crosslinking. It gives rise to the creation of new macromo-
lecular assemblies that frequently reveal physicochemical
properties and functionalities different from those of the
sole parent compounds. As illustrated by Wong and
Jameson (2012), the terms “protein crosslinking”and
“protein conjugation”are sometimes used to discriminate
between the covalent linkage of proteins that have a natural
affinity in vivo and the covalent coupling of two unrelated
protein species. To avoid confusion in the terminology, we
shall use the term “protein crosslinking”in a general sense
throughout this review article to specify the formation of
one or more covalent bonds between proteins.
Consequently, we will cover approaches that result either
in the targeted fusion of distinct protein molecules by a
single covalent bond or in the formation of protein networks
by multiple covalent bonding.
Modifying the properties of proteins by site-directed
fusion or network formation is of great significance for
applications in many fields such as food processing, leather
and textile fabrication, tissue engineering as well as biochem-
ical and biomedical research. The generation of covalent
bonds between proteins can be induced physicochemically
by application of heat, alkaline conditions, mechanical agita-
tion, or photooxidative treatment (Gerrard 2002;Singh1991)
by addition of chemical crosslinkers or by enzyme catalysis.
Chemical crosslinking methodologies take advantage of the
enormous diversity of available crosslinking reagents that
differ in chemical functionality, reactivity, and size (Wong
and Jameson 2012). Homobifunctional and heterobifunctional
chemical crosslinkers carry two reactive groups to target pro-
teins at the same or at different functional groups, respectively.
Among these, glutaraldehyde probably represents the most
commonly used crosslinking reagent (Migneault et al. 2004).
Additionally, there are a few examples of multifunctional
crosslinkers that can be used to target proteins at more than
two functional groups simultaneously. Protein crosslinking
with bi- and multifunctional crosslinkers leads to the incorpo-
ration of molecular spacer groups of defined length and com-
position between the reaction partners. By contrast,
monofunctional crosslinkers (e.g., formaldehyde) react such
that with the exception of the reactive moiety no additional
T. Heck :G. Faccio :M. Richter :L. Thöny-Meyer (*)
Empa, Swiss Federal Laboratories for Materials Science and
Technology, Laboratory for Biomaterials, Lerchenfeldstrasse 5,
CH-9014 St. Gallen, Switzerland
e-mail: linda.thoeny@empa.ch
Appl Microbiol Biotechnol
DOI 10.1007/s00253-012-4569-z

linker is introduced into the final product. Furthermore, acti-
vating agents such as carbodiimides are widely used to direct-
ly connect proteins without incorporating a spacer (Wong and
Jameson 2012).
In addition to the prevalent chemical crosslinking
reagents, enzymes are increasingly employed as catalysts
to promote the introduction of covalent bonds between
protein molecules. In this article, we review the diversity
of enzymatic approaches that can be used for protein cross-
linking in vitro. By addressing the physiological back-
grounds of the enzymes and their underlying coupling
mechanisms, we illustrate how these biomimetic approaches
have emerged to complement the toolbox of protein cross-
linking strategies. Eventually, we discuss advantages and
limitations of choosing and utilizing enzyme-based cross-
linking strategies for different practical scenarios.
Enzymatic protein crosslinking in vivo
The posttranslational covalent modification of proteins is
essential to pro- and eukaryotic cells in order to increase
the structural and functional diversity of the proteome
(Walsh 2006; Walsh et al. 2005). Most of these modifica-
tions are catalyzed by specific enzymes that have evolved
for their respective tasks. Enzymatically introduced modifi-
cations of proteins occur at the functional groups of amino
acid side chains, which are, in some cases, embedded in the
context of sequence-specific recognition motifs. They com-
prise (1) the addition of organic molecules including cofac-
tors (http://www.ebi.ac.uk/thornton-srv/databases/
CoFactor), oligosaccharides, nucleotides, lipids, and small
moieties such as methyl, acetyl, and phosphoryl groups; (2)
intramolecular transformations such as disulfide bond for-
mation and proteolytic processing; and (3) intermolecular
crosslinking by covalent bond formation between individual
protein molecules (Fig. 1).
One of the most extensively studied cellular protein
crosslinking events is the enzyme-catalyzed covalent tether-
ing of ubiquitin to target proteins. Ubiquitin represents a
small protein of 76 amino acid residues with a size of
~8 kDa. The process of ubiquitin attachment, designated
as “ubiquitylation”or “ubiquitination,”is traditionally asso-
ciated with protein trafficking to the eukaryotic 26S protea-
some for specific degradation (Hershko and Ciechanover
1998; Hershko et al. 1982). Proteins directed to the protea-
some carry a minimum of four covalently linked ubiquitin
molecules that are connected through highly proteolysis-
resistant isopeptide bonds between the ε-amine of Lys48
and the C-terminus of the successive subunit. The activation
and transfer of ubiquitin to target proteins is catalyzed by a
cascade of three enzymes (E1, E2, and E3) in an adenosine
triphosphate (ATP)-dependent manner (Fig. 2). Nowadays,
it is evident that modifications of proteins with ubiquitin and
other ubiquitinlike proteins influences a great variety of
cellular signaling and regulatory processes depending on
the type and length of the attached ubiquitin chain (mono-
or polyubiquitination) and on the lysine residues involved in
the connection between the individual ubiquitin subunits
(Hochstrasser 2009; Spasser and Brik 2012; Weissman et
al. 2011). Only recently, even a prokaryotic ubiquitin-like
protein (Pup) from Mycobacterium tuberculosis has been
described (Darwin 2009), which is coupled to target proteins
by the action of the ligase PafA (Guth et al. 2011).
