Biocatalysis and Biotransformation

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Application of immobilized enzyme technologies

for the textile industry: a review

José C. Soares, Patrícia R. Moreira, A. Catarina Queiroga, José Morgado, F.

Xavier Malcata & Manuela E. Pintado

To cite this article: José C. Soares, Patrícia R. Moreira, A. Catarina Queiroga, José Morgado,
F. Xavier Malcata & Manuela E. Pintado (2011) Application of immobilized enzyme technologies
for the textile industry: a review, Biocatalysis and Biotransformation, 29:6, 223-237, DOI:
10.3109/10242422.2011.635301

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Published online: 14 Nov 2011.

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Biocatalysis and Biotransformation, November–December 2011; 29(6): 223–237

REVIEW

Application of immobilized enzyme technologies for the

textile industry: a review

JOSÉ C. SOARES1, PATRÍCIA R. MOREIRA1, A. CATARINA QUEIROGA1,

JOSÉ MORGADO2, F. XAVIER MALCATA1§* & MANUELA E. PINTADO1‡
1CBQF/Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino
de Almeida, Porto, Portugal and 2CITEVE - Centro Tecnológico das Indústrias Têxtil e do Vestuário de Portugal,
Quinta da Maia – Rua Fernando Mesquita, Famalicão, Portugal

Abstract
A number of enzymes have applications in the textile sector, as several natural fibres (e.g. cotton, wool and flax) can be
subjected to processing using such natural biocatalysts. In the latest two decades, demand for industrial enzymes has
increased considerably; however, the textile industry requires highly stable enzymes, and good performance at extreme
values of pH and temperature. New strategies are continuously emerging for the immobilization of enzymes with superior
efficiency and usage. Enzymatic immobilization can stabilize enzymes and extend their useful life. Additionally, reduction
in effluent treatment costs and improvement of efficiency is possible. This paper briefly reviews the most recent research
efforts pertaining to immobilization of enzymes with potential application in the textile industry.

Keywords: Cotton, effluent treatment, enzyme immobilization, dye, textile, wool

Enzymes as industrial catalysts

Most of the recent scientific developments in the
textile industry have focused on biotechnological
tools – with particular emphasis on enzymology.
Enzymes have a long history in textile processing and
fibre modification, and one of the most important
criteria for specific application at industrial scale is
resistance to high temperature, alkaline pH and the
presence of denaturing agents normally employed
during bleaching, washing or dyeing (Tzanov et al.
2003). Utilization of highly specific enzymes for
various textile-processing applications is becoming
increasingly accepted because of their ability to
replace harsh chemicals currently used in the indus-
try, and consequent reduction of water, chemicals
and energy usage (Yachmenev et al. 2002). Enzymes
can be applied to virtually all manufacturing steps in
textile processing, from fibre and fabric processing, to
laundry detergents and effluent treatment (Queiroga
et al. 2007). Enzymatic treatment of cotton fibres is
a non-toxic, environmentally benign process used for
a large variety of cotton processing conditions. Such
applications includes: desizing or removal of starch
size with amylases; bioscouring or dissolution of
waxes, proteins, pectins and natural fats from the sur-
face of cotton fibres with pectinase, lipase and cellu-
lase solutions; bleaching cleanup or effluent treatment
to remove residual H2O2 with catalases; biofinishing
which improves the appearance of cotton fabrics and
garments by removal of fibre fuzz and pills from the
substrate surface with cellulases; biostoning or ‘stone-
washing’ of denim fabrics to produce the fashionable
aged appearance with cellulases; laundering of gar-
ments or removal of various types of stain with mix-
tures of enzymes (Yachmenev et al. 2002); and
biobleaching or in situ generation of H2O2 for cotton
bleaching with glucose oxidase. In the case of wool
fibre, there are efforts to substitute the conventional

*These authors had equal contributions to the work.

§Current addresses: ISMAI – Instituto Superior da Maia, Avenida Carlos Oliveira Campos, P-4475-690 Avioso S. Pedro, Portugal & Instituto de Tecnologia

Química e Biológica, Universidade Nova de Lisboa, Avenida da República, P-2780-157, Oeiras, Portugal.
Correspondence: Manuela E. Pintado, Escola Superior de Biotecnologia - Universidade Católica Portuguesa, CBQF, Rua Dr. António Bernardino de Almeida,
Porto, 4200-072 Portugal. Tel: ⫹ 351-225580097. E-mail: mmpintado@esb.ucp.pt

(Received 26 January 2011; revised 19 July 2011; accepted 22 October 2011)

ISSN 1024-2422 print/ISSN 1029-2446 online © 2011 Informa UK, Ltd.
DOI: 10.3109/10242422.2011.635301
224 J. C. Soares et al.
chlorine treatment by an enzymatic process with pro-
teases capable of providing the fabric with the same
characteristics, like anti-shrinking and anti-pilling
behaviour (Silva et al. 2005).
Furthermore, textile and several other industries
also generate a number of visual pollutants, which
they discharge to the surrounding environment with-
out any further treatment. These pollutants not only
add colour to water but they also cause extensive
toxicity to the aquatic and other forms of life. There-
fore, the treatment of industrial effluents containing
aromatic compounds becomes necessary prior to
their final discharge to the environment. Several per-
oxidases and/or phenoloxidases (laccases and tyrosi-
nases) have been used for the treatment of
wastewater containing dyes (Duran & Esposito 2000;
Opwis et al. 2004b).
Enzymes from extremophiles
The industrial application of enzymes that can with-
stand the harsh environments prevailing in textile
processing has greatly increased over the past
decades. This is mainly a result of the discovery of
novel enzymes from extremophilic microorganisms
which have found the most practical commercial use
because of their overall intrinsic stability (Paar et al.
2001). Extremophilic microorganisms are adapted
to survive in ecological niches such as at high tem-
peratures, extremes of pH, high salt concentrations
and high pressure (Demirjian et al. 2001). Therefore,
technical applications of enzymes in the industry are
feasible only if such biocatalysts are stabilized against
temperature (e.g. washing from 60 to 70°C, textile
desizing from 80 to 90°C, etc.), pH (e.g. bleaching
at pH 10 or even more) and in the presence of salts,
alkalis and surfactants usually employed in the tex-
tile processing conditions (Iyer & Ananthanarayan
2008). Thus, the increase in the number of newly
isolated extremophiles and related enzymes docu-
ment the enormous potential within this scientific
field. Furthermore, modern techniques like site-di-
rected mutagenesis will lead to in vitro tailored
enzymes that are highly specific for numerous indus-
trial applications.
Immobilization of enzymes
Besides the benefits of selecting enzymes from extre-
mophiles, it seems rational to consider enzyme immo-
bilization to improve stabilization. The industrial
application of enzymes requires, in many instances,
its recovery and reuse to make an economically fea-
sible process. Moreover, the use of an immobilized
enzyme permits simplification of the reactor design
and easy control of the reaction. Thus, immobiliza-
tion is typically a requirement for the use of an
enzyme as a realistic industrial biocatalyst (Mateo
et al. 2007). Thus, several of the enzyme-mediated
process limitations may be dramatically alleviated by
enzyme immobilization (Takahashi et al. 2001; Durán
et al. 2002; Yachmenev et al. 2002), essentially due
to tailor-made improvements in enzyme performance
(Sheldon et al. 2005). Enzyme immobilization can be
defined as the attachment of soluble enzymes to dif-
ferent types of support resulting in reduction or loss
of mobility of the enzyme (Khan & Alzohairy 2010).
Many methods of immobilization, coupled to a wide
spectrum of supports have been tested, and are well-
documented in the literature (Viswanath et al. 1998;
Costa et al. 2001; Peralta-Zamora et al. 2003; Tzanov
et al. 2002; Krajewska 2004; Delanoy et al. 2005;
Opwis et al. 2005). Support materials play an impor-
tant role in the usefulness of an immobilized enzyme
as it should be low-cost and provide adequate large
surface area together with the least diffusion limita-
tion in the transport of substrate and product
(Krajewska 2004).
Enzyme orientation on the support surface may
be critical if the active centre needs to be in appropri-
ate association with the support. Thus, in many
examples, the combined use of site-directed muta-
genesis and immobilization has been used to control
the orientation of the protein on the support surface.
There are several reports showing the advantages of
site-directed mutagenesis to improve enzyme proper-
ties directly, and how enzyme immobilization can be
a powerful tool to improve the properties of biocata-
lysts (Turková 1999; Camarero 2008; Hernandez &
Fernandez-Lafuente 2011).
Another complex problem is the stabilization of
multimeric proteins (e.g. catalases), where dissocia-
tion of the subunits produces enzyme inactivation
and even product contamination. Moreover, in the
case of multimeric enzymes, not all the subunits may
be covalently bound to the support, and thus, they
are free to dissociate under certain circumstances
promoting enzyme inactivation. These unbound sub-
units may be prevented from dissociating using
defined experimental conditions that assist in the
handling of enzymes (ions, polymers, etc.) or by
genetic tools that might be used to crosslink (via
disulfide bonds) or reinforce the subunit–subunit
interactions (Mateo et al. 2007).
Additionally, rational application of immobilized
enzymes specifically to textile processing conditions
should focus on interactions between the enzyme
protein, textile chemicals and additives – which often
destabilize the enzyme, and hence reduce its activity
(Tzanov et al. 2003). This review critically updates
advances in basic and applied science covering this

