Food Research International 140 (2021) 109979

Contents lists available at ScienceDirect

Food Research International

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

Review

Biopolymers and nanostructured materials to develop pectinases-based

immobilized nano-biocatalytic systems for biotechnological applications
Shuangshuang Zhang a, Muhammad Bilal b, *, Jakub Zdarta c, Jiandong Cui d, Ashok Kumar e,
Marcelo Franco f, Luiz Fernando Romanholo Ferreira g, h, Hafiz M.N. Iqbal i, *
a
School of Food Science and Technology, Jiangsu Food and Pharmaceutical Science College, Huai’an 223003, China
b
School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huai’an 223003, China
c
Institute of Chemical Technology and Engineering, Faculty of Chemical Technology, Poznan University of Technology, Berdychowo 4, PL-60965 Poznan, Poland
d
State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of Science and
Technology, No 29, 13th, Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, PR China
e
Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, Himachal Pradesh 173 234, India
f
Department of Exact and Technological Sciences, State University of Santa Cruz, 45654-370 Ilhéus, Brazil
g
Graduate Program in Process Engineering, Tiradentes University, Murilo Dantas Avenue, 300, Farolândia, 49032-490 Aracaju, Sergipe, Brazil
h
Institute of Technology and Research, Murilo Dantas Avenue, 300, Farolândia, 49032-490 Aracaju, Sergipe, Brazil
i
Tecnologico de Monterrey, School of Engineering and Sciences, Monterrey 64849, Mexico

A R T I C L E I N F O A B S T R A C T

Keywords: Pectinases are the emerging enzymes of the biotechnology industry with a 25% share in the worldwide food and
Pectinases beverage enzyme market. These are green and eco-friendly tools of nature and hold a prominent place among the
Enzyme biotechnology commercially produced enzymes. Pectinases exhibit applications in various industrial bioprocesses, such as
Polymers
clarification of fruit juices and wine, degumming, and retting of plant fibers, extraction of antioxidants and oil,
Nanomaterials
Immobilization
fermentation of tea/coffee, wastewater remediation, modification of pectin-laden agro-industrial waste materials
Fruit juice clarification for high-value products biosynthesis, manufacture of cellulose fibres, scouring, bleaching, and size reduction of
Bioactive compounds fabric, cellulosic biomass pretreatment for bioethanol production, etc. Nevertheless, like other enzymes, pecti­
nases also face the challenges of low operational stability, recoverability, and recyclability. To address the above-
mentioned problems, enzyme immobilization has become an eminently promising approach to improve their
thermal stability and catalytic characteristics. Immobilization facilitates easy recovery and recycling of the
biocatalysts multiple times, leading to enhanced performance and commercial feasibility. In this review, we
illustrate recent developments on the immobilization of pectinolytic enzymes using polymers and nanostructured
materials-based carrier supports to constitute novel biocatalytic systems for industrial exploitability. The first
section reviewed the immobilization of pectinases on polymers-based supports (ca-alginate, chitosan, agar-agar,
hybrid polymers) as a host matrix to construct robust pectinases-based biocatalytic systems. The second half
covers nanostructured supports (nano-silica, magnetic nanostructures, hybrid nanoflowers, dual-responsive
polymeric nanocarriers, montmorillonite clay), and cross-linked enzyme aggregates for enzyme immobiliza­
tion. The biotechnological applications of the resulted immobilized robust pectinases-based biocatalytic systems
are also meticulously vetted. Finally, the concluding remarks and future recommendations are also given.

1. Introduction
Pectinolytic enzymes are a diversified group of biocatalysts that
mediate the depolymerization and biodegradation of pectinaceous ma­
terials, occurring as structural heteropolysaccharides in the primary cell
walls and middle lamella of terrestrial plants (Ridley, O’Neill, &
Mohnen, 2001; Jayani, Saxena, & Gupta, 2005). Pectinases are catego­
rized into depolymerases and pectin esterase depending on their action
on pectinaceous substances. Depolymerases show catalytic action on
pectin, polygalacturonic acid, and rhamnogalacturonan. Depolymerases
can be further classified into two groups, one group of an enzyme that
acts on pectin and 2nd one that acts on polygalacturonic acid. The first

* Corresponding authors.
E-mail addresses: bilaluaf@hotmail.com (M. Bilal), hafiz.iqbal@tec.mx (H.M.N. Iqbal).