Lipidation
N-Myristoylation
S-Palmitoylation
S-Isoprenylation
(farnesyl or geranylgeranyl)
Glycosyl phosphatidylinositol
(GPI) anchorin
g
Intermolecular protein crosslinking
Ubiquitination
Fibrin crosslinking
Collagen and elastin crosslinking
Protein attachment to bacterial surfaces
Bacterial pilus assembly
Intramolecular protein
modification
Disulfide-bond formation
Proteolytic processing
(e.g. signal-peptide removal,
pro-protein maturation)
Attachment of small
functional groups
N-, O-, S-andC-Methylation
N-Acetylation
Phosphorylation
Sulfurylation
Hydroxylation
Cofactor attachment
Biotinylation
Phosphopantetheinylation
Lipoic-acid attachment
Tet r a
py
rrole/heme li
g
ation
TARGET
PROTEIN
Glycosylation
O-, N-andC-Glycosylation
ADP-Ribosylation
Fig. 1 Schematic illustration of
enzyme-catalyzed covalent
modifications of proteins in
vivo; for a more comprehensive
overview we refer to Walsh
(2006) and Walsh et al. (2005).
The cartoon depicted in the
center was created with the
program PyMOL and shows the
structure of ubiquitin (PDB ID:
1ubq) (Schrodinger 2010)
Appl Microbiol Biotechnol

Many physiologically important protein crosslinking reac-
tions in higher eukaryotes are accomplished by transglutami-
nases. Eukaryotic transglutaminases are calcium-dependent
enzymes that catalyze the formation of protein networks by
introducing glutamyl-lysyl isopeptide bonds between target
proteins (Fig. 3). The most prominent member of the mamma-
lian tissue transglutaminases is the fibrin-stabilizing factor XIII,
which participates in blood coagulation by crosslinking anti-
parallel fibrin chains to mechanically stable clots. For addition-
al information on the functional diversity of eukaryotic
transglutaminases, we refer to a review article by Lorand and
Graham (2003). In the late 1980s, the first calcium-independent
transglutaminase from the microbial strain Streptomyces
mobaraensis (formerly classified as Streptoverticillium mobar-
aense) was described in the patent and peer-reviewed literature
(Ando et al. 1989; Motoki et al. 1993). Diverse physiological
functions including mycelium growth and differentiation in S.
mobaraensis (Pasternack et al. 1998) as well as spore coat
formation in Bacillus subtilis (Zilhão et al. 2005) are associated
with microbial transglutaminases.
Another naturally occurring crosslinking reaction between
proteins is catalyzed by sortases that constitute a group of
calcium-dependent enzymes embedded in the membrane of
Gram-positive bacteria (http://nihserver.mbi.ucla.edu/Sortase/).
Based on their primary amino acid sequence, sortases are
currently assigned to six different classes (A–F) that exert
highly site-specific transpeptidation reactions (Fig. 3)atthe
bacterial cell surface (Spirig et al. 2011). These include the
anchoring of diverse functional proteins to the growing cell
wall by sortase A (Marraffini et al. 2006; Mazmanian et al.
1999) and the assembly of pili from individual pilin subunits by
sortase C (Hendrickx et al. 2011).
In contrast to the transamidation reactions catalyzed by
transglutaminases and sortases, lysyl oxidases induce the
oxidative crosslinking of collagen and elastin chains in the
extracellular matrix of eukaryotic cells (Lucero and Kagan
2006). The enzymatic step catalyzed by lysyl oxidases com-
prises the deamination of lysyl side chains in collagen and
elastin with the concomitant formation of hydrogen perox-
ide (Fig. 4). The resulting aldehydes can react spontaneous-
ly either with a second aldehyde to yield the corresponding
aldol condensation product or add to the primary amine of a
neighboring lysine residue to generate a Schiff base. The so-
formed crosslinked chains of collagen and elastin constitute
fibrous network structures that largely contribute to the
stabilization of eukaryotic tissues.
Enzymes used for protein crosslinking in vitro
The capability of enzymes to crosslink proteins in vivo
immediately suggests their use for diverse applications in
vitro. Of the aforementioned enzymes, mainly transglutami-
nases have been successfully applied for the introduction of
OH
O
+ ATP AMP + PPi
S
O
SH
NH
On
E 1
E 2
S
O
E 2
E 3
NH2
SH
E 1
Protein 1
Protein 1
Ubiquitin
Ubiquitin
Ubiquitin
Ubiquitin
Fig. 2 Simplified schematic
illustration of the enzymatic
cascade leading to
ubiquitination of target proteins
in eukaryotic cells (Spasser and
Brik 2012)
Fig. 3 Protein crosslinking through transamidation reactions catalyzed by transglutaminase and sortase A. The reactive thioester intermediates
generated at the active site of the enzymes are framed
Appl Microbiol Biotechnol

crosslinks into various protein matrices (Kuraishi et al.
2001; Zhu and Tramper 2008), and also, the crosslinking
reactions catalyzed by sortase A and lysyl oxidase have
recently gained increasing attention. By contrast, the enzy-
matic cascade responsible for protein ubiquitination has not
been investigated for crosslinking applications in vitro, and
indeed, its practical usefulness seems very limited due to the
ATP dependence of the reaction and the complex interplay
of the enzymes E1, E2, and E3 (Fig. 2). Interestingly, further
oxidoreductases that catalyze reactions with nonproteino-
genic substrates in vivo have been investigated for the
synthesis of new covalent bonds between proteins in vitro.
Regarding the enzymatic mechanisms, two types of cross-
linking reactions can be distinguished, i.e., (1) direct cova-
lent bonding catalyzed by transferases (EC 2) and
hydrolases (EC 3) via proteinyl–enzyme–thioester inter-
mediates and (2) enzyme-mediated covalent bonding via
reactive species that are enzymatically generated by oxidor-
eductases (EC 1) and spontaneously react further with pro-
teins to form protein networks. In this section, we outline the
physiological functions and biochemical properties of
enzymes relevant to protein crosslinking applications in
vitro. For summaries of the enzymes discussed herein and
the proposed underlying crosslinking reactions, we refer to
Table 1and to Figs. 3and 4, respectively.
Transferases and hydrolases
The common crosslinking reaction catalyzed by transferases
and hydrolases entirely occurs at the active site of the
Fig. 4 Crosslinking of proteins mediated through oxidation by oxidor-
eductases. The reactive species generated by the enzymes are framed.