important field of biotechnology – with a focus on
textile processing, and referring mainly to hydrolases
and oxidoreductases.
Advantages of soluble bioconjugates over insoluble forms
In several textile processing conditions, enzymes
need to act on macromolecular (insoluble) substrates
particularly in fibre processing viz. starch, cellulose,
protein and pectins. Thus, poor contact between the
insoluble substrate and the immobilized enzyme, as
well as incomplete separation of the immobilized
enzyme from unreacted solid substrates is a common
problem in heterogeneous reaction systems. There-
fore, when enzymes are to be used with macromo-
lecular substrates, the use of smart polymers as
carriers has been suggested as a good solution to
these problems (Dourado et al. 2002). Enzymes
linked to smart polymers combine the benefits of
using soluble carriers (reduced mass transfer con-
straints) with the advantage of reusability. Smart
materials respond to chemical or physical changes in
their environment in a predictable fashion and can
be used to design reversible soluble-insoluble bio-
catalysts. Stimuli that are used to recover smart
polymer-enzyme conjugates for reuse include
changes in pH, temperature, ionic strength and addi-
tion of chemical species. End-group conjugation and
site-specific conjugation are further strategies to to
obtain catalysts with improved design. Such poly-
mers occur naturally (e.g. alginate and chitosan), but
can also be synthesized chemically (e.g. methyl
methacrylate polymers, hydroxypropyl methylcellu-
lose phthalate and hydroxypropyl methylcellulose
acetate succinate). These smart biocatalysts have
already been used in the bioconversion of starch,
cellulose, chitin/chitosan, hydrolysis of xylan and for
protein hydrolysates (Roy et al. 2004). Hence, the
potential of smart polymers for employment in tex-
tile fibre processing establishes them as very attrac-
tive carriers.
Effect of enzyme immobilization on interaction with the
surrounding medium
In wet processing conditions many interactions occur
between immobilized enzymes, dyes, chemical addi-
tives and processing aids, which may be of a great
importance. Therefore, a balance between stabilizing
and destabilizing interactions should be attained.
Stability of laccases in immobilized form depends
both on the composition of the dyeing liquor and the
chemical structure of the dye itself (Zille et al. 2003).
Anionic sulphonic dyes are known to protect enzymes
from inactivation by forming ionic pairs between
Immobilized enzyme technologies 225
negatively charged sulphonic groups and positively
charged amino acid residues (Matulis et al. 1999).
The extremely heterogeneous character of textile
fibres requires surfactants as auxiliary agents in tex-
tile processing – in which they serve as wetting, anti-
foaming, dispersing, washing or level-dyeing agents
(Opwis et al. 1999). Ionic surfactants undergo a
strong hydrophobic interaction with globular pro-
teins in aqueous solution (Morén et al. 1997; Tzanov
et al. 2003). Unfortunately, inconsistent findings
have been reported in the literature regarding the use
of surfactants (Mozhaev et al. 1989; El-Batal et al.
2005). Vasconcelos et al. (2006) and Zhang et al.
(2006) demonstrated that the biological activity of
immobilized proteases was not affected by exposure
to anionic or non-ionic surfactants. Conversely,
Costa et al. (2001) reported a notable reduction in
activity of catalase immobilized on alumina during
treatment of textile bleaching effluents with anionic
and non-ionic surfactants; such a reduction reached
96%, and the type and size of the carbon chain were
two important factors affecting enzyme stability.
The solution ionic strength is also an important
factor for biocatalyst behaviour (Zille et al. 2003). Salt
addition affects the prevailing electrostatic interac-
tions in solution, and operations in textile mills are
usually performed at salt concentrations above 0.5 M.
Zille et al. (2003) observed a higher stability of laccase
in the presence of relatively high levels of salt in the
dyeing bath, probably due to enhancement of electro-
static coupling of anionic dyes, and consequent for-
mation of stable aggregates between positively charged
enzyme and dyes. Nevertheless, the degree of stabili-
zation/destabilization by salts depends to a great
extent on the protein:salt ratio (Tzanov et al. 2003).
Another important issue in textile baths is the
influence of bleaching stabilizers. Costa et al. (2001)
concluded that such stabilizers exert some degree of
inhibition upon immobilized catalase. Finally, the issue
of interaction with organic solvents has been gaining
importance, since wet treatment of textile materials is
carried out using several of these solvents. Increasing
the concentration of organic solvents leads to faster
inactivation of enzymes due to reversible changes in
their protein structure, while longer incubation pro-
motes irreversible inactivation (Azevedo et al. 2001).
Current and potential applications of enzyme
immobilization for textile processing
Several techniques may be applied to immobilize
enzymes on solid supports. The basic aspects of
enzyme immobilization in respect to their potential
applications for the textile processing conditions are
summarized in Table I.
Table I. Enzyme immobilization for textile industry applications.
226

Enzyme Origin Support Immobilization Comments References

Cellulase Aspergillus niger Polyvinyl alcohol coated chitosan beads Epichlorohydrin- Acid cellulase became a neutral cellulase Dinçer and Telefoncu (2006)
Adsorption
Commercial acid Chemically modified pumices particles Zr OCl2 - Adsorption Immobilized cellulase can efficiently Pazarlioglu et al. (2005)
cellulase abrade indigo dyed denim fabrics
(Rotta)
Pectinase Aspergillus niger Sodium bis-2-ethylhexysulpho-succinate Encapsulation Good efectiveness in terms of cotton Sawada and Ueda (2001)
J. C. Soares et al.

bioscouring
Amylase Aspergillus niger Alkylamine glass beads coated with Adsorption followed Immobilized enzymes with better washing Dhingra et al. (2006)
(Diastase) zirconia by GLUTAL properties and used 100 times without
any considerable loss of activity
Non specified Fe3O4/Poly(styrene-co-maleic anhydride) Covalent binding Excellent operational stability and bound Qiu et al. (2005)
Magnetic composite microspheres enzyme was separated from reaction
medium by an magnetic field
Protease Subtilisin Eudragit S-100 Covalent linked by Shift in optimum pH to alkaline side, Silva et al. (2006)
(Novozymes) carbodiimide reduction of weight and fibre tensile
coupling strength losses with immobilized
enzymes
Subtilisin Eudragit S-100 and L-100, Kollicoat Covalent linked by Less fibre damage was observed for all six Smith et al. (2010)
(Novozymes) MAE 100P, HPMCP-55, cellulose carbodiimide modified enzymes and was less than
acetate phthalate and AQOAT AS-LG coupling native enzyme of a similar activity level
Subtilisin (Sigma) PEG Covalent binding Diffusion of proteses into wool is Silva et al. (2005)
dependent on the size of the protease
Subtilisin (Sigma) Eudragit S-100 Covalent linked by Enzyme immobilization enable the Shen et al. (2006)
carbodiimide reaction of the enzyme with wool to be
coupling controlled, and the dyeing properties
appear to be unaffected
Subtilisin (Sigma) Eudragit S-100 Covalent linked by An anionic and non-ionic surfactant was Zhang et al. (2006)
carbodiimide compatible with immobilized proteases
coupling in the wool treatment
Glucose Aspergillus niger Alumina and glass Covalent binding Sufficient bleaching levels with low Tzanov et al. (2002)
oxidase enzyme concentration; good operational
stability of alumina support
Commercial Cotton Covalent binding Recycling of desizing liquors into Opwis et al. (1999)
(Serva GmbH) bleaching liquors
Catalase Bacillus SF Alumina pellets Covalent-GLUTAL Higher stabilities and surfactant Costa et al. (2001)
inactivation
Bacillus SF Alumina pellets Covalent-GLUTAL 93% protein bound and 87% activity Paar et al. (2001)
retained
Catalase Bacillus SF α- and γ-Alumina balls, Novalox saddles Covalent-GLUTAL Porosity and shape of the carriers Fruhwirth et al. (2002)
and Raschigrings
Bovine liver Cotton fabric or Nylon 6 Adsorption and Low cost and flexible construction Opwis et al. (2004)
covalent-GLUTAL
 Bovine liver Poly(ethylene terephthalate) or Covalent -Photo After 20 applications, the immobilized Opwis et al. (2005)
polyamide 6.6 chemical enzyme showed a integral activity
around 3.5 higher than free catalase
Bovine liver Poly(ethylene terephthalate) Chemical and Enzyme modification before the Opwis et al. (2007)
Covalent- immobilization; photochemical
Photochemical technique may be able to compete with
conventional immobilization procedures
Peroxidase Horseradish Ion exchange resins Covalent binding Amberlite IRA-400 ion exchange resin Peralta-Zamora et al. (1998)
showed the best results for enzyme
immobilisation
Horseradish Eupergit®C Covalent binding High degrees of phenol removal using Pramparo et al. (2010)
about one hundredth of the enzyme
used in the soluble form
Momordica Con A-Sephadex Adsorption Higher TOC and color removal with Akhtar et al. (2005)
charantia immobilized enzyme
Momordica Con A-layered calcium aginate-starch Adsortion followed by Immobilized peroxidase decolorized more Matto and Husain (2009)
charantia beads GLUTAL than 90% effluent after 3 h of
incubation in a batch process
Saccharum Polyethylene Covalent-GLUTAL Reusabilty was studied for 15 cycles and Shaffiqu et al. (2002)
spontaneum the half-life was found to be 60 h
Laccase Myceliophthora Sepabeads EC-EP3 and Dilbeads NK Covalent binding Very attractive for their application on the Kunamneni et al. (2008)
thermophila decolorization of dyes in batch and
continuous fixed-bed reactors
Rhus Vernificera PEG Covalent binding Activity of modified laccases on fibre Schroeder et al. (2006)
bound was reduced preventing dye
re-deposition
Sclerotium rolfsii Alumina Covalent-APTES- Acid stable and potential for wool dye Ryan et al. (2003)
GLUTAL decolorization
Sclerotium rolfsii Alumina Covalent-APTES- Potential for the treatment of dyeing Campos et al. (2001)
and GLUTAL effluents and to obtain
Trametes hirsuta various bleaching appearances in denim
garments
Laccase Trametes hirsuta Alumina Covalent-APTES- T reated effluents could be used for Abadulla et al. (2000)
GLUTAL re-dyeing
Trametes hirsuta Alumina Covalent-GLUTAL Laccase activity of the coated laccase was Rodríguez Couto et al.
followed by coating higher than that of the non-coated one (2007)
with polyelectrolytes
Trametes hirsuta poly amide 6,6 Cross linking- Potential for application in the continuous Silva et al. (2007)
GLUTAL and decolorization of textile effluents, where
spacer it can be applied into a membrane
reactor
Trametes pubescens Alumina Covalent-APTES- The decolorization of a simulated textile Osma et al. (2010)
GLUTAL effluent was achieved using different
laboratory-scale bioreactors
Immobilized enzyme technologies

(Continued)
227
228 J. C. Soares et al.