https://doi.org/10.1016/j.foodres.2020.109979
Received 16 June 2020; Received in revised form 27 November 2020; Accepted 8 December 2020
Available online 17 December 2020
0963-9969/© 2020 Elsevier Ltd. All rights reserved.
S. Zhang et al.
group can be further subcategorized into (i) polymethylgalacturonase
(PMG, E.C. 3.2.1.1), which cleaves pectin exolytically (Exo PMG) or
endolytically (Endo-PMG); and (ii) pectin lyase (PL, E.C. 4.2.2.10),
which carries out the trans-elimination reaction of pectin (Endo PL)
endolytically only resulting in unsaturated oligosaccharides. Depoly­
merases acting on the substrate, polygalacturonic, acid can be further
divided into two groups based on their mode of action (i) poly­
galacturonase (PG), which exolytically carries out the hydrolysis of
polygalacturonic acid (Exo PG1, E.C. 3.2.1.67 and Exo PG1 E.C.
3.2.1.82), or endolytically (Endo PG, E.C.3.2.1.15) and (ii) pectate lyase
(PGL), which cause trans-elimination of polygalacturonic acid by its
exoaction (Exo PGL, E.C. 4.2.2.9) and endolytic catalysis (Endo PGL, E.
C. 4.2.2.2) (Lü, Wang, Shao, & He, 2016; Patidar, Nighojkar, Kumar, &
Nighojkar, 2018). Depolymerases that show action by taking rhamno­
galacturonan as a substrate are additionally categorized into (i) rham­
nogalacturonan hydrolyzing enzyme rhamnogalacturonase (RG, E.
C.3.2.1.171), and (ii) rhamnogalacturonan endolyase (RGL, E.C.
4.2.2.23), which caused trans-elimination reaction to depolymerize
rhamnogalacturonan. Esterases also show action on pectinaceous ma­
terials and are classified into (i) pectin acetyl esterase (E.C. 3.1.1.6) and
(ii) pectin methylesterase (E.C. 3.1.1.11) (Amin et al., 2017a; 2017b,
Amin, Mohsin, Bhatti, & Bilal, 2020). These enzymes exhibit applica­
tions in various industrial bioprocesses, such as clarification of fruit
juices and wine, degumming and retting of plant fibers, extraction of
antioxidants and oil, fermentation of tea/coffee, wastewater remedia­
tion, valorization of pectin-laden agro-industrial wastes into high-value
Fig. 1. The exploitation of various polymeric cues and nanostructured constructs for pectinase immobilization and their effective deployment for different
applications.
2
Food Research International 140 (2021) 109979
products, manufacture of cellulose fibres, scouring, bleaching, and size
reduction of fabric, cellulosic biomass pretreatment for bioethanol
production, etc. (Alkorta, Garbisu, Llama, & Serra, 1998; Henriksson,
Akin, Slomczynski, & Eriksson, 1999; Hoondal et al., 2002; Rehman
et al., 2013; Rehman, Aman, Zohra, & Qader, 2014; Cerreti, Liburdi,
Benucci, & Esti, 2016; Amin, Bhatti, & Bilal, 2019; Amin et al., 2020).
Unlike chemical treatment, pectinolytic-assisted degradation and
depolymerization of pectinaceous materials is an environmentally
realistic and economical approach, as biocatalysts possess marked ac­
tivity, high selectivity, and specificity under the milder reaction envi­
ronment (Amin, Bhatti, Bilal, & Asgher, 2017c). Therefore, in the
current biotechnological era, pectinases have gained global attention as
emerging biocatalysts that comprise of 25% share in the global food and
beverage enzyme market (Jayani et al., 2005; Nakkeeran,
Umesh-Kumar, & Subramanian, 2011). There has been an accelerating
rise in the pectinases market, maintaining the average annual growth
rate of 2.86% from 27.6 million $ in 2013 to 30 million $ in 2016, and
expected to increase up to 35.5 million $ by 2021 (Global Pectinase
Market Research Report, 2017). Among the entire global market for
enzymes with industrial potentialities, approximately half of the share
goes to food applications. There have been three main legislation gov­
erning bodies, i.e., (1) the Joint Food and Agriculture Organization of
the United Nations/World Health Organization Expert Committee on
Food Additives, (2) the European Food Safety Authority, and (3) the U.S.
Food and Drug Administration, which regulate the production and uti­
lization of enzymes in food related applications.
S. Zhang et al.
A high potential to catalyze a wide variety of reactions renders mi­
crobial pectinases very useful biocatalysts for application in biotech­
nological sectors (Hoondal et al., 2002). The rational elucidation for
their enormous applicability is their broad-substrate specificity, versa­
tility, inducibility, and ability to act on a large number of pectic sub­
stances Pedrolli & Carmona, 2010; Amin et al., 2020). The catalytic
features and the stability of pectinases under various physicochemical
conditions are important for their commercialization. The catalytic at­
tributes and molecular weight of pectinase vary in different microbial
strains. However, yeasts and molds produce pectinases, which can
function in acidic conditions (Ahmed & Sohail, 2020). Fig. 1 shows the
exploitation of various polymeric cues and nanostructured constructs for
pectinase immobilization and their effective deployment for different
applications.
Notwithstanding all these advantages and proven potential, the in­
dustrial deployment of native enzymes is often restrained due to lack of
sufficient operational stability, challenging recoverability and recycla­
bility (Sheldon, 2007; Liu, Zhao, He, Chen, & Huang, 2012; Bilal & Iqbal,
2020a). To deal with these insufficiencies, considerable attention has
been directed to find new pectic enzymes and devising novel approaches
to augment the biocatalytic features of existing enzymes for industrial
syntheses and biotransformation (Li, Li, Wang, & Tain, 2008). Several
techniques including chemical modification, protein engineering, use of
additives, and immobilization have been recognized for modifying en­
zymes from their pristine forms to industrial biocatalysts (Rehman et al.,
2016; de Oliveira, Dias, da Silva, & Porto, 2018; Benucci et al., 2019;
Bilal & Iqbal, 2019a; Bilal & Iqbal, 2019b). Enzyme immobilization has
been proved an efficient approach to overcome the above-mentioned
drawbacks (Hanefeld, Gardossi, & Magner, 2009; Tran and Balkus Jr
2011; Garcia-Galan et al., 2011; Sheldon & van Pelt, 2013; Bilal et al.,
2019a, 2019b, 2019c; Bilal et al., 2020a, 2020b) (Fig. 2). In addition to
convenient handling of the enzyme, it offers easy separation from the
product, thereby reducing protein contamination of the product.
Immobilization also enables the efficient recovery and re-cycle of the
biocatalyst, thus promoting its cost-effective use in continuous opera­
tions (Homaei, Sariri, Vianello, & Stevanato, 2013; Li et al., 2020; Bilal
& Iqbal, 2019c; Bilal & Iqbal, 2020b; Bilal & Iqbal, 2020c). Additional
benefits are generally improved storage and operational stability, and
resistant to denaturation by heat or organic solvents or by autolysis.
Enhanced enzyme biocatalytic performance and repeated re-use are
reflected in higher catalyst productivities (kg product per kg enzyme),
which subsequently determine the enzyme costs per kg product (Shel­
don, 2007). Investigators have chemically modified the pectinases using
immobilization technologies to improve their thermal stability and
Fig. 2. Comparative evaluation of challenging issues and improved features of crude and immobilized pectinase in/on nanostructured constructs.
3
Food Research International 140 (2021) 109979
catalytic characteristics. Immobilized pectinases may render enzymes
less vulnerable to harsh conditions; circumvent product contamination
and improve process control. Researchers have proposed many methods
for pectinases immobilization. Among these, entrapment and covalent
binding to a carrier are the most popular and frequently used methods
for the immobilization of pectinases. In this review, we present recent
developments on the immobilization of pectinolytic enzymes using
polymers and nanomaterials-based carrier supports to constitute novel
biocatalytic systems. The biotechnological applications of the resulted
immobilized robust pectinases-based biocatalytic systems are also
thoroughly vetted. This review offers new opportunities to develop
enzyme-mediated promising and novel technologies for application in
the food industry.
2. Immobilization of pectinases on polymers-based supports
2.1. Pectinase immobilization on Ca-alginate
A variety of techniques such as entrapment, adsorption, and covalent
binding has been proposed for pectinases immobilization. Entrapment is
an immobilization method, which physically restricts enzyme molecules
within the structured polymer network. Though a plethora of different
support matrices is available, porous immobilization matrix, in partic­
ular, calcium alginate is among the most widely used matrix for the
entrapment of pectinase enzymes due to low cost, good biocompati­
bility, and abundant availability than other methods (Talekar & Cha­
vare, 2012; Zhang et al., 2013). Alginate is a naturally occurring anionic
polymer that is obtained from marine algae. This polysaccharide can
generate biocompatible and thermally stable hydrogel network in the
presence of calcium cations. Through a simple approach, the non-toxic,
inexpensive, and mechanically stable spherical particles can be formed
(Flores-Maltos et al., 2011). de Oliveira et al. (2018) immobilized
Aspergillus aculeatus pectinase in alginate beads and investigated their
thermodynamics and kinetics characteristics in comparison to the free
form of the enzyme. Both the free and entrapped pectinases exhibited