The illustration of the laccase- and peroxidase-catalyzed oxidation
reactions is simplified and does not show the stoichiometry of
substrates and products (laccase oxidizes four substrate molecules per
molecule of oxygen, whereas peroxidase oxidizes only two substrate
molecules per molecule of hydrogen peroxide)
Appl Microbiol Biotechnol

Table 1 Overview of enzymes used for protein crosslinking in vitro
Enzyme
(EC number)
Main
function
in vivo
Cofactor/metal
ion dependence
Main applications of enzymatic
crosslinking reaction
Target moiety
for crosslink
formation
Mechanism of
crosslink formation
Recent review articles
Transferases
Transglutaminase
(EC 2.3.2.13)
Crosslinking of
diverse proteins
Calcium (only in
eukaryotic trans-
glutaminases)
Network formation of various food
proteins; production of protein
hydrogels; modification of protein
fibers and leather; site-specific
fusion; and immobilization of
tagged target proteins
Glutamyl side chains
of proteins
Formation of glutamyl-
lysyl isopeptide bonds
Griffin et al. (2002),
Lorand and Graham
(2003), Serafini-Fracassini
and Del Duca (2008),
Yokoyama et al. (2004)
Hydrolases
Sortase A
(EC 3.4.22.70)
Attachment of cell-
surface proteins
Calcium Site-specific coupling and immobilization
of engineered target proteins
Threonine–glycine
peptide bond of
LPXTG sorting motif
Formation of threonine–
glycine peptide bonds
Popp and Ploegh (2011),
Spirig et al. (2011)
Subtilisin
(EC 3.4.21.62)
Extracellular proteolysis –Peptide fragment ligation C-terminally esterified
amino acids of
polypeptides
Formation of peptide bonds Gupta et al. (2002)
Oxidoreductases
Tyrosinase
(EC 1.14.18.1)
Melanin formation Copper Network formation of food proteins
(e.g., in meat, fish, yoghurt, wheat-
based products, and edible protein
films); enzyme crosslinking
Tyrosyl side chains
of proteins; phenolic
compounds
Quinone formation;
subsequent spontaneous
1,4-additions
Claus and Decker (2006),
Faccio et al. (2012), Fairhead
and Thöny-Meyer (2012),
Halaouli et al. (2006)
Laccase
(EC 1.10.3.2)
Lignin degradation
and biosynthesis
Copper Network formation of food proteins
(e.g., in meat, fish, wheat-based
products, and edible protein films)
Tyrosyl side chains of
proteins; phenolic
compounds
Radical formation;
subsequent spontaneous
radical couplings
Mayer and Staples (2002),
Quintanar et al. (2007),
Santhanam et al. (2011),
Witayakran and Ragauskas
(2009)
Peroxidase
(EC 1.11.1.x)
Oxidation of diverse
compounds
Mostly heme Network formation of food proteins
(e.g., in wheat-based products);
production of protein hydrogels;
site-specific fusion of tagged target
proteins
Tyrosyl side chains
of proteins; phenolic
compounds
Radical formation;
subsequent spontaneous
radical couplings
Koua et al. (2009), Veitch
(2004)
Lysyl oxidase
(EC 1.4.3.13)/amine
oxidase (EC 1.4.3.6)
Crosslinking of
collagen and elastin
Lysine tyrosinyl
quinone (LTQ);
copper
Production of protein hydrogels Lysyl side chains
of proteins
Aldehyde formation;
subsequent spontaneous
formation of aldol
condensation and Schiff
base products
Lucero and Kagan (2006)
Appl Microbiol Biotechnol

enzymes and proceeds via formation of a covalent pro-
teinyl–enzyme–thioester intermediate. Nucleophilic attack
by an incoming amine nucleophile at the carbonyl group
of the thioester intermediate releases the bound protein
moiety from the enzyme, leading to formation of a new
peptide bond between the target molecules (Fig. 3).
Transglutaminases (EC 2.3.2.13)
As outlined previously, transglutaminases catalyze a trans-
amidation reaction between glutamyl and lysyl side chains
of target proteins. The catalytic reaction proceeds via gluta-
mine deamination and formation of a protein–glutamyl–
thioester at the active site of the enzyme. Nucleophilic attack
by a lysyl ε-amino group of a second protein at the carbonyl
moiety of the thioester intermediate generates isopeptide-
crosslinked proteins that are largely resistant to proteolysis
by common peptidases (Mariniello et al. 2007). In contrast
to eukaryotic transglutaminases, transglutaminases from mi-
crobial origin are calcium-independent, which represents a
major advantage for their practical use (Griffin et al. 2002;
Yokoyama et al. 2004).
Peptidases (EC 3.4.x)
An exceptional enzyme, which crosslinks proteins in a highly
site-specific manner, is the sortase SrtA (EC 3.4.22.70)
from Staphylococcus aureus (Mazmanian et al. 1999).
Although SrtA is formally assigned to the class of pepti-
dases, the enzyme in vivo catalyzes a transpeptidation reac-
tion to tether surface proteins to the cell wall of Gram-
positive bacteria. SrtA recognizes target proteins containing
a conserved LPXTG amino acid motif, where X represents
any proteinogenic amino acid (Kruger et al. 2004). The
catalytic reaction proceeds via nucleophilic attack by the
enzyme's catalytic cysteinyl thiol group at the threonine–
glycine amide bond within the sorting motif of the target
protein. Subsequently, a proteinyl–enzyme–thioester inter-
mediate is formed with concomitant release of the C-
terminal target protein moiety. An amine nucleophile, the
terminal amino group of an oligoglycine polypeptide in
vivo, eventually releases the covalently bound protein moi-
ety from the enzyme under formation of a peptide bond
crosslink.
Although common peptidases are associated with peptide
bond hydrolysis, examples of enzymes are described that
can, under certain conditions, catalyze peptide bond forma-
tion between polypeptide fragments (Bordusa 2002). In the
so-called kinetically controlled approach, a synthetic poly-
peptide ester serves as the substrate for enzymatic coupling
by peptidases. The crosslinking reaction is initiated by for-
mation of a covalent reaction intermediate between the
catalytic nucleophile of the peptidase and the carboxy
terminus of the substrate. Eventually, nucleophilic attack
by the terminal amino group of an acceptor polypeptide
leads to release of the bound acyl moiety from the enzyme
and crosslink formation between the polypeptide fragments
through a newly established peptide bond.