Textile fibre processing

Peralta-Zamora et al. (2002)

Khan and Husain (2007)

Bayramoglu et al. (2010)
Cellulases. Cellulases constitute a group of enzymes
References that are able to catalyze cellulose hydrolysis via de-
grading β-(1-4) linkages of the biopolymer, and

Zille et al. (2003)

concomitantly release reducing sugars (Sinegani et
al. 2005; Dinçer & Telefoncu 2006). In nature, cel-
lulolytic systems belong to three major classes, which
act in a synergistic fashion: endoglucanases, exog-
lucanases and β-glucosidases (Azevedo et al. 2002).
These enzymes achieved worldwide success in textile
large-scale biotechnological applications processing because of their ability to modify cellulosic
Reusability of the magnetic beads can

Treatment of wastewater/dye effluent,

fibres in a controlled manner, producing better qual-

immobilized tyrosinase wa s more

effective than soluble counterpart
provide economic advantages for

Decolorization of Reactive Black 5

ity fabrics without excessive inner structural damage
(Cavaco-Paulo 1998; Pazarlioglu et al. 2005). The
Photochemical pre-treatment

multicomponent nature of the cellulase system finds

Comments

major application in biopolishing (removal of fluffy

industrial effluent

and protruding fibres from cotton fabrics resulting

in clear fabric surface), as well as in denim garment
treatment to achieve the desired stonewashed final
look (Gusakov et al. 2001).
Various methods and supports have been reported
to date for immobilization of cellulases for different
purposes (Kajiuchi & Park 1992; Dourado et al.
2002; Wu et al. 2005; Mao et al. 2006; Tu et al.
2006). Pazarlioglu et al. (2005) used a commercial
covalent-APTES-
Immobilization

Covalent-imidazol,

Covalent-APTES-
GLUTAL and

cellulase immobilized onto ZrOCl2-activated (pum-

carbodiimide-

ice) particles for denim washing. They concluded

GLUTAL

GLUTAL
covalent-

Adsorption

Adsorption

that such immobilized enzymes could efficiently

abrade indigo-dyed denim fabrics, and because
strongly bound enzymes remained on the pumice,
staining was detected at lower levels after the second
usage with immobilized cellulase than its free coun-
Silica, amberlite IRA-400, glass ceramic

terpart. Dinçer & Telefoncu (2006) improved the

Poly(4-VP) grafted and/or Cu(II) ions

stability of an acidic cellulase in the neutral pH range

by immobilizing it on chitosan beads coated with
maleic anhydride-modified polyvinyl alcohol. The
chelated magnetic beads

major reason behind replacing acid cellulases with

Support

and montmorillonite

neutral ones is that the former are more aggressive

and produce more abrasion that reduces the tensile
strength of the fabric.
The applications of cellulases in immobilized
Celite 545

form for textile fibre processing (Table I) are still

Alumina

scarce, probably because of substantial constraints

associated with limited substrate diffusion, as dis-
cussed above (Taniguchi et al. 1992; Zhang et al.
Solatium tuberosum

2010). Eudragit L-100 a soluble–insoluble reversible

Trametes versicolor

Trametes versicolor

Trametes villosa

polymer was successfully used for cellulase immobi-

Origin

lization and could be an alternative to apply in bio-

polishing and/or bioscouring (Dourado et al. 2002).
Table I. Continued

Pectinases. Pectinases are enzymes able to breakdown

complex pectins, which occur as structural polysac-
Tyrosinase

charides in plant tissues, into simpler molecules, viz.

Enzyme

galacturonic acids. They include polygalacturonases,

pectin esterases, pectin lyases and pectate lyases,

depending on their mode of action (Kashyap et al.
2001; Hoondal et al. 2002). Various reviews are avail-
able on the production and application of pectinases
(Sakai et al. 1993; Naidu & Panda 1998; Kashyap et
al. 2001; Hoondal et al. 2002), but these enzymes are
still mainly used in the food industry and in wood
preservation, and only recently employed in textile
fibre processing (Gummadi & Panda 2003).
Many non-cellulosic impurities are found in the
cellulose matrix of the primary wall, and to a lesser
extent the secondary wall of a cotton fibre. This bar-
rier limits the dyeing and finishing processes (Hoondal
et al. 2002). Hence, scouring, which remove the
cuticle and the primary wall compounds from the
cotton fibre is essential to achieve satisfactory wet-
tability. Bioscouring has become a feasible alternative
to classic chemical scouring, because of the far better
eco-friendliness of the former. Besides pectinases,
several other types of enzyme can be used for bio-
scouring – viz. lipases, proteases, cellulases, xylanases
and cutinases (Wang et al. 2007).
Despite several advantages of using pectinases in
immobilized form, relatively little research has been
performed specifically on this topic (Roy & Gupta
2003; Lei & Bi 2007a,b). However, Sawada & Ueda
(2001) successfully attempted to expose cotton to
scouring via a pectinase immobilized in a reverse
micellar system (Table I). The effectiveness of scour-
ing was equivalent or better than that achieved by
conventional alkaline processes or bioscouring in
aqueous media. The enzyme demonstrated excellent
activity, even in organic media.
Amylases. Amylases are enzymes that cleave starch
molecules and related compounds, by hydrolysing α-
1,4- and/or α-1,6-glucosidic linkages, in either endo-
or exo-locations (Hamilton et al. 2000). Several re-
views are available covering these enzymes, which
discuss their sources, applications, catalytic perfor-
mance and biochemical properties (Pandey et al.
2000; van der Maarel et al. 2002; Gupta et al. 2003;
Sivaramakrishnan et al. 2006). Modern production
processes for textiles introduce a considerable strain
on the warp during weaving. The yarn must, there-
fore, be prevented from breaking. For this purpose
a removable protective layer (size) is applied to the
threads. Starch is a very attractive size, because it is
cheap, easily available in most regions of the world,
and it can be removed quite easily. Good desizing of
starch sized textiles is achieved by the application of
amylases, which selectively remove the size and do
not attack the fibres (Gupta et al. 2003).
Despite the various forms, and many studies on
immobilized amylases (Tümtürk et al. 1999; Chang
& Juang 2005; Kara et al. 2005; Pandya et al. 2005;
Kahraman et al. 2006), the general enzymatic
Immobilized enzyme technologies 229
actitivity is usually reduced, so practical applications
of immobilized amylases in textile desizing are rare,
since they act upon macromolecular substrates. Nev-
ertheless, Qiu et al. (2005) reported the immobiliza-
tion of α-amylase on magnetic composite microspheres.
Owing to easy separation under a magnetic field for
repeated use, a potential for a wide range of industrial
applications was claimed – including textiles. Further-
more, an amylase from Aspergillus niger was immobi-
lized via glutaraldehyde-mediated coupling onto
zirconia-coated alkylamine glass beads. The useful-
ness in removal of starch stains from cotton was tested
using various detergents. The immobilized enzyme
was used in removal of starch stain from cotton clothes
along with detergents. All the detergents gave better
washing in presence of immobilized amylase than that
with detergent alone (Dhingra et al. 2006).
Proteases. Proteases are a class of enzymes which
bring about protein degradation via hydrolysis of the
peptide bonds in the polypeptide backbone (Singh
et al. 2001; Gupta et al. 2002). The conventional wool
processing techniques to achieve shrink-resistant
wool uses chlorination steps which pose ecological
problems. Replacing these harsh chemicals with pro-
teases for processes such as shrink-resistance, while
simultaneously improving whiteness, dyeability and
handle are well recognised (Smith et al. 2010). How-
ever, despite some beneficial effects, such enzymatic
treatments may cause excessive damage to the fibre
cuticle, with a consequent high degree of weight and
strength losses of the wool fibres (Cortez et al. 2004;
Silva et al. 2005; Queiroga et al. 2007).
Table I tabulates some of the latest references for
different immobilized proteases with potential appli-
cation in the textile industry. Immobilization of pro-
teases (or chemical modification thereof) typically
increases their molecular size, constraining prote-
olytic attack to the cuticle and thus reducing the
problem. To elucidate the pattern of diffusion of ser-
ine proteases into wool fabrics and yarns, free sub-
tilisin and subtilisin covalently linked to polyethylene
glycol (PEG) were studied (Silva et al. 2005). The
modified protease remained on the surface of the
cuticle layer region (hydrolyzing just the cuticle layer
of wool), hence producing a higher tensile strength
and a lower felting of the fibres. Silva et al. (2006)
used a commercial protease (Esperase) covalently
linked to Eudragit S-100. This novel approach is a
promising alternative for wool shrink-resist finishing,
replacing the conventional chlorine treatments.
Under optimised conditions (Eudragit, 2.5% w/v,
carbodiimide, 0.2% w/v, coupling time, 1 h and
blocking agent concentration, 0.05%), the conjugate
activity yield was about 45% and its operational sta-
bility at 60°C was increased by 1.7 times. These data
230 J. C. Soares et al.
agreed with those by Shen et al. (2006), and the
dyeing properties of the wool fabric appeared to be
unaffected by treatment with immobilized Esperase.
Zhang et al. (2006) also used Eudragit S-100 via
carbodiimide coupling; their results made it appar-
ent that the ethoxylated alkyl phosphate (i.e. an
anionic surfactant) acts synergistically with the
immobilized enzyme, in the bioscouring processes.
More recently, Smith et al. (2010) demonstrated that
different enteric polymers can also be successfully
coupled with Esperase using carbodiimide coupling.
Treatment on wool showed that such enzymes mod-
ified with Kollicoat, hypromellose acetate succinate,
hydroxylpropyl methylcellulose phthalate or cellu-
lose acetate phthalate produced effects similar to
those of Esperase modified with Eudragit S-100 and
L-100, in terms of improved shrink-resistance, but
with less fibre damage in terms of weight loss and
tensile strength.
Textile bleaching and treatment of the bleaching liquor
Hydrogen peroxide is a substrate (catalase and per-
oxidase) or product (glucose oxidase) of several
interesting industrial textile enzymes. These biocata-
lysts may have to act in the presence of high H2O2
concentrations during textile bleaching or in the
treatment of the bleaching liquor. Under these con-
ditions, such enzymes may be sensitive to H2O2,
because it can oxidize some residues in the proteins
and denature them. Hence, an increase in the resis-
tance to this chemical reagent has major practical
relevance. This might be addressed from different
perspectives: (i) multipoint and multisubunit cova-
lent immobilization, which reduces any conforma-
tional change involved in enzyme inactivation and
greatly increase the enzyme stability. Supports like
agarose beads, zeolites, porous glass, epoxy resins,
offer large areas for enzyme-support interactions
(Betancor et al. 2003, 2006; Mateo et al. 2007); (ii)
immobilization on porous supports with large inter-
nal surface areas stabilizes the enzyme against inter-
action with molecules from the surrounding medium;
and (iii) chemical modification by crosslinking with
polyfunctional-polymers, which could reduce the
effective concentration of H2O2 in the proximity of
the enzyme (Iyer & Ananthanarayan 2008).
Glucose oxidase. Glucose oxidase is a dimeric glycosy-
lated flavoprotein – which is able to catalyze oxidation
of glucose to glucolactone, which in turn spontane-
ously yields gluconic acid, as well as H2O2 as a side-
product (Betancor et al. 2006). In the last decade,
due its biodegradability, H2O2 has almost entirely re-
placed conventional chlorine treatments in the textile
bleaching processes. Therefore, glucose oxidase has
been considered to be a possible method for produc-
ing H2O2 for biobleaching, aimed at improving fibre
performance before dyeing, and destroying pigments
initially present in the fibres that account for cotton
greyness. Implementation of this enzymatic system
in cotton pretreatment is beneficial for reusing the
desizing waste baths as an additional source for glu-
cose, and to decrease waste water pollution and water
consumption (Tzanov et al. 2001, 2002). As reported
previously by Tzanov et al. (2001), higher quantities
of enzymatically-produced peroxide were needed to
accomplish the same bleaching effect as in the stan-
dard procedure. Probably, the added glucose protects
the cotton from being bleached, acting as a substrate
for bleaching and oxidation, thereby consuming the
peroxide, or as a stabilizer. On the other hand, in-
creasing the bleaching temperature causes the protein
to denaturate and deposit on the fabric surface, due to
hydrophobic fabric/enzyme interactions. These prob-
lems could be overcome by using immobilized instead
of free enzyme. The stability of glucose oxidase has
been improved via several immobilization procedures
on various supports (Quinto et al. 1998; Tzanov
et al. 2002; Blin et al. 2005; Betancor et al. 2006;
Godjevergova et al. 2006). The first textile application
(Table I) was reported by Opwis et al. (1999), with
cyanuric chloride-mediated attachment to cotton –
with subsequent release of H2O2 at levels high enough
to support adequate bleaching. This biotechnological
approach allowed important economic and ecological
savings – in terms of water and chemicals, as desiz-
ing liquors include glucose from cotton pre-treatment
with amyloglycosidase. Glucose oxidase from Asper-
gillus niger was also covalent immobilized on two in-
expensive and commercially available supports, viz.
alumina and glass (Tzanov et al. 2002). High protein
immobilization yields, as well as sufficient bleaching
levels were attained. Glass permitted a higher rate of
generation of H2O2 (0.35 g/L) due to its more porous
morphology and thus bound more protein, however,
alumina produced a higher operational stability and
permitted reuse for at least three consecutive cycles.
Catalases. Catalases are ubiquitous enzymes in aero-
bic organisms, in which they catalyze breakdown of
H2O2 into water and oxygen (Chelikani et al. 2005).
Several studies covering the main aspects of catalase
function and properties are described in the litera-
ture (Loewen 1996; Jouve et al. 1997; Zámocky &
Koller 1999; Hersleth et al. 2006).
As a strong oxidizing agent, H2O2 oxidizes reac-
tive dyes – which constitute a major reason for dye
loss and non-uniformity in dyeing. Consequently,
residual H2O2 in bleaching baths must be removed
from the fabric prior to dyeing (Thompson et al.
2003). A critical problem is that bleaching is carried