better stability at 30 and 40 ◦ C since they took longer times for a 10%
activity reduction of the original values. At a higher temperature value
beyond 50 ◦ C, the thermal tolerance was markedly reduced, but the
immobilized biocatalyst was observed to be more stable compared to the
free counterpart as evidenced by the thermodynamic parameters (acti­
vation energy, entropy, enthalpy, and Gibbs free energy). Deng et al.
(2019) adopted an endogenous emulsification approach, for the first
time, to synthesize Ca-alginate beads and used for the immobilization of
polygalacturonase. Under optimal preparation conditions [sodium
S. Zhang et al.
alginate concentration (4.5%), pH value (4.0), enzyme quantity (39.0
mg), time (6.5 h), stirring speed (400 rpm), and W(Ca): W(Alg) of
13.0%], the resultant microspheres with an average particle diameter of
38.0 ± 0.2 μm presented appreciable sphericity, narrow particle-size
distribution, surface area, and storage stability. The Ca-alginate beads
entrapped immobilized polygalacturonase displayed about 10 ◦ C higher
temperature optimum (at 60 ◦ C) compared to the free form of the
enzyme and the catalytic activity was less vulnerable to pH variations. It
also demonstrated high operative stability retaining more than 60% of
its preliminary activity after three consecutive reaction cycles. In
another study, three different biopolymers including Ca-alginate mi­
crospheres, agar-agar, and polyacrylamide hydrogels were assessed for
polygalacturonase immobilization via an easy and simple entrapment
approach. Among these ascertained supports, polyacrylamide gel turns
out to be the most prospective immobilization support yielding the
highest immobilization efficiency (89%) relative to Ca-alginate (46%)
and agar-agar (80%). In contrast to the free enzyme, which showed
maximum pectinolytic activity after 5.0 min of reaction, the polymers-
entrapped polygalacturonase prolonged the reaction time and exhibi­
ted the best biocatalytic activity after 10 min of reaction. After immo­
bilization, the temperature was improved from 45 ◦ C to 50 ◦ C and 55 ◦ C
in the case of agar-agar and Ca-alginate microspheres, respectively.
Temperature stability was substantially enhanced after biopolymers
entrapment and enzyme presented greater resistance towards various
temperature values than that to the free enzyme. It also showed
remarkable repeatability maintaining over 80% of its primary activity
after two batch reactions (Rehman et al., 2016). The entrapment of
crude pectinolytic enzymes produced by Aspergillus niger on Na-alginate
polysaccharide extracted from Cystoseira Caespitosa was reusable for up
to four consecutive cycles keeping more than 40% of its actual activity
(Hachemi-Benmalek, Nouani, & Benchabane, 2019). Rehman et al.
(2013) optimized the immobilization process for a Bacillus sp. pectinase
on Ca-alginate support. Not only the optimal reaction time was
increased but also the temperature optima for the best catalytic per­
formance showed an increment. Nonetheless, the optimal working pH
remained the same for both counterparts. Compared to the free pecti­
nase (30% activity), the immobilized enzyme presented over 80% of its
preliminary performance after incubating at 30 ◦ C for 5 days. Good
operational stability was also observed for the immobilized enzyme and
65% activity was retained during the 3rd cycle of use. Table 1 illustrates
polymers-based materials as support matrices for pectinases immobili­
zation and their improved biocatalytic features.
2.2. Pectinase immobilization on chitosan
Chitosan-based immobilization is a promising strategy for enzyme
modification owing to a set of unique functional and structural merits
such as degradability, biocompatibility, physiological inertness, non-
toxicity, and cost-effectiveness (Monier, Ayad, Wei, & Sarhan, 2010).
Chitosan is an amine-carrying linear chain biopolymer that is obtained
by alkaline hydrolysis of chitin. The presence of reactive amino groups
in chitosan structure can readily act as chemical alteration sites and thus
making chitosan as one of the few naturally occurring cationic poly­
electrolytes (pKa-6.5) (Bilal et al., 2017a). Pectinolytic consortia
including polygalacturonase (PG), pectin methylesterase (PME), and
pectin lyase (PL) were extracted from Aspergillus ornatus, purified and
separately immobilized on the surface of high-quality chitosan micro­
spheres using dextran polyaldehyde as a crosslinker. After immobiliza­
tion, the thermal and pH stability profile of the enzyme was improved,
while an insignificant difference between the Km and Vmax of soluble and
immobilized biocatalysts revealed no conformational alteration and
preservation of biologically functional three-dimensional structure
(Irshad et al., 2017). Bibi, Irshad, Anwar, Bhatti, and Raza (2017)
extracted active fractions of pectin lyase (PL) and polygalacturonase
(PG) from Neurospora crassa-fermented citrus peel waste and surface-
attached on chitosan support in the presence of glutaraldehyde. The
4
Food Research International 140 (2021) 109979
chitosan-bound enzyme fractions were found stable over a wide tem­
perature (45–85 ◦ C) and pH (4.0–9.0). Immobilized biocatalysts pre­
served their conformational flexibility that was indicated by a negligible
difference between the Km and Vmax of both fractions. After immobili­
zation, the pectin lyase and polygalacturonase maintained over 70% of
their original catalytic activities after processing in seven successive
reaction cycles. Immobilization of Aspergillus niger pectinase on
chitosan-grafted chitin support was reported by adsorption on glutar­
aldehyde activated supports as well as covalent binding in the presence
of glutaraldehyde. After immobilization, the heat endurance or thermal
resistance was improved, and the immobilized biocatalyst showed high
stability maintaining 100% and 70% of its original activity after 9 and
15 repeated cycles (Ramirez, Gómez Brizuela, Úbeda Iranzo, Arevalo-
Villena, & Briones Pérez, 2016).
2.3. Pectinase immobilization on hybrid polymers-based supports
Hybrid polymeric networks, the combination of two or more poly­
mers, are preferred immobilization support matrices in the biomedical
and biotechnological applications because of their superior biophysical
characteristics such as high stability, easy construction to various
geometrical shapes and forms, and soft, firm and rubbery texture (Bilal
et al., 2017b). The combination of different polymers can provide an
improvement in the physical–chemical properties of biomaterials such
as mechanical and chemical resistance, biocompatibility, biodegrad­
ability, permeability, and processability (de Barros Nunes et al., 2019;
Thai et al., 2020; Soares et al., 2020). Inadequacies such as enzyme
leaching, and greater pore size can also be addressed using hybrid
polymers for enzyme immobilization. Several researchers have reported
the immobilization of pectinases on hybrid polymers-based supports.
Wahab et al. (2018) attempted the covalent immobilization of pectinase
by A. awamori KX943614 fungus onto hydrogel microspheres composed
of two biopolymers including agar and alginate. The prepared beads
were immersed in polyethyleneimine solution for 3 h followed by
glutaraldehyde soaking to integrate the new aldehyde group and then
used for immobilization (Fig. 3). Characterization revealed that the
immobilization process broadened the optimum working range of pH
and the optimal temperature was also displaced from 55 ◦ C to 60 ◦ C,
which is considered favorable to prevent microbial contamination. The
thermodynamics profile depicted an extensive improvement in thermal
stability against high temperatures combined with an increase in D-
values and half-lives. The immobilized bioconjugate presented appre­
ciable reusability retaining over 60% of its actual catalytic activity after
eight reuse cycles. Polymer inorganic-hybrid materials exhibit exquisite
properties of organic as well as an inorganic matrix (Lu, Zhang, Ma,
Song, & Gu, 2012). These hybrid biocomposites produced by the bio­
mimetic mineralization process offer a biocompatible and environ­
mentally friendlier surface for enzyme attachment. In addition, it is an
easy and simple method and thus conceived as next-generation host
materials for the encapsulation and steadiness of enzymes (Shen et al.,
2011). Bustamante-Vargas et al. (2016) fabricated a biomimetic poly­
meric network of alginate/gelatin/calcium oxalate (AGOCa) for encap­
sulating crude pectinase extract produced by Aspergillus niger ATCC
9642. For performance evaluation, the AGOCa support matrix was
compared with control matrices of alginate/water (ACa) and alginate/
calcium oxalate (AOxal). pH and temperature characterization revealed
an improvement in pectinases activity by immobilization on both
ascertained matrices by increasing pH and temperature. After storage for
over one month, AGOCa-immobilized biocatalyst, among three different
matrices, better storage stability with a relative activity of 160%. The
pectinase encapsulated on Aca, AOxal, and AGOCa could be recycled in
7, 8, and 10 cycles, respectively, retaining about 40% of its original
catalytic activity.
Sometimes, the reaction catalyzed by a single enzyme is not satis­
factory, particularly, in living entities, the use of multienzyme driven
reactions with high efficiency encourages the conception of artificial
S. Zhang et al. Food Research International 140 (2021) 109979