Oxidoreductases
With the exception of lysyl oxidase, most oxidoreductases
are not explicitly destined by nature to promote the forma-
tion of covalent bonds between proteins. Nevertheless,
members of the oxidoreductases have been widely investi-
gated for their potential to crosslink proteins in vitro because
they lack pronounced substrate specificity. Many oxidore-
ductases react with a broad range of small molecules such as
low molecular weight phenols and/or macromolecular sub-
strates including functional amino acid side chains of pro-
teins. Covalent bond formation between proteins initiated by
the action of oxidoreductases involves at least two sequen-
tial chemical steps (Fig. 4). Of these, only the initial redox
reaction with the primary substrate is directly catalyzed by
the enzyme. The resulting reactive species, i.e., quinones,
radicals, or aldehydes may subsequently undergo nonenzy-
matic conversions to form various types of covalent bonds.
In this article, we focus on oxidoreductases that have been
experimentally shown to catalyze redox reactions directly at
functional amino acid side chains of target proteins. Hence,
we do not include glucose oxidases (Wong et al. 2008) and
sulfhydryl oxidases (Faccio et al. 2011) that are proposed to
induce intermolecular crosslinking of food proteins mainly
by producing reactive hydrogen peroxide from small-
molecule sugars and thiols, respectively.
Tyrosinases (EC 1.14.18.1)
Tyrosinases are dicopper enzymes that are widespread in
pro- and eukaryotic organisms (Claus and Decker 2006;
Faccio et al. 2012; Halaouli et al. 2006). They initiate the
biosynthetic production of melanins by converting L-tyro-
sine to dopaquinone, which subsequently undergoes spon-
taneous reactions to yield melanin. Furthermore, they are
attributed to other distinct functions including phenol detox-
ification in bacteria and cuticle sclerotization in inverte-
brates. Activated tyrosinases have both monophenolase
and diphenolase activity as they are able to catalyze the
hydroxylation of monophenolic compounds to o-diphenols
as well as the subsequent oxidation of these o-diphenols to
the respective o-quinones with concomitant reduction of
molecular oxygen to water (Espín et al. 2000). In addition
to many low molecular weight mono- and diphenolic mol-
ecules, surface-exposed tyrosyl side chains of proteins may
also serve as substrates for tyrosinases that convert them to
the respective o-quinones (Ito et al. 1984;Matheisand
Appl Microbiol Biotechnol

Whitaker 1984a). These are proposed to react spontaneously
mainly via 1,4-additions with the side chains of lysine, tyro-
sine, histidine, and cysteine residues, depending on their
abundance and accessibility on the target protein, to form
covalent protein–protein crosslinks (Bittner 2006). Proteins
with weakly defined three-dimensional structure and unfolded
proteins are the preferred targets for crosslinking by tyrosi-
nases, whereas globular proteins are poorly, if at all, converted
by the enzyme (Hellman et al. 2011;Selinheimoetal.2007b).
However, it has been shown that crosslink formation between
proteins that are not accessible to tyrosinases can be induced
by the addition of small-molecule phenolic compounds
(Fairhead and Thöny-Meyer 2010; Jus et al. 2012;
Thalmann and Lötzbeyer 2002). These molecules likely func-
tion as crosslinking mediators to overcome the absence of
surface-exposed tyrosine residues on the target proteins.
Laccases (EC 1.10.3.2)
Laccases are “blue”multicopper oxidases that have been
discovered in fungi, plants, and bacteria (http://www.lcce
d.uni-stuttgart.de/). Most of the characterized laccases orig-
inating from fungi contribute to the degradation of lignins,
whereas laccases derived from plants are mainly related to
lignin and cell wall biosynthesis (Mayer and Staples 2002;
Singh Arora and Kumar Sharma 2010). Prokaryotic laccases
have been proposed to be involved in many intra- and
extracellular processes, but their exact physiological func-
tions remain mostly elusive (Santhanam et al. 2011). The
active sites of laccases harbor a total of four copper atoms
organized at three distinct sites (T1, T2, and T3) that can be
distinguished by their spectral properties (Quintanar et al.
2007). They catalyze single-electron abstractions from a
wide range of phenols and anilines among other substrates
(Reiss et al. 2011; Witayakran and Ragauskas 2009). The
oxidation of four substrate molecules by laccases goes along
with the concomitant reduction of one equivalent of molec-
ular oxygen to water that is the sole by-product of the
reaction. The formed radicals may undergo subsequent cou-
pling reactions of various types leading to the formation of
different covalently linked products. In proteins, mainly
exposed tyrosyl side chains serve as substrates for oxidation
by laccases, and the resulting phenoxy radicals may sponta-
neously initiate subsequent protein crosslinking reactions
(Mattinen et al. 2006). As for tyrosinases, the addition of
phenolic compounds as crosslinking mediators can be neces-
sary to promote protein crosslinking by laccases (Steffensen et
al. 2008).
Peroxidases (EC 1.11.1.x)
The group of peroxidases encompasses miscellaneous
enzymes that universally occur in microorganisms, plants,
and animals. Presently, members of the peroxidases are
assigned to 20 classes with different EC numbers. Due to
the great complexity and diversity of peroxidases, the data-
base PeroxiBase has been created to summarize information
on properties, functions, and regulation of these enzymes
(https://peroxibase.toulouse.inra.fr/) (Koua et al. 2009).