out at temperatures above 60°C and under alkaline
conditions (pH 9 and above), so most commercial
catalases cannot withstand these harsh conditions
(Spiro & Griffith 1997; Paar et al. 2001; Oluoch
et al. 2006). An alternative is to use catalases from
alkalothermophilic and thermophilic microorganisms
(Kagawa et al. 1999; Gudelj et al. 2001; Fruthwirth
et al. 2002; Hidalgo et al. 2003; Thompson et al.
2003; Oluoch et al. 2006). However, direct applica-
tion of free catalase promotes interaction between the
protein and the dye, which in turn reduces dye uptake
by the fabric (Paar et al. 2001).
Immobilization of catalase overcomes this con-
straint, and concomitantly enables reuse of the
enzyme. Recycling bleaching baths, pre-treated with
immobilized catalase, for subsequent dyeing leads to
savings up to 50% in overall water consumption.
Catalase immobilization has been attempted by sev-
eral authors (Emine & Leman 1995; Cetinus &
Öztop 2000; Costa et al. 2001; Betancor et al. 2003;
Opwis et al. 2004b; Wang & Caruso 2005; Jürghen-
Lohmann & Legge 2006), using organic and inor-
ganic materials such as porous glass, cellulose,
alumina, silica gel and hydrogels. Some natural poly-
mers – e.g. gelatin and chitosan, as well as some
synthetic polymers – e.g. polyacrylamide, were found
not to be appropriate for bleaching treatments
(Fruhwirth et al. 2002).
Several authors employed alumina-based supports,
due to their greater mechanical stability at high pH
and temperatures, for immobilization of catalase
obtained from Bacillus SF for the treatment of textile
bleaching effluents (Costa et al. 2001; Paar et al. 2001;
Fruhwirth et al. 2002). The treated liquid was reused
for dyeing fabrics with various dyes, resulting in accept-
able colour differences for all the dyes used.
However, Costa et al. (2001) confirmed that
immobilized catalase could be inhibited by anionic
stabilizers or surfactants added to the H2O2 substrate
solution. Opwis et al. (2004a, 2005 and 2007) used
low-cost synthetic textile fabrics as alternative carrier
materials for covalent catalase immobilization by a
photochemical process (UV light) in the presence of
different cross-linking agents. The great advantage of
using fabrics as carriers is that they can be removed
very quickly from the reactor without a filtration step
and without protein residues after the enzymatic reac-
tion. Even after 20 (Opwis et al. 2005) or 30 (Opwis
et al. 2007) applications the immobilized enzyme still
showed a distinct activity, and the integral activity was
higher by a factor of 3.5 and 1.5, respectively.
Decolorization of dyes and effluent treatment
Peroxidases. Peroxidases are oxidoreductases that con-
sume H2O2 to catalyze oxidation of a wide variety of
Immobilized enzyme technologies 231
organic and inorganic chemicals, including a large va-
riety of aromatic compounds. Several studies covering
general properties, biochemical and molecular char-
acterization, as well as industrial and environmental
applications have been discussed and reviewed else-
where (Durán & Esposito 2000; Moreira et al. 2001;
Barceló & Pomar 2002; Conesa et al. 2002; Azevedo
et al. 2003; Moreira et al. 2005; Atack & Kelly 2006;
Matto & Husain 2006; Moreira et al. 2006).
As mentioned before, huge volumes of potentially
hazardous compounds are released at various stages
of textile manufacturing including dyes, alkalis, chro-
mium, phenol, oils and waxes (Asamudo et al. 2005).
Therefore, transformation of such potential toxic
compounds is essential, prior to their disposal. Most
of the physico-chemical methods currently used have
technical and economic restrictions, since they are
expensive, produce large amounts of sludge and are
inefficient for some soluble dyes. Therefore, white-rot
fungal peroxidases have attracted particular interest
due to the low yield and high cost of production of
plant peroxidases (Shaffiqu et al. 2002; Khan &
Husain 2007; Blánquez et al. 2008) and since they
participate in the degradation of a broad range of
substrates.
Peroxidase immobilization, for wastewater treat-
ment, was successfully achieved on various natural
and synthetic carriers (Duran & Esposito 2000;
Duran et al. 2002; Torres et al. 2003). However, only
a few reports have been linked with direct decon-
tamination of textile industry effluents. The funda-
mental features of peroxidase immobilization with
regard to potential applications for the textile indus-
try are summarized in TableI.
The majority of the matrices currently used for
the immobilization of enzymes such silica, controlled
pore glass, polyvinyl alcohol, polyacrylamide and
chitosan beads were not suitable, because of dye
adsorption onto the matrices, probably inactivating
the enzyme (Shaffiqu et al. 2002). Therefore, immo-
bilization of horseradish peroxidase (HRP) on vari-
ous supports, and application in treatment of textile
effluents, was assessed by Peralta-Zamora et al.
(1998). Among them, Amberlite IRA-400 ion
exchange resin produced the best results for enzyme
immobilization, efficiency of colour removal and
degradation of phenol species. The decolorization
process was more efficient with combined photo-
enzymatic procedures which appear to be an efficient
decontamination method for industrial effluent
treatment. Moreover, the peroxidase from the plant
Saccharum spontaneum can be used as an alternative
to the commercial HRP and soybean peroxidase
for the decolorization of textile dyes. Four textile
dyes at initial concentration of 50 mg/L were com-
pletely degraded within 6–8 h at pH 3 by the enzyme
232 J. C. Soares et al.
immobilized on a modified polyethylene matrix. The
immobilized enzyme was also used in a batch reactor
and the reusabilility was studied for 15 cycles,
demonstating a half-life of around 60 h (equivalent
to 10 cycles) (Shaffiqu et al. 2002).
Bioaffinity-based immobilization can be exploited
for the direct immobilization of enzymes from par-
tially purified preparation or even from crude homo-
genates. Simplicity of immobilization, lack of chemical
modification and an enhancement in stability is usu-
ally achieved (Akhtar et al. 2005). In this way, Matto
& Husain (2009) immobilized bitter gourd peroxidase
on concanavalin A layered calcium alginate-starch
beads for the effective decolorization of textile indus-
trial effluents. They evaluated its efficiency at large
scale by using a continuous two-reactor system, one
reactor containing immobilized enzyme and the other,
activated silica. Immobilized enzyme could remove
more than 90% of the colored compounds over 10
days of operation. The reactor was capable of remov-
ing 40% of the color from the textile effluent even
after 60 days of operation. An enhancement in decol-
orization was probably due to increased adsorption
on adsorbent activated silica.
Nevertheless, amongst the large number of sup-
ports available for enzyme immobilization, epoxy-
activated carriers are being widely used and have
received great attention for large scale applications
such as textile industry effluents. One of the most
interesting epoxy-activated supports is Eupergit®C,
because it is commercially available, resistant to
mechanical and chemical stress and adaptable to a
variety of configurations and specific processes car-
ried out in reactors. Pramparo et al. (2010) reported
direct HRP binding on Eupergit®C as an effective
method for the preparation of stable biocatalysts in
removing phenol compounds from wastewater. In
this case, a maximum immobilised enzyme activity
of 127 U/g support was found using an enzyme load-
ing on the support of 35.2 mg/g support.
Laccases. Laccases are copper-containing oxidoredu-
ctases, which belong to the group of small blue oxi-
dases. They are widely distributed in higher plants,
fungi and bacteria (Thurston 1994; Karam & Nicell
1997; Alexandre & Zhulin 2000; Durán et al. 2002).
Such enzymes are able to oxidize various aromatic
compounds (particularly phenols) and inorganic
compounds, with concomitant reduction of oxygen
to water (Durán et al. 2002). Laccases have been
extensively studied, and fundamental aspects have
been duly reviewed (Burton 1994; Giardina et al.
1996; Xu 1996; Gianfreda et al. 1999; Durán &
Esposito 2000; Durán et al. 2002; Wesenberg et al.
2003; Rodríguez-Couto & Herrera 2006). They have
found industrial and biotechnological applications
in the food, pulp and paper industries, as as well as
the textile industry. The use of laccase in this sector
is growing fast, since besides decolorization of tex-
tile effluents, laccase is used to bleach textiles and
even to synthetise dyes (Setti et al. 1999; Rodríguez-
Couto & Herrera 2006). Applied aspects of immo-
bilized laccases, using a wide range of methods and
substrates, have been described (Lante et al. 2000;
Durán et al. 2002; Peralta-Zamora et al. 2003). The
basic aspects of laccase immobilization with respect
to potential applications for textile processing condi-
tions are summarized in Table I.
Most laccases that are efficient in decolorization
of dyeing effluents are produced by white-rot fungi
(Kandelbauer et al. 2004). Therefore, laccases from
Trametes hirsuta, Sclerotium rolfsii, T. villosa, T. modesta
and T. pubescens have been successfully immobilized
on alumina pellets, and could efficiently decolorize
several synthetic dyes (Abadulla et al. 2000; Campos
et al. 2001; Ryan et al. 2003; Zille et al. 2003;
Kandelbauer et al. 2004; Rodriguez-Couto et al.
2007; Osma et al. 2010). Research has shown that the
subsequent coating of the alumina-immobilised lac-
case with polyelectrolyte layers considerably increased
laccase stability (Osma et al. 2010). However, although
studies have been performed with immobilized lac-
case at laboratory-scale using reactors with small vol-
umes, these have not addressed the large wastewater
volumes generated by the textile industry.
Peralta-Zamora et al. (2003) immobilized laccase
from T. versicolor on various supports viz. silica,
amberlite IRA-400, glass-ceramic and montmoril-
lonite. Imidazole modified silica (SiIm) allowed
almost total laccase immobilization. In the initial
stages, decolorization was mainly due to adsorption
of the dyes onto the support surface, but later the
support became saturated and enzymatic decoloriza-
tion was apparent. The use of a brief photochemical
pre-treatment permitted a significant increase in the
efficiency of the enzymatic decolorization process.
For the treatment of wool dyeing effluents at low
pH, an acid stable laccase would be desirable. Ryan
et al. (2003) confirmed that an acidic laccase could
be used for continuous decolorization of wool dyeing
effluents which allowed water recycling. Eighty-three
percent of the protein was bound to the carrier (0.13
mg/g alumina) and 72% of the laccase activity was
retained on that carrier material.
Silva et al. (2007) demonstrated the potential of
woven nylon (polyamide 6,6) as a carrier for laccase
immobilization and application in a membrane reac-
tor. Woven nylon offers several advantages such as
low cost, chemical inertness, non-toxicity, mechani-
cal stability, insolubility in water, ready availability
and availability in a number of forms. Thus, it seems
to be a promising system about future application.