Table 1
Illustration of polymers-based materials as support matrices for pectinases immobilization, and their improved biocatalytic features.
Source of Enzyme name Polymeric support Type of Improved biocatalytic performance References
pectinase immobilization

Aspergillus niger Pectinase Calium alginate beads Entrapment Recyclability in 4 cycles retaining over Hachemi-Benmalek et al.
40% of its original activity. (2019)
Schizophyllum Pectin lyase Chitosan beads Adsorption High thermal stability in the range of Mehmood et al. (2019)
commune 45–55 ◦ C and pH 8.0–9.0.
Stable up to six consecutive cycles of
reuse.
Schizophyllum Pectin lyase Sodium alginate beads Entrapment Elevated pH and thermal stability of Mehmood et al. (2019
commune immobilized enzyme.
Aspergillus niger Polygalacturonase Ca-alginate beads Entrapment Immobilized enzyme displayed about Deng et al. (2019)
10.0 ◦ C higher temperature optimum (at
60.0 ◦ C) compared to the free form. High
operative stability retaining more than
60% of its preliminary activity after three
consecutive cycles.
Aspergillus Polygalacturonase Ca-alginate beads Covalent Improved kinetic and thermodynamic de Carvalho Silva, de
aculeatus immobilization features França, Converti, and Porto
URM4953 (2019)
Bacillus cereus Pectin lyase Ca-alginate Entrapment Enhanced stability and biocatalytic Kohli and Gupta (2019)
properties
Aspergillus Pectinase Calcium alginate beads Encapsulation Increased stability in the pH range Cardoso and KOBLITZ, M.
oryzae between 7 and 8 and above 10 to 12. G. B., ORTIZ, G. M. D.,
CARVALHO, J. L. V. D., &
CARVALHO, L. M. J. D.
(2019)
Aspergillus Pectinase Calcium alginate beads Entrapment Improved stability at a higher de Oliveira et al. (2018)
aculeatus temperature value beyond 50 ◦ C.
Commercial Pectinase Alginate Entrapment Increased specific activity Gür et al. (2018)
Commercial Pectinase Glutaraldehyde- Covalent High activity that free form of enzyme Gür et al. (2018)
functionalized chitosan immobilization
microspheres
Aspergillus Polygalacturonase Ca-alginate Entrapment Elevated thermal stability of the de Carvalho Silva, de
aculeatus entrapped biocatalyst França, Converti, and Porto
URM4953 Resistance to heating than free enzyme (2018)
Exceptional recycling ability in multiple
cycles.
Aspergillus Pectinase Agar and alginate Covalent Immobilization process broadened the Wahab et al. (2018)
awamori immobilization optimum working range of pH and the
KX943614 optimal temperature was also shifted
from 55 ◦ C to 60 ◦ C.
A substantial enhancement in thermal
stability against high temperatures
combined with increase in D-values and
half-lives.
The immobilized bioconjugate presented
appreciable reusability retaining over
60% of its initial activity after eight
repeated reuse cycles.
Neurospora crassa Pectin lyase Chitosan beads Covalent Stable working over a wider temperature Bibi et al. (2017)
attachment and pH range from 45 to 85 ◦ C, and
4.0–9.0, respectively.
High reusability maintaining over 70% of
the original catalytic activity after seven
reaction cycles.
Neurospora crassa Polygalacturonase Chitosan beads Covalent Excellent pH and temperature stability. Bibi et al. (2017)
attachment Recycling and reuse for seven cycles.
Aspergillus Pectinolytic consortia Chitosan beads Covalent binding The pH and temperature stability profile Irshad et al. (2017)
ornatus including polygalacturonase, of the enzyme was considerably
pectin methylesterase, and improved,
pectin lyase Immobilized biocatalysts revealed no
conformational alteration and
preservation of biologically functional
three-dimensional structure.
Bacillus Polygalacturonase calcium alginate beads Entrapment After entrapment, the temperature Rehman et al. (2016)
licheniformis stability was substantially enhanced.
KIBGE-IB21 Immobilized pectinase presented greater
resistant towards various temperature
values than that to the free enzyme.
It also showed remarkable repeatability
maintaining over 80% of its primary
activity after two batch reactions.
Bacillus Polygalacturonase Polyacrylamidegel Entrapment High thermal stability Rehman et al. (2016)
licheniformis Immobilized biocatalyst showed well
KIBGE-IB21
(continued on next page)

5
S. Zhang et al. Food Research International 140 (2021) 109979

Table 1 (continued )
Source of Enzyme name Polymeric support Type of Improved biocatalytic performance References
pectinase immobilization

reusability over two-batch reaction,

preserving over 80% of its initial activity.
Bacillus Polygalacturonase Agar-agar matrix Entrapment Entrapped pectinase maintained over Rehman et al. (2016)
licheniformis 80% of its primary catalytic activity after
KIBGE-IB21 two consecutive batch reaction cycles.
Improved thermal stability after agar-agar
immobilization.
Aspergillus niger Pectinase extract Biomimetic polymeric Encapsulation Improvement in pectinases activity by Bustamante-Vargas et al.
ATCC 9642 network of alginate/ increasing pH and temperature. (2016)
gelatin/calcium oxalate Immobilized biocatalyst better showed
storage stability after storage of one
month.
Excellent recycling ability in multiple
cycles.
Aspergillus Pectinase Epoxy-activated acrylate Covalent binding Improved performance in a wide working Rajdeo et al. (2016)
aculeatus copolymer range from pH 3.0 to 7.0.
It can be reused and recycled more than
10 times without loss of any noticeable
activity.
Aspergillus niger Pectinase Chitosan-grafted chitin Covalent binding The thermal resistance was improved, and Ramirez et al. (2016)
support the immobilized biocatalyst showed high
stability maintaining 100% and 70% of its
original activity after 9 and 15 repeated
cycles.

Fig. 3. Schematic representation of alginate-agar gel activation and enzyme immobilization. Reprinted from Wahab et al. (2018) with permission from Elsevier.
Copyright © 2018 Elsevier B.V. License Number: 4956910763123.