Most peroxidases contain a prosthetic heme group that is
required for the electron transfer reaction, and also, perox-
idases with other cofactors including flavin adenine dinu-
cleotide (FAD) are described. Peroxidases abstract single
electrons from a broad range of substrates of mostly aro-
matic nature (Gumiero et al. 2010), including surface-
accessible tyrosine residues of proteins (Matheis and
Whitaker 1984b). The oxidation of two substrate molecules
is accompanied by the concomitant reduction of hydrogen
peroxide to water. The phenoxy radicals resulting from
tyrosine oxidation by peroxidases may undergo various
spontaneous reactions including radical couplings, which
mainly leads to the formation of different dityrosyl-type
crosslinks between proteins (Heijnis et al. 2011; Matheis
and Whitaker 1984a).
Lysyl oxidases (EC 1.4.3.13)
As described above, lysyl oxidases initiate the crosslinking
of collagen and elastin chains in the extracellular matrix of
higher eukaryotes (Lucero and Kagan 2006). Hence, they
are among the few enzymes that promote the formation of
protein crosslinks in their physiological environments and
likewise have been used for practical crosslinking applica-
tions in vitro (Bakota et al. 2011). Lysyl oxidases require
two cofactors for catalysis, namely, a covalently bound lysine
tyrosinyl quinone (LTQ) cofactor and a copper ion. They
oxidize the primary amine groups of accessible lysyl side
chains on target proteins to the corresponding aldehydes,
which may undergo subsequent reactions to form the cova-
lently crosslinked aldol condensation or Schiff base product.
Formation of protein networks
The covalent assembly of proteins into macromolecular net-
works by enzyme catalysis has been extensively investigat-
ed in various areas of applications where biological protein
matrices with material-like structural and mechanical prop-
erties are required. The ability of enzymes to form cross-
linked protein networks has, for instance, been exploited to
modify the texture and appearance of food products, to
develop new biomimetic tissue scaffolds, or to strengthen
protein-based fibers for textile fabrication. In this section,
we give an overview of enzymatic approaches that are used
in different fields to create products composed of covalent
protein networks.
Appl Microbiol Biotechnol

Food processing
The modification of food proteins by enzymatic crosslink-
ing can affect the long-term stability required for storage or
the organoleptic properties such as structure, texture, ap-
pearance, and flavor of food products. There is extensive
literature available on the enzymatic crosslinking of proteins
in foods, and many aspects of this field have been covered in
a comprehensive book and review articles (Whitehurst and
van Oort 2010; Buchert et al. 2010; Gerrard 2002; Singh
1991). For this reason, we restrict our discussion to the gen-
eral aspects of enzyme-catalyzed crosslinking approaches that
are used to modify the properties of proteins in food products,
and we explicitly highlight selected studies from this rapidly
progressing field of research.
Enzyme preparations suitable for applications in the food
industry must be tested for toxicity and immunogenicity and
certified with the status Generally Recognized As Safe
(GRAS) defined by the US Food and Drug Association
(FDA). For instance, a GRAS status has been assigned to
transglutaminase preparations from S. mobaraensis for pro-
tein crosslinking in seafood, meat, dairy, and cereal products
(FDA/CFSAN agency response letters: GRAS notice numb-
ers 000004 (1998), 000029 (1999), 000055 (2001), and
000095 (2002)). Nowadays, microbial transglutaminase is
produced on large scale and distributed under the trade name
ACTIVA® by Ajinomoto US, Inc. (http://www.transgluta
minase.com). Transglutaminase is commonly referred to as
“meat glue”due to its ability to assemble pieces of meat by
crosslink formation, thereby restoring the structure of meat
from low-quality fresh meat cuts (Kuraishi et al. 1997)or
improving the textural properties of processed meat gels
(Sun and Arntfield 2011). The transglutaminase-catalyzed
crosslinking reaction in meat and fish products is predomi-
nantly directed towards the myofibrillar protein myosin
(Chanarat et al. 2012; Huang et al. 1992; Lantto et al.
2005), which represents the primary constituent of thick
muscle filaments. In addition to muscle proteins, microbial
transglutaminase shows high crosslinking activity with
caseins from milk (Sharma et al. 2001) and glutenins from
wheat grains (Autio et al. 2005; Basman et al. 2002). This
makes the use of the enzyme attractive for applications in
other areas of the food industry such as the dairy and the
baking sectors (Jaros et al. 2006;Joyeetal.2009). For
example, transglutaminase has been used to induce protein
crosslinking during yoghurt making with the aim of super-
seding the addition of dry matter stabilizers that are com-
monly added to enhance the strength of acidified caseinate
gels (Bönisch et al. 2007; Jaros et al. 2006). In noodle and
bread making, transglutaminase-catalyzed crosslinking has
been shown to change the rheological properties and the
microstructure of dough preparations as compared to un-
treated doughs (Kim et al. 2008; Steffolani et al. 2010;Wu
and Corke 2005). For more detailed summaries on the
applications of transglutaminase in the food sector, we refer
to review articles by Motoki and Kumazawa (2000)and
Kuraishi et al. (2001).
The generation of protein crosslinks by oxidative
enzymes including tyrosinases, laccases, and peroxidases
hasalsobeeninvestigatedinseveralareasofthefood
industry (reviewed in Buchert et al. (2010); Whitehurst
and van Oort (2010)). However, in comparison to trans-
glutaminase, relatively little is known on how exactly these
enzymes affect the chemical compositions of protein matri-
ces in foods. It should be noted here that the reaction of
quinones generated by tyrosinases with free amino acids
may influence the color and aroma of the food product,
which may limit the use of tyrosinases in certain applica-
tions of the food industry (Bittner 2006). Nevertheless,
tyrosinase from the filamentous fungus Trichoderma reesei
has been shown to induce the gelation of acidified milk gels.
In contrast to transglutaminase, the studied tyrosinase did
not require preheating of the milk to allow the enzymatic
crosslinking reaction, which could be advantageous for the
production of certain milk products (Ercili Cura et al. 2010).
It has been proposed that the allergenic properties of the
milk protein casein might be mitigated by enzymatic cross-
linking (Stanic et al. 2010). Selinheimo et al. (2007a) eval-
uated the application of tyrosinase from T. reesei and laccase
from Trametes hirsuta on dough preparations from wheat
flour for bread making. Although protein crosslinking of
gliadins and glutenins induced by these enzymes made the
doughs harder and less extensible, the resulting breads were
more voluminous and had softer crumbs than ordinary
breads.