Recently, Kunamneni et al. (2008) investigated
decolorization of synthetic dyes by laccase from
Myceliophthora thermophila covalently immobilized
on polymethacrylate-based polymers activated with
epoxy-groups. The enzyme immobilized in Sepa-
beads EC-EP3 exhibited notable activity (203 U/g)
and retained 41% of its activity in the decolorization
of methyl green in a fixed-bed reactor after five
cycles. As mentioned before, high mechanical stabil-
ity and the absence of swelling in water make them
very interesting alternative carriers for dye degrada-
tion in both batch and continuous fixed-bed bioreac-
tors. Interesting findings were also reported by
Bayramo lu et al. (2010) using magnetic beads for
laccase immobilization. Magnetic particles have
gained significant attention for large-scale applica-
tions due to their potential for rapid separation in a
magnetic field. The immobilized laccase was more
stable during operation (degradation of three indi-
vidual dyes in batch systems) and storage compared
to the free enzyme.
Besides their potential for detoxification of textile
effluents, laccases are also effective in preventing dye
redeposition on fabrics and garments – during wash-
ing and finishing processes, by degradation of the
solubilized dye. However, the action of those enzymes
must be restricted to solubilized dyes to avoid decol-
orization of fabrics. In order to prevent dye redepo-
sition on already dyed fabrics, laccases have been
covalently attached to PEG; this led to enhanced
stability and limited adsorption onto fibres and gar-
ments, thus preventing their enzyme-mediated
decolorization (Schroeder et al. 2006).
Tyrosinases. Tyrosinases also known as polyphenol
oxidases or catecholases, catalyze two sequential
oxygen-dependent reactions (Burton 1994; Durán
et al. 2002): the o-hydroxylation of monophenols to
o-diphenols (i.e. a cresolase activity), and the subse-
quent oxidation to o-quinones (i.e. a catecholase ac-
tivity) which undergo spontaneous (non-enzymatic)
polymerization to yield water-insoluble substances
(Karam & Nicell 1997). Aspects of the molecular
mechanisms of tyrosinases have been reviewed and
discussed elsewhere (Decker et al. 2000; Decker &
Tuczek 2000; Fenoll et al. 2000; Durán et al. 2002).
At present, several peroxidases and laccases are
used specifically for the treatment of textile dyeing
effluents – but decolorization promoted by the former
adds considerably to the costs of industrial process-
ing, because of the (expensive) H2O2 required as co-
substrate (Akhtar et al. 2005). Furthermore, the
limited availability and high purification cost of lac-
cases constrains their use for this purpose.Tyrosinases,
mainly extracted from plants, are simpler to use, and
less expensive when decolorization of aromatic-rich
Immobilized enzyme technologies 233
effluents is considered, as only molecular oxygen is
required as oxidant (Khan & Husain 2007).
The use of tyrosinases in immobilized form is
well documented in the literature, particularly for
phenol removal from synthetic and industrial efflu-
ents, using a wide range of methods and with various
supports, e.g. chitosan, chitosan with alginate-filled
pore space sodium and calcium aluminosilicates,
silica gel, polysulfone capillary membrane and poly-
clar (Wada et al. 1993; Edwards et al. 1999; Krastanov
2000; Seetharam & Saville 2003; Ensuncho et al.
2005). Khan & Husain (2007) took advantage of a
tyrosinase preparation from Solanum tuberosum,
immobilized on Celite in view of its high stability
and cost of preparation, for treatment of wastewater/
dye effluents contaminated with reactive textile and
non-textile dyes; this was more effective in terms of
decolorization efficiency, pH tolerance and total
organic carbon removal than the corresponding free
form, for treatment of either individual or complex
mixtures of dyes. The maximum decolorization was
found between pH 3.0 and 4.0. These observations
suggested that industrial effluents could be success-
fully treated with immobilized tyrosinase.
Concluding remarks
The moderate stability of enzymes is one of the main
drawbacks that hinder general implementation of
these interesting biocatalysts on an industrial scale.
Nevertheless, enzymes have already a long history in
textile processing. Regulatory enforcement of pollu-
tion reduction directives upon textile manufacturers
has led them to expand the use of enzymes in actual
processing of fibres and textiles; this trend has rap-
idly gained wide recognition, because of their non-
toxic and eco-friendly characteristics.
Immobilized enzymes have attracted particular
interest since this area has continued to develop
into an ever-expanding and multidisciplinary field
over recent years, especially, because it permits
recovery of enzymes and increases their stability,
both of which contribute to lower operational costs.
Recent advances in the design of immobilization
supporting materials with tailorable pore size and
surface functionality has enabled more precise
control of immobilization enzymes. New simula-
tions of the surface characteristics of the target
enzymes can be used to aid in the design of appro-
priate support materials. However, no single
method and support is best for all enzymes and
their applications. This is because of the widely dif-
ferent chemical characteristics and composition of
enzymes, the different properties of substrates and
products and the different uses to which the prod-
uct can be applied.
234 J. C. Soares et al.
The future prospects for enzyme immobilization
procedures could involve site-directed mutagenesis.
There are several reports showing that the use of
site-directed mutagenesis can improve how enzymes
are immobilized on a support, and can be a powerful
tool to improve the properties of immobilized
biomolecules. Therefore, perfectly oriented and very
rigid enzyme molecules could be prepared. Exten-
sive scientific research is still needed, that should
focus on process integration.
Acknowledgements
This work was supported by the project GOBLUE –
Biotecnology applied to textile processing financed
by ADI– Agência de Inovação, S.A., and by a doc-
toral fellowship granted to author A. C. Queiroga
(SFRH/BD/19212/2004) and a post-doctoral fellow-
ship granted to author P. R. Moreira (SFRH/
BPD/26527/2005), both fellowships were granted by
Fundação para a Ciência e a Tecnologia (Portugal).
Declaration of interest: The authors report no
conflicts of interest. The authors alone are respon-
sible for the content and writing of the paper.
References
Abadulla E, Tzanov T, Costa G, Robra KH, Cavaco-Paulo A,
Gübitz GM. 2000. Decolorization and detoxification of textile
dyes with a laccase from Trametes hirsuta. Appl Environ Micro-
biol 66:3357–3362.
Akhtar S, Khan AA, Husain Q. 2005. Potential of immobilized
bitter gourd (Momordica charantia) peroxidases in the decolor-
ization and removal of textile dyes from polluted wastewater
and dyeing effluent. Chemosph 60:291–301.
Alexandre G, Zhulin IB. 2000. Laccases are widespread in bac-
teria. Trends Biotechnol 18:41–42.
Asamudo NU, Daba AS, Ezeronye OU. 2005. Bioremediation of
textile effluent using Phanerochaete chrysosporium. Afr J Biotech-
nol 4:1548–1553.
Atack JM, Kelly DJ 2006. Structure, mechanism, and physiolog-
ical roles of bacterial cytochrome c peroxidases. Adv Microb
Physiol 52:73–106.
Azevedo AM, Martins VC, Prazeres DMF, Vojinovic V, Cabral
JMS, Fonseca PL. 2003. Horseradish peroxidase: a valuable
tool in biotechnology. Biotechnol Ann Rev 9:199–247.
Azevedo AM, Prazeres DMF, Cabral JMS, Fonseca LP. 2001.
Stability of free and immobilized peroxidase in aqueous-organic
solvent mixtures. J Mol Catal B Enzymatic 15:147–153.
Azevedo H, Bishop D, Cavaco-Paulo A. 2002. Possibilities for
recycling cellulases after use in cotton processing. Appl Bio-
chem Biotechnol 101:61–75.
Banci L. 1997. Structural properties of peroxidases. J Biotechnol
53:253–263.
Barceló AR, Pomar F. 2002. Plant peroxidases: versatile catalysts
in the synthesis of bioactive material products. Stud Nat Prod
Chem 27:735–791.
Bayramoğlu G, Yilmaz M, Arica MY. 2010. Reversible immobili-
zation of laccase to polbioresouy(4-vinylpyridine) grafted and
Cu(II) chelated magnetic beads: biodegradation of reactive
dyes. Bioresour Technol 101: 6615–6621.
Betancor L, Hidalgo A, Fernández-Lorente G, Mateo C,
Fernández-Lafuente R, Guisán JM. 2003. Preparation of a sta-
ble biocatalyst of bovine liver catalase using immobilization and
postimmobilization techniques. Biotechnol Prog 19:763–767.
Betancor L, López-Gallego F, Hidalgo A, Alonso-Morales N,
Dellamora-Ortiz G, Guisán JM, Fernández-Lafuente R. 2006.
Preparation of a very stable immobilized biocatalyst of glucose
oxidase from Aspergillus niger. J Biotechnol 121:284–289.
Blánquez P, Sarrà M, Vicent T. 2008. Development of a continu-
ous process to adapt the textile wastewater treatment by fungi
to industrial conditions. Process Biochem 43:1–7.
Blin JL, Gèrardin C, Carteret C, Rodeüser L, Selve C, Stébé MJ.
2005. Direct one-step immobilization of glucose oxidase in
well-ordered mesostructured silica using a nonionic fluorinated
surfactant. Chem Mater 17:1479–1486.
Burton SG. 1994. Biocatalysis with polyphenol oxidase: a review.
Catal Today 22:459–487.
Camarero JA. 2008. Recent developments in the site-specific
immobilization of proteins onto solid supports. Biopolymers
90:450–458.
Campos R, Kandelbauer A, Robra KH, Cavaco-Paulo A, Guebitz
GM. 2001. Indigo degradation with purified laccase from Tram-
etes hirsuta and Sclerotium rolfsii. J Biotechnol 89:131–139.
Cavaco-Paulo A. 1998. Mechanism of cellulase action in textile
processes. Carbohydr Polym 37:273–277.
Cetinus SA, Öztop HN. 2000. Immobilization of catalase on chi-
tosan film. Enzyme Microb Technol 26:497–501.
Chang MY, Juang RS. 2005. Activities, stabilities, and reaction
kinetics of three free and chitosan-clay composite immobilized
enzymes. Enzyme Microb Technol 36:75–82.
Chelikani P, Ramana T, Radhakrishnan TM. 2005. Catalase: a rep-
ertoire of unusual features. Ind J Clin Biochem 20:131–135.
Conesa A, Punt PJ, van del Hondel CA. 2002. Fungal peroxi-
dases: molecular aspects and applications. J Biotechnol 93:143–
158.
Cortez J, Bonner PLR, Griffin M. 2004. Application of trans-
glutaminases in the modification of wool textiles. Enzyme
Microb Technol 34:64–72.
Costa SA, Tzanov T, Paar A, Gudelj M, Gübitz GM, Cavaco-
Paulo A. 2001. Immobilization of catalases from Bacillus SF on
alumina for the treatment of textile bleaching effluents. Enzyme
Microb Technol 28:815–819.
Couto SR, Osma JF, Saravia V, Gübitz GM, Toca Herrera JLT.
2007. Coating of immobilised laccase for stability enhancement:
a novel approach. Appl Catal A-Gen 329:156–160.
Decker H, Dillinger R, Tuckez F. 2000. How does tyrosinase
work? Recent insights from model chemistry and structural
biology. Angew Chem Int Ed 39:1591–1595.
Decker H, Tuczek F. 2000. Tyrosinase/catecholoxidase activity of
hemocyanins: structural basis and molecular mechanism.
Trends Biochem Sci 25:392–397.
Delanoy G, Li Q, Yu J. 2005. Activity and stability of laccase in
conjugation with chitosan. Int J Biol Macromol 35:89–95.
Demirjian DC, Morís-Varas F, Cassidy CS. 2001. Enzymes from
extremophiles. Curr Opin Chem Biol 5:144–151.
Dhingra S, Khanna M, Pundir CS. 2006. Immobilization of
α-amylase onto alkylamine glass beads affixed inside a plastic
beaker: kinetic properties and application. Indian J Chem
Technol 13:119–121.
Dinçer A, Telefoncu A. 2006. Improving the stability of cellulase
by immobilization on modified polyvinyl alcohol-coated
chitosan beads. J Mol Catal B Enzymatic 45:10–14.