multienzyme-based biocatalytic systems (Li et al., 2014; Ren et al.,
2020). Intending to construct robust biocatalysis, the designing of arti­
ficial multienzyme systems that mimic multiple or cascaded reactions is
of paramount importance. Two or more enzymes, which are capable of
working together, are integrated to construct a multi-enzyme system to
catalyze a cascade reaction (Zhang, Lyu, Ge, & Liu, 2014; Wu, Ge, Yang,
Hou, & Liu, 2015). In this juncture, the co-immobilization of multien­
zymes emerges as a potential alternative to traditional multi-step syn­
thetic methods. Gür, İdil, and Aksöz (2018) produced a co-immobilized
biocatalytic system adopting two different immobilization strategies.
For this, alginate and glutaraldehyde-functionalized chitosan micro­
spheres were used for co-immobilizing three enzymes pectinase,
α-amylase, and protease. Immobilization of enzymes on both support
materials results in increased specific activities, in which the
glutaraldehyde-activated chitosan beads co-immobilized enzymes a
particularly high activity that free forms of enzymes.
2.4. Pectinase immobilization on other polymers
Pectinase from Aspergillus aculeatus was effectively immobilized on
biocompatible and recyclable polymeric support matrix prepared by
reacting polyethyleneimine with epoxy-functionalized acrylate copol­
ymer DILBEAD-VWR. The enzyme was first immobilized on the polymer
by simple adsorption at neutral pH through ion exchange and subse­
quently cross-linked by dextran aldehyde for stabilization (Fig. 4)
(Rajdeo, Harini, Lavanya, & Fadnavis, 2016). In comparison with the
non-immobilized enzyme, which presents an optimal pH of 5.0, the
polymer-conjugated pectinase showed activity in a wide working range
from pH 3.0 to 7.0. It can be recycled and reused more than 10-times
without dropping any noticeable activity. Interestingly, the immobi­
lized dextran aldehyde and bio-conjugate can be easily separated from
the polymeric support by HCl treatment and thus can be again exploited
for immobilizing new batch of the enzymes (Rajdeo et al., 2016).

6
S. Zhang et al.
Fig. 4. Reversible immobilization of pectinase on PEI-modified DILBEAD-VWR. Reprinted from Rajdeo et al. (2016) with permission from Elsevier. Copyright © 2016
Institution of Chemical Engineers. Published by Elsevier B.V. License Number: 4956911063376.
Rehman et al. (2014) immobilized a pectinolytic enzyme from
B. licheniformis KIBGE IB-21 in the agar-agar support matrix using an
entrapment strategy. Maximum entrapment efficiency was observed
using a 3.0% agar-agar with an enzyme activity of 80%. In contrast to
the free enzyme, immobilization increased the temperature optima 5 ◦ C
higher and reaction duration from 5 to 10 min. After 2 h incubation, the
agar-agar-coupled biocatalyst preserved up to 82% and 71% of its ac­
tivity at 30 and 40 ◦ C, respectively. It was reusable for 10 consecutive
cycles maintaining 69.21% of activity after the 3rd cycle. Esawy, Gamal,
Kamel, Ismail, and Abdel-Fattah (2013) entrapped an Aspergillus niger
pectinase in polyvinyl alcohol (PVA) sponge. After immobilizing the
enzyme in PVA sponge, a shift in pH from 4 to 6 and a temperature shift
to 50 ◦ C from 40 ◦ C was observed. pH stability and thermal stability
profile were greatly improved than the free counter form. The immo­
bilized counterpart could be used in 12 cycles with a 9% loss in activity
only. Kinetic parameters i.e. Vmax and Km of the enzyme were signifi­
cantly changed after immobilization.
3. Pectinase immobilization on nanosupports
3.1. Pectinase immobilization on silica-based supports
In recent years, the use of nanoscale materials as host matrices for
enzyme immobilization has gained fascinating research interest to
improve their stability and catalytic performance owing to their sub­
stantial specific surface area (Table 2) (Min, Kim, Park, & Yoo, 2012;
Mukhopadhyay, Bhattacharyya, Dasgupta, & Chakrabarti, 2015;
Kumari, Kaur, Srivastava, & Sangwan, 2015). Silica-based nano­
structures are being widely employed as enzymatic carrier support due
to the facile handling, fabrication, and functionalization, high me­
chanical and thermal resistance, and steadiness in acidic settings or
organic solvents (Tallury, Payton, & Santra, 2008). Lei et al. (2006)
reported that the covalent immobilization of pectinase onto amino and
carboxylic acid-modified nanoporous silica showed improved activity
and stability. Likewise, the immobilization results in a substantial
improvement in stability and catalytic performance of pectinase onto
amino-functionalized silica-coated MNPs, which were surface activated
7
Food Research International 140 (2021) 109979
by glutaraldehyde (Seenuvasan et al., 2013). Pectinase enzyme isolated
from Aspergillus aculeatus was immobilized onto nano-silica and Florisil
supports in the presence of 3-glyoxypropyltrietoxysilane and glutaral­
dehyde as spacer arms. Immobilized pectinase onto both supports
showed complete inactivation when 3-glyoxypropyltrietoxysilane was
used as a spacer arm. However, it retained 75 and 10% of activity by
immobilization using glutaraldehyde spacer arm onto nano-silica, and
Florisil, respectively. Results revealed that the nano-silica immobilized
pectinase via glutaraldehyde spacer arm exhibited 6.3-folds greater
biocatalytic efficacy compared to the enzyme coupled on Florisil. The
temperature resistance ability of pectinase at 35 ◦ C was 2.3-fold
increased following attachment onto silica nanosupport (Alagöz,
Tükel, & Yildirim, 2016).
3.2. Pectinase immobilization on magnetic nanostructures
Though nanosupports have garnered increasing attention since they
provide elevated specific surface area for the binding of pectinolytic
enzymes. Nonetheless, the separation of immobilized formulation from
the reaction system remains a challenging step, because the clarification
process of fruit juices produces insoluble sediments. Therefore, the
effective recovery of immobilized enzymes and sediment separation is
not possible by simple centrifugation and filtration methods. Magnetic
particles stand out as a prospective kind of immobilization support due
to notable features such as biodegradability, biocompatibility, low
toxicity, high surface area, amenability to surface modification, simple
fabrication, and superparamagnetic properties (Sojitra et al., 2017;
Defaei, Taheri-Kafrani, Miroliaei, & Yaghmaei, 2018; Soozanipour,
Taheri-Kafrani, Barkhori, & Nasrollahzadeh, 2019). Their magnetic core
permits rapid, easy, and highly efficient retrieval of the immobilized bio-
conjugate from the reaction combination by applying an external mag­
netic field. The size of the MNPs can be modified to furnish high surface
area and, eventually greater catalytic performance (Laurent et al., 2008;
Ansari & Husain, 2012). Notwithstanding all these desired merits, the
lack of adequate functional moieties on their surface, and vulnerability
to oxidative and acidic conditions necessitate functionalization of bare
MNPs by some biocompatible compounds for stability maintenance
S. Zhang et al. Food Research International 140 (2021) 109979

Table 2
Illustration of nanostructured materials as catalytic supports for pectinases immobilization, and their improved biocatalytic features.
Source of pectinase Enzyme name Polymeric support Type of Improved biocatalytic performance References
immobilization