Food packaging
The development of polysaccharide- and protein-based
polymeric films has recently received attention due to the
need for biocompatible and biodegradable materials that
possess mechanical, swelling, and barrier properties suitable
for food packaging. Edible thin films from various different
proteins have been prepared by enzyme-catalyzed crosslink-
ing with microbial transglutaminase (Di Pierro et al. 2006;
Mariniello et al. 2010), and recently, also with tyrosinase
and laccase (Juvonen et al. 2011). For example, films con-
taining transglutaminase-crosslinked whey proteins embed-
ded in a chitosan matrix showed (1) low solubility in water
(2) reduced permeability for oxygen, carbon dioxide, and
water vapor, and (3) enhanced elongation to break but lower
deformability than noncrosslinked films (Di Pierro et al.
2006). Similar changes regarding the barrier properties were
observed in thin film preparations of grapefruit albedo
homogenates combined with the bean protein phaseolin
after transglutaminase-catalyzed crosslinking. However, in
Appl Microbiol Biotechnol

contrast to the whey protein films studied by Di Pierro et al.
(2006), the transglutaminase-treated albedo homogenate–
phaseolin films not only revealed higher tensile strength
but also an approximately twofold increase in their elasticity
(Mariniello et al. 2010).
Biomimetic materials
Protein- and polysaccharide-based hydrogels (i.e., highly
water-absorbent polymeric networks) have emerged as bio-
logical scaffolds to support three-dimensional tissue formation
because their mesh structures represent good mimics of the
natural extracellular matrix and offer the possibility to entrap
bioactive molecules such as growth factors. Extensive re-
search has been directed towards the development of enzy-
matic approaches for hydrogel network formation (Moreira
Teixeira et al. 2012). The use of enzymes has gained increas-
ing attention particularly for the synthesis of injectable in situ-
forming hydrogels because enzymes usually react under phys-
iological conditions and, in contrast to many chemicals, are
mostly regarded as biocompatible. Protein hydrogels have
been created by enzyme-catalyzed crosslinking from various
protein sources, mostly from collagen or its denatured form
gelatin. For example, Yung et al. (2007) used microbial trans-
glutaminase to generate crosslinked gelatin hydrogels that are
thermally stable in saline solution at body temperature, allow
proliferation of encapsulated HEK293 cells, and facilitate the
transport of secreted therapeutic proteins such as interleukin-2
(Yung et al. 2010). Moreover, synthetic glutamine- and lysine-
rich oligopeptides have been crosslinked with two eukaryotic
transglutaminases to yield modular cytocompatible protein
hydrogels (Davis et al. 2010). Besides hydrogels, a microfluidic
device composed of a transglutaminase-crosslinked gelatin
mold has been designed to analyze attached cell cultures in an
in vivo-like environment mimicking the extracellular matrix
(Paguirigan and Beebe 2006).
In addition to transglutaminases, horseradish peroxidase
has been exploited to induce the gelation of mainly
polysaccharide-based building blocks including chitosan or
dextran derivatives for applications in tissue engineering
(Jin et al. 2009; Jin et al. 2010). Furthermore, Sofia et al.
(2002) demonstrated the peroxidase-catalyzed crosslinking
of protein hydrogels from differently substituted polyas-
partic acid derivatives. Fetal bovine serum, which contains
lysyl oxidase as a natural enzymatic component, and com-
mercially available plasma amine oxidase have been
employed as catalysts for the production of hydrogels by
oxidative crosslinking of lysine-rich synthetic peptide nano-
fibers (Bakota et al. 2011). Gradually advancing gelation
with time was observed, going along with a steady increase
of robustness of the formed peptide hydrogels as determined
by rheology. Lau et al. (2006) reported the development of a
gene delivery system that allowed gene uptake and local
expression of lysyl oxidase by fibroblasts embedded in a
collagen layer mimicking the extracellular matrix. After
3 weeks of culturing, collagen gels with lysyl oxidase-
transfected cells showed threefold increased mechanical
strength over gels harboring control transgenic cells. This
effect could be attributed to additional crosslinking of the
collagen fibers induced by expression of lysyl oxidase in the
matrix-embedded cells.
Textile and leather manufacturing
The treatment of wool proteins, i.e., mainly keratins, with
proteolytic enzymes is a field of active research in textile-
processing applications. Limited enzymatic hydrolysis of
keratin fibers has been shown to improve the shrink resis-
tance and antifelting behavior of woolen fabrics (Wang et al.
2011). However, the breakdown of keratin has to be tightly
controlled because excessive proteolysis causes drastic fiber
damage and reduces tensile strength. The introduction of
additional crosslinks into the keratin network by the use of
enzymes represents an approach to compensate for the loss
of tensile strength caused by peptidase-catalyzed hydrolysis.
Several studies indicated that keratin fibers crosslinked by
microbial transglutaminase maintain higher fabric strength
after (1) proteinase treatment (Cortez et al. 2004; Du et al.
2007), (2) repeated washing cycles with proteinase-
containing detergents (Cortez et al. 2005), or (3) bleaching
with hydrogen peroxide (Montazer et al. 2011) as compared
to the corresponding fibers without enzyme treatment.
Furthermore, transglutaminase has been employed to graft
casein (Cui et al. 2011), gelatin (Cui et al. 2009), and silk
proteins (Cortez et al. 2007) onto wool yarns, generating
products with improved physicomechanical properties such as
increased tensile strength and higher smoothness. In a similar
approach, wool fibers have been functionalized with ε-linked
polylysine, an FDA-approved biomolecule with antibacterial
properties (Wang et al. 2010). Recent studies demonstrated
that tyrosinase can be employed to oxidize tyrosyl side chains
of keratin, allowing crosslinking of proteins such as collagen
andelastintowoolfibers(Jusetal.2009;Lanttoetal.2012).