Dourado F, Bastos M, Mota M, Gama FM. 2002. Studies on the
properties of Celluclast/Eudragit L-100 conjugate. J Biotechnol
99:121–131.
Duran N, Esposito E. 2000. Potential applications of oxidative
enzymes and phenoloxidase-like compounds in wastewater and
soil treatment: a review. Appl Catal B Environ 28: 83–99.
Duran N, Rosa MA, d’Annibale A, Gianfreda L. 2002. Applications
of laccases and tyrosinases (phenoloxidases) immobilized on dif-
ferent supports: a review. Appl Catal B Environ 28:83–99.
Edwards W, Bownes R, Leukes WD, Jacobs EP, Sanderson R, Rose
PD, Burton SG. 1999. A capillary membrane bioreactor using
immobilized polyphenol oxidase for the removal of phenols from
industrial effluents. Enzyme Microb Technol 24:209–217.
El-Batal AI, Atia KS, Eid M. 2005. Stabilization of α-amylase by
using anionic surfactant during the immobilization process.
Radiat Phys Chem 74:96–101.
Emine A, Leman T. 1995. Characterization of immobilized cata-
lases and their application in pasteurization of milk with H2O2.
Appl Biochem Biotechnol 50:291–303.
Ensuncho L, Alvarez-Cuenca M, Legge RL. 2005. Removal of
aqueous phenol using immobilized enzymes in a bench scale
and pilot scale three-phase fluidized bed reactor. Biopr Biosyst
Bioeng 27:185–191.
Fenoll LG, Rodriguez-Lopez JN, Varon R, Garcia-Ruiz PA,
Garcia-Canovas F, Tudela J. 2000. Action mechanism of tyro-
sinase on meta- and para-hydrolxylated monophenols. Biol
Chem 381:313–320.
Fruhwirth GO, Paar A, Gudelj M, Cavaco-Paulo A, Robra K-H,
Gübitz GM. 2002. An immobilised catalase peroxidase from
the alkalothermophilic Bacillus SF for the treatment of textile-
bleaching effluents. Appl Microb Biotechnol 60:313–319.
Gianfreda L, Xu F, Bollag J-M. 1999. Laccases: a useful group of
oxidoreductive enzymes. Bioremed J 3:1–25.
Giardina P, Aurelia V, Cannio R, Marzullo L, Amoresano A,
Siciliano R. 1996. The gene, protein and glycan structure of
laccase from Pleurotrus ostreatus. Eur J Biochem 235:508–515.
Godjevergova T, Nenkova R, Konsulov V. 2006. Immobilization
of glucose oxidase by acrylonitrile copolymer-coated silica sup-
ports. J Mol Catal B Enzymatic 38:59–64.
Gudelj M, Fruhwirth GO, Paar A, Lottspeich F, Robra K-H,
Cavaco-Paulo A, Gübitz GM. 2001. A catalase-peroxidase from
a newly isolated thermoalkaliphilic Bacillus sp. with potential
for the treatment of textile bleaching effluents. Extremophiles
5:423–429.
Gummadi SN, Panda T. 2003. Purification and biochemical prop-
erties of microbial pectinases – a review. Process Biochem
38:987–996.
Gupta R, Beg QK, Lorenz P. 2002. Bacterial alkaline proteases:
molecular approaches and industrial applications. Appl Micro-
biol Biotechnol 59:15–32.
Gupta R, Gigras P, Mohaptra H, Goswami VK, Chaulan B. 2003.
Microbial α-amylases: a biotechnological perspective. Process
Biochem 38:1599–1616.
Gusakov AV, Sinitsyn AP, Markov AV, Sinitsyna OA, Ankudimova
NV, Berlin AG. 2001. Study of protein adsorption on indigo
particles confirms the existence of enzyme-indigo interaction
sites in cellulase molecules. J Biotechnol 87:83–90.
Hamilton LM, Kelly CT, Fogarty WM. 2000. Review: cyclodex-
trins and their interaction with amylolytic enzymes. Enzyme
Microb Technol 26:561–567.
Hersleth HP, Ryde U, Rydberg P, Görbitz CH, Andersson KK.
2006. Structures of the high valent metal-ion heme-oxygen
intermediates in peroxidases, oxygenases and catalases. J Inorg
Biochem 100:460–476.
Hidalgo A, Betancor L, Lopez-Gallego F, Moreno R, Berenguer J,
Fernández-Lafuente R, Guisán JM. 2003. Design of an
Immobilized enzyme technologies 235
immobilized preparation of catalase from Thermus thermophilus
to be used in a wide range of conditions. Structural stabilization
of a multimeric enzyme. Enzyme Microb Technol 33:278–285.
Hernandez K, Fernandez-Lafuente R. 2011. Control of protein
immobilization: coupling immobilization and site-directed
mutagenesis to improve biocatalyst or biosensor performance.
Enzyme Microb Technol 48:107–122.
Hoondal GS, Tiwari RP, Tewari R, Dahiya N, Beg QK. 2002.
Microbial alkaline pectinases and their industrial applications:
a review. Appl Microbiol Biotechnol 59:409–418.
Iyer PV, Ananthanarayan L. 2008. Enzyme stability and stabiliza-
tion—Aqueous and non-aqueous environment. Process Bio-
chem 43:1019–1032.
Jouve HM, Andreoletti P, Gouet P, Hajdu I, Gagnon J. 1997.
Structural analysis of compound I in hemoproteins: study on
Proteus mirabilis catalase. Biochimie 79:667–671.
Jürghen-Lohmann DL, Legge RJ. 2006. Immobilization of bovine
catalase in sol-gels. Enzyme Microb Technol 39:626–633.
Kagawa M, Murakoshi N, Nishikawa Y, Matsumoto G, Kurata Y,
Mizobata T, Kawata Y, Nagai J. 1999. Purification and cloning
of a thermostable manganese catalase from a thermophilic bac-
terium. Arch Biochem Biophys 15:346–355.
Kahraman MV, Kayaman-Apohan N, Ogan A, Güngör A. 2006.
Soybean oil based resin: a new tool for improved immobiliza-
tion of α-amylase. J Appl Polym Sci 100:4757–4761.
Kajiuchi T, Park JW. 1992. Characteristics of cellulase modified
with a copolymer of polyethylene glycol derivative and maleic
acid anhydride. J Chem Eng Japan 25:202–206.
Kandelbauer A, Maute O, Kessler RW, Erlacher A, Gübitz GM.
2004. Study of dye decolorization in an immobilized laccase
enzyme-reactor using online spectroscopy. Biotechnol Bioeng
87:552–563.
Kara A, Osman B, Yavuz H, Besirli N, Denizli A. 2005. Immobi-
lization of α-amylase on Cu2 ⫹ chelated poly(ethylene glycol
dimethacrylate-n-vinyl-imidazole) matrix via adsorption. React
Funct Polym 62:61–68.
Karam J, Nicell JA 1997. Potential applications of enzymes in
waste treatment. J Chem Technol Biotechnol 69:141–143.
Kashyap DR, Vohra PK, Chopra S, Tewari R. 2001. Applications
of pectinases in the commercial sector: a review. Bioresour
Technol 77:215–227.
Khan AA, Alzohairy MA. 2010. Recent advances and applications
of immobilized enzyme technologies: a review. Res J Biol Sci
5:565–575.
Khan AA, Husain Q. 2007. Decolorization and removal of textile
and non-textile dyes from polluted wastewater and dyeing efflu-
ent by using potato (Solanum tuberosum) soluble and immobi-
lized polyphenol oxidase. Bioresour Technol 98:1012–1019.
Krajewska B. 2004. Application of chitin- and chitosan-based
materials for enzyme immobilizations: a review. Enzyme Microb
Technol 35:126–139.
Krastanov A. 2000. Removal of phenols from mixtures by co-
immobilized lacase/tyrosinase and Polyclar adsorption. J Ind
Microbiol Biotechnol 24:383–388.
Kunamneni A, Ghazi I, Camarero S, Ballesteros A, Plou FJ, Alcalde
M. 2008. Decolorization of synthetic dyes by laccase immobi-
lized on epoxy-activated carriers. Process Biochem 43:169–178.
Lante A, Crapisi A, Krastanov A, Spettoli P. 2000. Biodegradation
of phenols by laccase immobilized in a membrane reactor. Proc-
ess Biochem 36:51–58.
Lei Z, Bi S. 2007a. Preparation and properties of immobilized
pectinase onto the amphiphilic PS-b-PAA diblock copolymers.
J Biotechnol 128:112–119.
Lei Z, Bi S. 2007b. The silica-coated chitosan particle from a