Bacillus subtilis Mutagenic Hybrid nanoflowers Covalent Immobilized nanoconjugate retained 33% of remaining Wu et al. (2020)
pectate lyase attachment activity after heating at 55 ◦ C for 24 h, whereas the soluble
PEL3 was inactivated after 18 h of incubation at the same
temperature.
Promising operational stability maintaining over 50% of
activity after four repeated use.
Commercial Pectinase Polyethylene glycol- Covalent binding Immobilized pectinase showed enhanced functional Kharazmi et al.
grafted magnetic stability, improved biocatalytic efficiency, and appreciable (2020)
nanoparticles reuse capability.
Elevated pH and thermal stability profile.
Exceptional storage stability and recyclability retaining
more than 94% and 55% of its preliminary activity after 125-
days storage at 25 ◦ C, and 10 repeated cycles, respectively.
Aspergillus aculeatus Pectinase Magnetic nanoparticles Covalent Two folds increased half-life in the temperature range of Nadar and
attachment 50–70 ◦ C as compared to native form. Rathod (2019)
Nanobiocatalyst retained up to 80% of residual activity even
after ten successive cycles.
– Pectinase Glutaraldehyde- Covalent Higher magnetic properties Dal Magro et al.
activated magnetic attachment High thermal stability and activity recovery. (2018)
particles
– Pectinase Magnetic CLEAs Covalent High performing and stable biocatalyst presenting elevated Dal Magro et al.
attachment recovered activities, thermal stabilities and enhanced (2018)
recyclability.
Clostridium Recombinant Magnetic nanoparticles Covalent Increased activity than the free enzyme. Chakraborty
thermocellum ATCC- pectate lyase immobilization 32-fold and 14-fold enhanced thermal stability at 80 ◦ C and et al. (2017)
27405 90 ◦ C, respectively, compared with free form of enzyme.
At high temperature, it retained activity for a longer
duration than free PL1B.
Reusability up to 5 cycles with retention of 70% of initial
activity.
Commercial Pectinase Chitosan magnetic Covalent As compared to free form, immobilized nanobiocatalyst Sojitra et al.,
nanoparticles attachment presented 2-folds enhancement in temperature resistance at (2017)
high temperature range of 55–75 ◦ C.
Robust stability and durability retaining over 80% of
remaining activity after seven reuse cycles. Up to 89% of
residual activity after storing for 15 days.
Aspergillus aculeatus Pectinase Nano-silica Covalent Immobilized pectinase retained 75 of its activity. Alagöz et al.
attachment Presented 6.3-folds greater biocatalytic efficacy compared to (2016)
the enzyme coupled on Florisil.
The thermal stability of pectinase was 2.3-fold increased at
35 ◦ C.
Aspergillus aculeatus Pectinase Florisil Covalent Immobilized pectinase onto Florisil support exhibited 10% Alagöz et al.
attachment of activity using glutaraldehyde spacer arm. Significant (2016)
improvement in the thermal stability and catalytic
performance.
Qing-dao Vland Pectinase Fe3O4 magnetic Covalent Enhanced storage stability and repeatability retaining more Fang et al.
Biotech Group nanometer particles immobilization than 60% of its initial activity after seven consecutive reuse (2016)
Company Limited. batches. It only dropped about 20% of activity after storage
for 30 days.

(Wu, He, & Jiang, 2008; Soozanipour et al., 2019; Defaei et al., 2018).
Generally, some functional groups as –NH2,–SH, –COOH, and –CH =
CH2, are inserted on the surface of MNPs for chemical modification
(White & Tripp, 2000; Shen, Fang, Zhou, & Liang, 2004; Bruce & Sen,
2005). For example, APTES (3-Aminopropyltriethoxysilane) is being
widely utilized as a promising silane-coupling molecule for amino
modification and silanization treatment on the surface of magnetic
particles and microspheres because APTES is enriched with numerous
amino- and many other functional groups, which take part in the reac­
tion (Etienne & Walcarius, 2003; Zhu, Yang, & Deng, 2009). Fang, Chen,
Zhang, and Chen (2016) successfully introduced NH2 groups on the
surface of Fe3O4@SiO2 NPs (30–40 nm) and wrapped in ammoniated
silicon dioxides, which were synthesized by sol–gel technique. The as
developed immobilized pectinase showed enhanced storage stability
and repeatability retaining over 60% of its initial bioactivity after seven
consecutive reuse batches. It only dropped about 20% of activity after
storage for 30 days. Seenuvasan et al. (2013) conducted a study for the
fabrication of nanobiocatalyst by covalent bonding of the pectinase onto
amino-functionalized silica-coated and glutaraldehyde-activated MNPs.
FTIR and TEM were used to characterize the particle size, morphology,
surface modifications, and binding of pectinase onto nanoparticles. The
immobilized biocatalytic system displayed augmented stability in the
acidic region of pH to act against pectin with greater affinity than the
free counterpart. Temperature optima of the free enzyme and synthe­
sized nanobiocatalyst were 50 ◦ C, if the temperature exceeded from this
optima, a decrease in activity was observed. However, at elevated
temperatures, a noticeable improvement in the relative activity of the
nanobiocatalyst was observed than that to the free state of the enzyme.
The use of a cross-linking agent plays a pivotal contribution during
cross-linking of the enzyme molecules on nano-support carriers since it
exhibits a direct impact on working stability and activity recovery. Amid
the potential cross-linking agents, glutaraldehyde is conceived as the
prevalent coupling molecule that has been widely attempted in enzyme
immobilization due to easy availability, inexpensive, amenable to
manipulation, and the ability to produce covalent linkages with proteins
and enzymes (Barbosa et al., 2014). Nevertheless, some inherent
drawbacks hinder the universal employment of glutaraldehyde as a
coupling agent. In some instances, the inclusion of glutaraldehyde as a