In the leather industry, transglutaminase-crosslinked prepara-
tions of gelatin and casein have been examined as low-cost
filling materials to stuff voids in animal hides. The crosslinked
fillers were shown to be homogeneously distributed in the
hides, to resist the washing steps during leather processing and
to improve properties of the leather such as grain smoothness
and fullness (Liu et al. 2011; Taylor et al. 2009; Taylor et al.
2007).
Stabilization of technical enzymes
Approaches using chemical crosslinking reagents such as
glutaraldehyde are extensively used for the preparation of
Appl Microbiol Biotechnol

crosslinked enzyme aggregates (CLEAs) from synthetically
relevant enzymes. CLEAs generally exhibit properties desir-
able for process development such as good operational stabil-
ity, high productivity, and recyclability (Sheldon 2011). To the
best of our knowledge, only one enzyme-based approach to
yield CLEAs has been reported in the scientific literature.
Using the tyrosinase from Verrucomicrobium spinosum in
the presence of phenol, Fairhead and Thöny-Meyer (2010)
reported the formation of catalytically active CLEAs from
Candida antarctica lipase B (CALB). A further evaluation
of enzymatically formed CLEAs is needed in order to assess
their usefulness for biocatalytic transformations as compared
to CLEAs produced by traditional chemical crosslinking strat-
egies. Especially factors including catalyst activity and stabil-
ity, production costs, sustainability, and possibility of
enzymatically introduced cross-contaminations should be
addressed.
Site-directed protein fusion
In accordance with the definition given in the “Introduction”,
protein crosslinking not only covers the formation of cova-
lently linked protein networks with material-like properties
but also includes the specific linkage of two proteins on the
molecular level. Traditionally, this is achieved by genetic
assembly of the respective encoding DNA regions and subse-
quent heterologous expression of the fusion protein. This
allows combination of the properties of distinct protein spe-
cies, for example, the binding specificity of an antibody with
the immunoregulatory function of a cytokine to create anti-
body–cytokine fusion proteins for anticancer therapy
(Kontermann 2012). However, if expression or correct folding
of the fusion protein fails, alternative methods are required to
link two separately expressed proteins in a site-specific man-
ner. Native chemical ligation (NCL) represents the most ro-
bust and widely applied strategy for the chemoselective
ligation of two synthetically produced, unprotected polypep-
tide chains (Dawson and Kent2000). In NCL, a native peptide
bond is formed between one polypeptide bearing a chemically
introduced C-terminal thioester and a second polypeptide
containing an N-terminal cysteine residue. NCL has been
complemented by intein-based methods, namely, expressed
protein ligation (EPL) and protein trans-splicing (PTS); for a
review on EPL and PTS, we refer to Muralidharan and Muir
(2006). Besides these synthetic and semisynthetic techniques,
hydrolytic enzymes have been used for the formation of native
peptide bonds between polypeptide fragments. The best
known example is the blockwise assembly of fully active
ribonuclease A from six esterified peptide segments in aque-
ous solution using a genetically optimized double mutant of
subtilisin BPN′(named “subtiligase”)fromBacillus amyloli-
quefaciens (Jackson et al. 1994).
Due to its site specificity for the LPXTG sorting motif,
the sortase SrtA from S. aureus has recently emerged as an
enzymatic tool for the site-directed assembly of proteins by
transpeptidation (Tsukiji and Nagamune 2009). Examples
for the SrtA-catalyzed fusion of proteins include the site-
specific coupling of various globular proteins to an IgG
antibody (Levary et al. 2011) and to a single-chain antibody
(Ta et al. 2011). Under optimized reaction conditions, prod-
uct yields of 40–85 % were reached and it was shown that
the antibody specificity was retained in the fusion proteins.
It is worth mentioning that SrtA can also be employed for
the oriented immobilization of proteins on glycine-modified
solid supports (Parthasarathy et al. 2007), for the site-
specific introduction of various molecular probes (Popp et
al. 2009), and for protein circularization (Antos et al. 2009).
In addition to SrtA, microbial transglutaminase (Takazawa
et al. 2004; Tanaka et al. 2004)aswellashorseradish
peroxidase (Minamihata et al. 2011) have been investigated
as catalysts to promote the site-specific coupling of proteins
carrying engineered peptide tags.
Advantages, challenges, and limitations of enzymatic
crosslinking strategies
The ability of enzymes to promote the formation of covalent
crosslinks between amino acid residues of proteins offers
versatile possibilities for protein modifications, thereby
complementing the toolbox of chemical crosslinking meth-
odologies. As shown in the previous sections, only a very
limited number of enzymes are frequently used for protein
crosslinking purposes. Because these enzymes differ funda-
mentally with regard to their biochemical properties, reac-
tion mechanisms, and substrate specificities (Table 1and
Figs. 3and 4), they can be exploited to access miscellaneous
crosslinked protein products ranging from fusion proteins
connected through a single covalent bond to protein net-
works linked through a multitude of intermolecular covalent
bonds.
Polymeric networks composed of proteins feature
material-like structural and mechanical properties that are
desirable in many fields of applications, as outlined above.
Unlike many chemicals, enzymes are most active under
mild aqueous reaction conditions and their crosslinking
reactions can often be controlled by modifying temperature,
pH, or ionic strength (as shown, for example, in Heijnis et
al. 2010). For the purpose of generating or extending cova-
lent protein networks, enzymes with a low degree of spec-
ificity towards the amino acid sequence of their target
proteins are preferred. In this respect, microbial transgluta-
minase from S. mobaraensis is by far the most extensively
studied and applied enzyme because (1) it is commercially
available in large quantities, (2) it is rated as nontoxic and
Appl Microbiol Biotechnol

nonimmunogenic by the FDA, (3) it does not require cofac-
tors, and (4) it is active over a wide pH range and resists
temperatures up to 50 °C (Motoki and Kumazawa 2000).