layer-by-layer approach for pectinase immobilization. Enzyme
Microb Technol 40:1442–1447.
236 J. C. Soares et al.
Loewen P. 1996. Probing the structure of catalase HP II of
Escherichia coli – a review. Gene 179:39–44.
Mao X, Guo G, Huang J, Du Z, Huang Z, Ma L, Li P, Gu L.
2006. A novel method to prepare chitosan powder and its appli-
cation in cellulase immobilization. J Chem Technol Biotechnol
81:189–195.
Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM,
Fernandez-Lafuente R. 2007. Improvement of enzyme activity,
stability and selectivity via immobilization techniques. Enzyme
Microb Technol 40:1451–1463.
Matto M, Husain Q. 2006. Entrapment of porous and stable con-
canavalin A-peroxidase complex into hybrid calcium alginate-
pectin gel. J Chem Technol Biotechnol 81:1316–1323.
Matto M, Husain Q. 2009. Decolorization of textile effluent by
bitter gourd peroxidase immobilized on concanavalin A layered
calcium alginate–starch beads. J Hazard Mater 164:1540–1546.
Matulis D, Wu C, van Pham T, Guy C, Lovrien R. 1999. Protection
of enzymes by aromatic sulfonates from inactivation by acid and
elevated temperatures. J Mol Catal B Enzymatic 7:21–36.
Moreira PR, Almeida-Vara E, Sena-Martins G, Polónia I, Malcata
FX, Duarte JC. 2001. Decolorisation of Remazol Brilliant Blue
R via a novel Bjerkandera sp. strain. J Biotechnol 89:107–111.
Moreira PR, Duez C, Dehareng D, Antunes A, Almeida-Vara E,
Frère JM, Malcata FX, Duarte JC. 2005. Molecular charac-
terisation of a versatile peroxidase from a Bjerkandera strain.
J Biotechnol 118:339–352.
Moreira PR, Bouillenne F, Almeida-Vara E, Malcata FX, Frère
JM, Duarte JC 2006. Purification, kinetics and spectral charac-
terisation of a new versatile peroxidase from a Bjerkandera sp.
Isolate. Enzyme Microb Technol 38:28–33.
Morén AK, Eskilsson K, Khan A. 1997. Phase behaviour of oppo-
sitely charged protein and surfactant mixtures: DOTAC-BLG-
WATER. Colloids Surf B Biointerfaces 9:305–314.
Mozhaev VV, Khmelnitsky YL, Sergeeva MV, Belova AB, Klyachko
NL, Levashov AV, Martinek K. 1989. Catalytic activity and
denaturation of enzymes in water/organic cosolvent mixtures:
α-chymotrypsin and laccase in mixed water/alcohol, water/glycol
and water/formamide solvents. Eur J Biochem 184:597–602.
Naidu GSN, Panda T. 1998. Production of pectolytic enzymes: a
review. Biopr Eng 19:355–361.
Oluoch KR, Welander U, Andersson MM, Mulaa FJ, Mattiasson
B, Hatti-Kaul R. 2006. Hydrogen peroxide degradation by
immobilized cells of alkaliphilic Bacillus halodurans. Biocatal
Biotransform 24:215–222.
Opwis K, Knittel D, Kele A, Schollmeyer E. 1999. Enzymatic recy-
cling of starch-containing desizing liquors. Starch 51:348–353.
Opwis K, Knittel D, Schollmeyer E. 2004a. Functionalization of
catalase for a photochemical immobilization on poly(ethylene
terephthalate). Biotechnol J 2:347–352.
Opwis K, Knittel D, Schollmeyer E. 2004b. Immobilization of
catalase on textile carrier materials. AATCC Rev 4:25–28.
Opwis K, Knittel D, Bahners T, Schollmeyer E. 2005. Photo-
chemical enzyme immobilization on textile carrier materials.
Eng Life Sci 1:63–67.
Opwis K, Knittel D, Schollmeyer E. 2007. Functionalization of
catalase for a photochemical immobilization on poly(ethylene
terephthalate). Biotechnol J 2:347–352.
Osma JF, Toca-Herrera JL, Rodríguez-Couto S. 2010. Biodegra-
dation of a simulated textile effluent by immobilised-coated
laccase in laboratory-scale reactors. Appl Catal A General
373:147–153.
Paar A, Costa S, Tzanov T, Gudelj M, Robra K-H, Cavaco-Paulo
A, Gübitz GM. 2001. Thermo-alkali-stable catalases from
newly isolated Bacillus sp. for the treatment and recycling of
textile bleaching effluents. J Biotechnol 89:147–153.
Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R.
2000. Advances in microbial amylases. Biotechnol Appl
Biochem 31:135–152.
Pandya PH, Jasra RV, Newalkar BL, Bhatt PN. 2005. Studies on
the activity and stability of immobilized α-amylase in ordered
mesoporous silicas. Micropor Mesopor Mater 77:67–77.
Pazarlioglu NK, Sariisik M, Telefoncu A. 2005. Treating denim
fabrics with immobilized commercial cellulases. Process
Biochem 40:767–771.
Peralta-Zamora P, Esposito E, Pelegrini R, Groto R, Reyes J,
Durán N. 1998. Effluent treatment of pulp and paper, and
textile industries using immobilized horseradish peroxidase.
Environ Technol 19:55–63.
Peralta-Zamora P, Pereira CM, Tiburtius ERL, Moraes SG, Rosa
MA, Minussi RC, Durán N. 2003. Decolorization of reactive dyes
by immobilized laccase. Appl Catal B Environ 42:131–144.
Pramparo L, Stüber F, Font J, Fortuny A, Fabregat A, Bengoa C.
2010. Immobilisation of horseradish peroxidase on Eupergit®C
for the enzymatic elimination of phenol. J Hazard Mater
177:990–1000.
Queiroga AC, Pintado MM, Malcata FX. 2007. Novel microbial-
mediated modifications of wool. Enzyme Microb Technol
40:1491–1495.
Qiu GM, Zhu BK, Xu YY. 2005. α-Amylase immobilized by
Fe3O4/poly(styrene-co-maleic anhydride) magnetic composite
microspheres: preparation and characterization. J Appl Polym
Sci 95:328–335.
Quinto M, Ciancio A, Zambonin PG. 1998. A molecular resolu-
tion AFM study of gold-adsorbed glucose oxidase as influenced
by enzyme concentration. J Electroanal Chem 448:51–59.
Rodríguez-Couto S, Herrera JLT. 2006. Industrial and biotech-
nological applications of laccases: a review Biotechnol Adv
24:500–513.
Roy I, Gupta MN. 2003. Repeated enzymatic hydrolysis of polyg-
alacturonic acid, chitosan and chitin using a novel reversibly-
soluble pectinase with the aid of κ-carrageenan. Biocatal
Biotransform 21:297–304.
Roy I, Sharma S, Gupta MN. 2004. Smart biocatalysts: design
and applications. Adv Biochem Engin/Biotechnol 86:159–189.
Ryan S, Schnitzhofer W, Tzanov T, Cavaco-Paulo A, Gübitz GM.
2003. An acid-stable laccase from Sclerotium rolfsii with poten-
tial for wool dye decolorization. Enzyme Microb Technol
33:766–774.
Sakai T, Sakamoto T, Hallaert J, Vandamme EI. 1993. Pectin,
pectinase and protopectinase: production, properties and appli-
cation. Adv Appl Microbiol 39:213–294.
Sawada K, Ueda M. 2001. Enzyme processing of textiles in
reverse micellar solution. J Biotechnol 89:263–269.
Schroeder M, Heumann S, Silva CJSM, Cavaco-Paulo A, Gübitz
GM. 2006. Specificities of a chemically modified laccase from
Trametes hirsuta on soluble and cellulose-bound substrates.
Biotechnol Lett 28:741–747.
Seetharam GB, Saville BA. 2003. Degradation of phenol using
tyrosinase immobilized on siliceous supports. Water Res 37:
436–440.
Setti L, Giuliani S, Spinozzi G, Pifferi PG. 1999. Laccase catalyzed-
oxidative coupling of 3-methyl 2-benzothiazolinone hydrazone
and methoxyphenols. Enzyme Microb Technol 25:285–289.
Shaffiqu TS, Roy JJ, Nair RA, Abraham TE. 2002. Degradation
of Textile Dyes Mediated by Plant Peroxidases. Appl Biochem
Biotechnol 102:315–326.
Sheldon RA, Schoevaart R, van Langen LM. 2005. Cross-linked
enzyme aggregates (CLEAs): a novel and versatile method for
enzyme immobilization: a review. Biocatal Biotransform
23:141–147.