8
S. Zhang et al.
cross-linker caused the deactivation of the enzyme, because of the
smaller size of cross-linker, which can easily penetrate the catalytic re­
gion and cross-links with amino acids residues responsible for catalysis
resulting in a significant reduction in enzyme activity (Mateo, Palomo,
Van Langen, Van Rantwijk, & Sheldon, 2004). Furthermore,
glutaraldehyde-mediated cross-linking of enzyme forms lumping of
enzyme molecules and thus hampering the catalytic regions. Therefore,
it suffers from severe mass transfer resistances for macromolecules and
substrates resulting in diminished bio-catalytic performance (Zhen,
Wang, Qi, Su, & He, 2013). Additionally, leakage of glutaraldehyde from
the engineered biocatalytic system poses hostile effects on human and
aquatic entities due to its toxicity (Arsenault et al., 2011).
Polysaccharides-based eco-friendly and biocompatible cross-linkers
have grabbed mounting interest for enzymes over the use of tradi­
tional glutaraldehyde. In a recent report, Nadar et al. (2016) demon­
strated pectin-based cross-linker as more promising for the
immobilization of glucoamylase onto APTES-functionalized MNPs
compared to the glutaraldehyde. Sojitra et al. (2017) ascertained
dextran polyaldehyde as a macromolecular cross-linker for pectinase
immobilization on chitosan-coated MNPs. As compared to free form,
immobilized nanobiocatalyst presented 2-folds enhancement in tem­
perature resistance at a high-temperature range of 55–75 ◦ C. It showed
robust stability and durability retaining over 80% of the remaining ac­
tivity after seven consecutive reuse cycles. On the other hand, up to 89%
of residual activity was recorded after storing for 15 days. In another
report, fabricated MNPs were activated with 1-ethyl-3-(3-dimethyl
aminopropyl) carbodiimide hydrochloride (EDC) for the covalent
immobilization of recombinant pectate lyase from family 1 poly­
saccharide lyase (PL1B). The MNPs-conjugated PL1B exhibited 18.2 and
20.3 U/mg of activity against citrus pectin, and polygalacturonic acid,
respectively, whereas the activity of free PL1B was 16.2 and 17.8 U/mg
towards the same substrates. With reference to the free form of PL1B, the
immobilized magnetic nanobiocatalyst presented 32– and 14-fold
higher temperature tolerance at 80 ◦ C and 90 ◦ C, respectively. Immo­
bilized MNPs-PL1B preserved its catalytic activity for an extended
period than that to the free PL1B at elevated temperature. It could be
recycled in five successive cycles retaining 70% of original activity and
can be readily separated from the reaction system by using a simple
magnet (Chakraborty, Rao, & Goyal, 2017). A rational elucidation for
the enhanced activity and thermal tolerance of MNPs-PL1B might be due
to enzyme rigidification following the immobilization process. The
presence of enormous hydroxyl groups on the MNPs surface can interact
with the enzyme molecules after carbodiimide activation resulting in the
tight packaging of the enzyme molecules, and thus restricting their free
movement. Therefore, PL1B immobilization caused a potent structural
rigidification of the enzyme molecules thereby are less vulnerable to any
distortion in their structure. On the other hand, due to lack of such
structural rigidness, the enzymes in the free form are susceptible to
distortion in the structure primarily at elevated temperature and thus
dropping the enzyme activity. The rigidification of multiple enzymes
following immobilization has been widely documented in the literature
reports (Mateo, Palomo, Fernandez-Lorente, Guisan, & Fernandez-
Lafuente, 2007; Rodrigues, Ortiz, Berenguer-Murcia, Torres, & Fernán­
dez-Lafuente, 2013).
Polyethylene glycol (PEG) is a highly biocompatible ethylene oxide-
derived polymers that have been utilized for MNPs stabilization. A list of
interesting merits such as hydrophilicity, non-toxicity, non-antigenicity,
flexibility, and biocompatible nature encourage its use as a commend­
able coating material in various biological and biocatalytic applications.
The PEG-capped nanostructured particles are chemically stable and
more dispersible compared to their pristine counterparts (Garcia-Galan
et al., 2011). Kharazmi, Taheri-Kafrani, and Soozanipour (2020) syn­
thesized PEG-grafted MNPs by chemical co-precipitation of FeCl3 and
FeCl2 solutions in the alkaline milieu, and modified with a coupling
agent, cyanuric chloride (CC), to incorporate active functionalities on
their surface. The modified MNP-PEG-CC were reacted with a
9
Food Research International 140 (2021) 109979
pectinolytic solution for the immobilization of pectinase enzyme via
covalent binding producing MNP/PEG/CC-pectinase (Fig. 5). The
resulted immobilized pectinase-based biocatalytic system presented
enhanced functional stability, improved biocatalytic efficiency, and
appreciable reuse capability. pH and thermal stability profile at extreme
points revealed the augmented performance of MNPs-immobilized
pectinase. Moreover, the immobilized nanobiocatalyst showed excep­
tional storage stability and recyclability retaining more than 94% and
55% of its primary activity after 125-days storage at 25 ◦ C, and 10
repeated cycles, respectively.
3.3. Pectinase immobilization on hybrid nanoflowers
Protein conjugated inorganic hybrid nanoflowers were first discov­
ered by Ge et al. (2012) by a rapid and facile synthesis method. In this
hybrid nanoflowers conjugate, the combination of proteins with metal
ions constitutes flower-shaped nanostructures in the presence of Cu3
(PO4)2 as an inorganic component under mild environments. This
fascinating method has been widely adopted to immobilize a large
number of industrially relevant enzyme including α-chymotrypsin (Yin
et al., 2015), lipase (Zhang et al., 2016b), α-amylase (Wang et al., 2013),
horseradish peroxidase (Lin et al., 2014a), catalase (Shi, Zhang, Wang,
Yang, & Jiang, 2014), papain (Zhang et al., 2016a), and trypsin (Lin
et al., 2014b) using various metals as the inorganic constituent. The
majority of as-synthesized inorganic hybrid nanoflowers immobilized
enzyme presented improved biocatalytic activity and thermal stability
characteristics. An alkaline triple-site mutagenic pectate lyase, PEL3,
was immobilized onto nanoflowers to develop a PEL3-inorganic hybrid
nanoflower biocatalytic system to improve its catalytic performance
(Wu, Luo, Lu, Zhan, & Zhang, 2020). Among various divalent ions (i.e.
Ca2C, Mn2C, Zn2C, Cu2C, and Co2C) as inorganic components, PEL3/
Cu3(PO4)2 hybrid nanostructured flowers exhibited the maximum ac­
tivity (2.5-fold increase) than that to free from of PEL3. The as-
fabricated PEL3/Cu3(PO4)2 nanoconjugate retained 33% of remaining
activity after heating at 55 ◦ C for 24 h, whereas the soluble PEL3 was
inactivated after 18 h of incubation at the same temperature. Addi­
tionally, maintenance of over 50% of activity after four repeated use
demonstrates promising functional steadiness of PEL3/Cu3(PO4)2
hybrid nanoflowers for large-scale applicability (Wu et al., 2020). In
many other studies, different nanoflowers-enzyme conjugates have been
generated with improved catalytic performance such as lipase/
Cu3(PO4)2 hybrid, α-lactalbumin/Cu3(PO4)2, carbonic anhydrase/
Cu3(PO4)2, laccase/Cu3(PO4)2, and papain/Zn3(PO4)2 hybrid nano­
flowers (Wang et al., 2013; Zhang et al., 2016a).
3.4. Pectinase immobilization on dual-responsive polymeric nanocarriers
and montmorillonite clay
Stimuli-responsive polymers that possess highly durable structural
morphology of amphiphiles in an aqueous system have been deemed as
excellent support matrices for encapsulating various classes of enzymes
(Qu, Chapman, Chen, Lu, & Stenzel, 2017; Wang, Zhang, Liu, Hu, & Liu,
2017) by modifying their structural organization in environmental
perturbation, such as pH, and extreme temperature (Betthausen,
Drechsler, Förtsch, Schacher, & Müller, 2011; Ma et al., 2013). Based on
the practicalities that, if the support matrix could regulate the in­
teractions between substrate and enzyme molecules by adjusting its
morphology, the biocatalytic activities and other properties of the
encapsulated enzyme could be commendably controlled. In this respect,
Lei, Liu, Ma, Yang, and Lei (2019) designed and developed a support
material polystyrene-b-polymethylacrylic/polystyrene-(5-propargyl
ther-2-nitrobenzyl bromoisobutyrate)-b-poly(diethylamino) ethyl
methacrylate-b-poly(polyethylene glycol methacrylate) with light and
pH-sensitive functionalities to constitute a novel strategy for effective
immobilization of pectinase enzyme. As portrayed in Fig. 6, carboxyl
groups of polymethacrylic acid provide abundant binding sites for
S. Zhang et al.