Transglutaminase shows residue, but not sequence specific-
ity, and forms defined isopeptide crosslinks between gluta-
mine and lysine residues of proteins. Similarly, phenol
oxidation by tyrosinases, laccases, and peroxidases can be
regarded as a residue-specific reaction in the context of the
22 proteinogenic amino acids because tyrosine represents
the only proteinogenic amino acid that contains a phenolic
moiety. Nevertheless, crosslink formation mediated by oxi-
doreductases is difficult to control because only the initial
activation of the tyrosyl side chains is enzyme-catalyzed,
and the resulting reactive species may undergo spontaneous
follow-up reactions to form a wide array of diverse covalent
crosslinks involving C–C, C–O, C–N, and C–S bonds
(Fig. 4). Transglutaminases as well as oxidoreductases ac-
cept diverse proteins as substrates for crosslinking, provided
they carry the respective amino acid residue required for the
enzymatic reaction at a surface-exposed position. On the
one hand, this implies that any protein lacking accessible
target amino acids will not be enzymatically crosslinked. On
the other hand, the enzyme itself may potentially serve as
substrate for the crosslinking reaction, which could conse-
quently cause its incorporation into the crosslinked protein
network. It should be noted here that oxidoreductases may
also abstract electrons from a wide range of phenols of low
molecular weight such as ferulic or caffeic acid. These
molecules, once activated to the corresponding quinone or
radical species, may act as mediators to crosslink target
proteins that are not directly converted by the enzyme due
to a lack of enzyme-accessible tyrosyl side chains.
While the generation of protein-based polymer networks
relies on the formation of multiple covalent bonds among
individual polypeptide chains, the oriented intermolecular
fusion of two distinct proteins requires the precise formation
of a single covalent bond at a defined position between the
reaction partners. Therefore, the coupling process has to be
tightly controlled and demands a reaction that ensures site
rather than residue specificity. Unlike densely crosslinked
protein matrices, which are commonly intended for indus-
trial bulk applications, site specifically linked fusion pro-
teins are usually produced on a comparably small scale.
Their applications are highly specialized and mostly restrict-
ed to the areas of biochemical and biomedical research. In
addition to the prevalent synthetic and semisynthetic liga-
tion strategies (NCL, EPL, and PTS) (Dawson and Kent
2000; Muralidharan and Muir 2006), proteolytic enzymes
may be employed to catalyze the site-specific formation of
peptide bonds. Although, in most cases, protease-catalyzed
approaches have been exploited to produce peptides of only
short-chain length (Bordusa 2002), the potential of commer-
cially available proteases to assemble intact proteins from
synthetic polypeptide esters has also been demonstrated
(Jackson et al. 1994; Machova et al. 2003). The sortase
SrtA from S. aureus catalyzes transpeptidation reactions of
proteins in vivo and probably represents the most straight-
forward enzymatic approach in vitro to generate site specif-
ically linked fusion proteins. While the aforementioned
chemical- and protease-based approaches to fusion protein
synthesis ensure site specificity by the presence of synthet-
ically introduced C-terminal ester or thioester groups, site
specificity of the SrtA-catalyzed coupling reaction is pro-
vided by the presence of a short LPXTG amino acid motif,
which can be introduced genetically near the C-terminus of
the recombinant target polypeptide (Fig. 3). Advantages
associated with the use of SrtA for the generation of fusion
proteins include (1) facile and efficient production of the
enzyme in E. coli, (2) minimal requirements for target
protein modification due to small recognition motifs, (3)
mild aqueous reaction conditions that sustain the native
conformation of most target proteins, and (4) high yields
of transpeptidation products. However, it is important to
mention that despite the site specificity of SrtA for the
LPXTG-sorting motif, unproductive side reactions may oc-
cur due to the presence of different nucleophiles competing
for the release of the enzyme-bound protein substrate. While
nucleophilic attack by the N-terminal oligoglycine amino
moiety of the fusion partner leads to formation of the direc-
tionally linked protein product (Fig. 3), hydrolysis of the
recognition motif and formation of isopeptidyl-crosslinked
fusion products may also occur as side reactions (Möhlmann
et al. 2011). Thus, thorough reaction optimization and a final
purification step may be necessary for obtaining the desired
fusion protein in a sufficiently pure form. Furthermore, the
need for engineered protein substrates may limit the applica-
tion of the SrtA-catalyzed reaction to the production of high-
value protein products that are difficult to access by chemical
fusion strategies.
Besides the enzymes described in this review, chemical
crosslinking reagents with a broad variety of chemical fea-
tures are available to target the functional groups present in
the amino acid side chains of proteins (Wong and Jameson
2012). While some chemical crosslinkers possess residue
specificity in the context of the naturally occurring amino
acids, others may selectively target different functional
groups in amino acids showing similar chemical reactivity.
Crosslinking strategies based on chemical crosslinkers are
widely used for diverse applications, ranging from funda-
mental studies of protein–protein interactions at the molec-
ular level to the large-scale production of crosslinked
aggregates of technical enzymes. Furthermore, crosslinking
reagents may be employed to provide target proteins with
new noncanonical functionalities for subsequent chemical
modifications, to attach molecular probes such as fluoro-
phores, or to covalently immobilize proteins on solid
Appl Microbiol Biotechnol

carriers. In contrast to enzymes that serve a catalytic func-
tion and thus are restored after each cycle of crosslink
formation, chemical reagents are consumed during the reac-
tion and hence are usually supplied in at least stoichiometric
amounts in order to allow the crosslinking reaction to pro-
ceed to completion.
Despite their broad commercial availability, reaction ef-
ficiency, and versatility, the use of chemical crosslinking
reagents can be unfavorable particularly when applied in
the areas of food processing and tissue engineering, because
many of these compounds are rated as toxic or may form
harmful by-products leaching from the crosslinked protein
matrix. To overcome these limitations, the study of enzymes
that catalyze the formation of protein crosslinks in vitro has
gained broad attention. However, except the established
approaches based on microbial transglutaminase, the field
of enzymatic protein crosslinking is still in its infancy. There
is a need not only to explore and further improve the protein
crosslinking technology using the currently known enzymes
but also to direct future investigations towards the discovery
of enzymes with new crosslinking activities in order to
broaden the range of target amino acids and protein sub-
strates accessible to enzymatic crosslinking.
Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited.
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