Shen J, Rushforth M, Cavaco-Paulo A, Guëbitz G, Lenting H.
2006. Development and industrialisation of enzymatic
shrink-resist process based on modified proteases for wool
machine washability. Enzyme Microb Technol 34:1–6.
Silva C, Silva CJ, Zille A, Guebitz GM, Cavaco-Paulo A. 2007.
Laccase immobilization on enzymatically functionalized polya-
mide 6,6 fibres. Enzyme Microb Technol 41:867–875.
Silva CJSM, Gübitz G, Cavaco-Paulo A. 2006. Optimisation of a
serine protease coupling to Eudragit S-100 by experimental
design techniques. J Chem Technol Biotechnol 81:8–16.
Silva CJSM, Prabaharan M, Gübitz G, Cavaco-Paulo A. 2005.
Treatment of wool fibres with subtilisin and subtilisin-PEG.
Enzyme Microb Technol 36:917–922.
Sinegani AAS, Emtiazi G, Shariatmadari H. 2005. Sorption and
immobilization of cellulase on silicate clay minerals. J Colloid
Interface Sci 290:39–44.
Singh J, Batra N, Sobti RC. 2001. Serine alkaline protease from
a newly isolated Bacillus sp. SSR1. Process Biochem 36:
781–785.
Sivaramakrishnan S, Gaugacharan D, Madhavan K, Nampoothiri
KM, Soccol CR, Pandey A. 2006. α-Amylases from microbial
sources – an overview on recent developments. Food Technol
Biotechnol 44:173–184.
Smith E, Schroeder M, Guebitz G, Shen J. 2010. Covalent bond-
ing of protease to different sized enteric polymers and their
potential use in wool processing. Enzyme Microb Technol
47:105–111.
Spiro MC, Griffith WP. 1997. The mechanism of hydrogen per-
oxide bleaching. Text Chem Color 20:12–13.
Takahashi H, Li B, Sasaki T, Miyazaki C, Kajino T, Inagaki S.
2001. Immobilized enzymes in ordered mesoporous silica
materials and improvement of their stability and catalytic activ-
ity in an organic solvent. Micropor Mesopor Mat 44–45:
755–762.
Taniguchi M, Hoshino K, Watanable K, Sugai K, Fujii M. 1992.
Production of soluble sugar from cellulosic materials by
repeated use of a reversely soluble-autoprecipitating cellulase.
Biotechnol Bioeng 39:287–292.
Thompson VS, Schaller KD, Apel WA. 2003. Purification and
characterization of a novel thermo-alkali-stable catalase from
Thermus brockianus. Biotechnol Prog 19:1292–1299.
Thurston CF. 1994. The structure and function of fungal laccases.
Microbiol 140:1994–1998.
Torres E, Bustos-Jaimes I, LeBorgne S. 2003. Potential use of
oxidative enzymes for the detoxification of organic pollutants.
Appl Catal B Environ 46:1–15.
Tu M, Zhang X, Kurabi A, Gilkes N, Mabee W, Saddler J. 2006.
Immobilization of β-glucosidase on Eupergit C for lignocellu-
lose hydrolysis. Biotechnol Lett 28:151–156.
Tümtürk H, Aksoy S, Hasirci N. 1999. Covalent immobilization
of α-amylase onto poly(methyl methacrylate-2-hydroxyethyl
methacrylate) microspheres and the effect of Ca2 ⫹ ions on the
enzyme activity. Starch 51:211–217.
Turková J. 1999. Oriented immobilization of biologically active
proteins as a tool for revealing protein interactions and func-
tion. J Chromatogr B 722:11–31.
Immobilized enzyme technologies 237
Tzanov T, Andreaus J, Gübitz GM, Cavaco-Paulo A. 2003.
Protein interactions in enzymatic processes in textiles. Electron
J Biotechnol 6:17–23.
Tzanov T, Calafell M, Güebitz GM, Cavaco-Paulo A. 2001.
Bio-preparation of cotton fabrics. Enzyme Microb Technol.
29:357–362.
Tzanov T, Costa SA, Gübitz GM, Cavaco-Paulo A. 2002. Hydro-
gen peroxide generation with immobilized glucose oxidase for
textile bleaching. J Biotechnol 93:87–94.
van der Maarel MJEC, van der Veen B, Uitdehaag JCM,
Leemhuis H, Dijkhuizen L. 2002. Properties and applications
of starch-converting enzymes of alpha amylase family. J Bio-
technol 94:137–155.
Vasconcelos A, Silva CJSM, Schroeder M, Guebitz GM, Cavaco-
Paulo A. 2006. Detergent formulations for wool domestic wash-
ings containing immobilized enzymes. Biotechnol Lett
28:725–731.
Viswanath S, Wang J, Bachas LG, Butterfield DA, Bhattacharya D.
1998. Site directed and random immobilization of subtilisin on
functionalized membranes: activity determination in aqueous
and organic media. Biotechnol Bioeng 60:608–616.
Wada S, Ichikawa H, Tsatsumi K. 1993. Removal of phenols from
wastewater by soluble and immobilized tyrosinase. Biotechnol
Bioeng 42:854–858.
Wang Q, Fan X, Hua Z, Gao W, Chen J. 2007. Degradation kinet-
ics of pectins by an alkaline pectinase in bioscouring of cotton
fabrics. Carbohydr Polym 67:572–575.
Wang Y, Caruso F. 2005. Mesoporous silica spheres as supports
for enzyme immobilization and encapsulation. Chem Mater
17:953–961.
Wesenberg D, Kyriakides I, Agathos SN. 2003. White-rot fungi
and their enzymes for the treatment of industrial dye effluents.
Biotechnol Adv 22:161–167.
Wu L, Yuan X, Sheng J. 2005. Immobilization of cellulase in
nanofibrous PVA membranes by electrospinning. J Memb Sci
250:167–173.
Xu F. 1996. Oxidation of phenols, anilines, and benzethiols by
fungal laccases: correlation between activity and redox poten-
tials as well as halide inhibition. Biochem 35:7608–7614.
Yachmenev VG, Bertoniere NR, Blanchard EJ. 2002. Intensifica-
tion of the bio-processing of cotton textiles by combined enzyme/
ultrasound treatment. J Chem Technol Biotechnol 77:559–567.
Zámocky M, Koller F. 1999. Understanding the structure and
function of catalases: clues from molecular evolution and in
vitro mutagenesis. Prog Biophys Mol Biol 72:19–65.
Zhang Q, Smith E, Shen J, Bishop D. 2006. An ethoxylated alkyl
phosphate (anionic surfactant) for the promotion of activities
of proteases and its potential use in the enzymatic processing
of wool. Biotechnol Lett 28:717–723.
Zhang Y, Xu J, Yuan Z, Xu H, Yu Q. 2010. Artificial neural
network-genetic algorithm based optimization for the immo-
bilization of cellulase on the smart polymer Eudragit L-100.
Bioresour Technol 101:3153–3158.
Zille A, Tzanov T, Gübitz GM, Cavaco-Paulo A. 2003. Immobi-
lized laccase for decolorization of Reactive Black 5 dyeing efflu-
ent. Biotechnol Lett 25:1473–1477.