Fig. 5. Schematic representation of the synthesis of 1,3,5-triazine-functionalized PEG-coated Fe3O4 nanoparticles and immobilization of pectinase. Reprinted from
Kharazmi et al. (2020) with permission from Elsevier. Copyright © 2020 Elsevier Ltd. License Number: 4956911283650.
pectinase attachment via multipoint ionic interactions between the
amino groups of enzyme and carboxyl groups in PMAA. The newly
synthesized stimuli-responsive carrier could regulate its activity by
governing the pectinase contact with the substrate. The immobilized
biocatalyst showed above 50% of its original activity after eight suc­
cessive reaction batches. Without UV irradiation, the relative bioactivity
of the nano-pectinase was 3.0% less than that with UV-irradiated sup­
port material at 60 ◦ C, highlighting the modification of enzyme activity
by altering external environments. The relative activity of the polymeric
nanocarrier-encapsulated enzyme was 11% greater than its free counter-
form after storage of 30 days at 4 ◦ C. Montmorillonite clay (MMC) is
often employed to improve the mechanical stability of polymeric sup­
ports, like alginate and chitosan (Mardani, Khiabani, Mokarram, &
Hamishehkar, 2018; Mohammadi et al., 2019). A pectinase enzyme from
A. aculeatus has been covalently immobilized on amino-silane-grafted
montmorillonite clay utilizing glutaraldehyde as a coupling agent.
MMC is considered as an inexpensive, simple, reasonably safe, and
provides better mechanical stability, and ample surface area for easy
penetration of product and substrate. Though the immobilization pro­
cess had no effect on the temperature optima and thermal profile of
pectinase for attaining the highest activity, the MMC-encapsulated
biocatalyst performed well in more acidic pH environments. It was
reusable for six consecutive cycles with the retention of higher than 60%
of operational activity (Mohammadi et al., 2020).
3.5. Pectinase immobilization on cross-linked enzyme aggregates
The development of carrier-free biocatalysts is one of the appealing
strategies for enzyme immobilization. Cross-linked enzyme aggregates
10
Food Research International 140 (2021) 109979
(CLEAs) can be synthesized by direct cross-linking of various enzyme
preparations (Sheldon, 2011). CLEAs provide many useful benefits such
as high stability, rigorous enzyme activity, and the relatively low pro­
duction and processing costs due to lack of additional carrier (Cao, van
Langen, & Sheldon, 2003). Dal Magro et al. (2018) designed two
different magnetic nanobiocatalysts by taking advantage of easy bio­
catalytic separation. For this, aminopropyltrimethoxysilane-
functionalized MNPs were used to develop nanobiocatalyst by two
different strategies, which include the covalent coupling of the enzyme
to glutaraldehyde-activated magnetic particles and magnetic CLEAs.
The activity recovery of MNPs-immobilized pectinase was improved
with increasing glutaraldehyde concentration up to 75 mM. At low
glutaraldehyde concentration, the binding of enzyme molecules might
be not efficient with the support. Increasing the amount of glutaralde­
hyde supplied a larger number of free aldehyde groups on the support
surface, thus strengthening the interactions between the support and
enzyme molecules, and circumventing the leaching of the enzyme
(Talekar et al., 2017). However, a further increase in glutaraldehyde
concentration results in a reduction in activity recovery due to the loss of
three-dimensional conformation, and extensive interactions of enzyme
with free aldehyde groups (López-Gallego et al., 2005). As compared to
CLEA-magnetic particles, MNPs-coupled pectinases showed higher
magnetic properties, whereas CLEA-MP* provided high pore volume
and surface area. CLEA-MP* were found to be high performing and
stable biocatalyst and presented elevated recovered activities, thermal
stabilities, and enhanced recyclability. The high enzyme thermal stabi­
lization is attributed to covalent bonding between amino and aldehyde
groups that hamper the molecular movability of the enzyme (Nadar &
Rathod, 2016). Multiples interactions are generated between enzyme-
S. Zhang et al.
Fig. 6. Representation of the self-assembly and structural changes of the micelles under the stimulations of pH, UV light, and tailoring the activity of immobilized
pectinases. Reprinted from Lei et al. (2019) with permission from John Wiley and Sons. Copyright © 2019 International Union of Biochemistry and Molecular
Biology, Inc. License Number: 4956920100928.
enzyme and enzyme-MP* after immobilization on CLEA-MP*, which
contributed to the conformational stability of proteins (Dal Magro et al.,
2016, 2016). A plenty of reports has revealed that larger and more
porous immobilization supports are capable of restricting the molecular
flexibility, thus are less vulnerable to inactivation leading to enhanced
enzymatic thermal stability (Klein et al., 2012).
4. Biotechnological applications of immobilized pectinases
4.1. Fruit juice clarification
Pectinases are being applied in many industrial bioprocesses, such as
food processing, textile, plant fiber processing, coffee, tea, oil extraction,
pectinaceous material-laden wastewater treatment, etc. (Garg et al.,
2016; Jayani et al., 2005). Based on their applications, pectinase is
mainly categorized into two types: acidic pectinase and alkaline pecti­
nase (Kashyap, Vohra, Chopra, & Tewari, 2001). Acidic pectinases are
used in the winemaking and fruit juice industries. They are often orig­
inated from yeast and fungal sources, where A. niger is the unanimously
known fungal species for the biosynthesis of acidic pectinase. Among the
yeast species, Saccharomyces and Kluyveromyces have been recognized
and widely used due to their GRAS status (Hoondal et al., 2002). The
extraction and clarification of fruit juice are the principal industrial
applicability of these pectinolytic enzymes. A consortium of pectinases
in combination with other depolymerases is often used to clarify fruit
juices. Pectinase-treated fruit pulps exhibited an increased fruit juice
volume and have been successfully used in the preparation of glistening
clear juices (Fig. 7).
11
Food Research International 140 (2021) 109979
Notwithstanding the prospective catalytic properties, the large-scale
application pectinases in the free form are often handicapped by mar­
ginal working stability and lack of adequate catalytic efficiency. Table 3
summarizes the application of immobilized pectinases in different in­
dustrial bioprocesses. Irshad et al. (2017) explored the potentialities of
chitosan-immobilized pectinase for clarification of different fruit juices
(apples, mango, peach, and apricot). After 1 h treatment with the
immobilized enzyme, a substantial reduction in viscosity, turbidity, and
color were obtained for apple juice. The results indicate the depoly­
merization of pectin, which was involved in deterring the extraction
process of juices by processing methods. Reports have demonstrated that
incorporating pectinolytic consortium in the extraction process can
improve the yield and reduce the viscosity of fruit juice by depolyme­
rizing the gelatinous structure (Bilal, Asgher, Iqbal, Hu, & Zhang, 2016,
Bilal et al., 2017b). Both the free and Ca-alginate immobilized poly­
galacturonases were found effective in decomposing pectin in apple
juice. However, in contrast to the free enzyme, the entrapped poly­
galacturonase treated apple juice exhibited low color value, high light
transmittance, and low concentration of soluble solids (Deng et al.,
2019). The potential of polyethylene glycol-coated MNPs-immobilized
pectinase was studied for pineapple juice clarification in terms of
turbidity reduction. After 2 h of treatment, the free pectinase (0.3 mg/
mL) and immobilized nanobiocatalyst reduced the tested juice turbidity
up to 48 and 59%, respectively. Moreover, the immobilized nano-
pectinase was operationally very stable and preserved over 60% of its
initial catalytic performance after nine successive reuse cycles (Khar­
azmi et al., 2020). Dal Magro et al. (2020) set-up continuous packed-bed
and fluidized-bed reactors (Fig. 8) by immobilization of commercial
S. Zhang et al.
Fig. 7. Schematic illustration of pectinase-assisted fruit juice clarification process.
enzyme preparation onto glutaraldehyde-functionalized superior qual­
ity chitosan microspheres and employed for the clarification of orange
juice. When applied in the fluidized-bed reactor, the developed nano­
biocatalyst (650 beads, 0.22 g dry mass, 1340 U.g− 1 enzyme) provided
clearer juices and retained 60% of its primary clarification performance
after continuous use for 72 h. On the other hand, the clarification effi­
ciency of the immobilized enzyme (0.22 g dry mass, 1340 U.g− 1) in the
packed-bed reactor was reduced gradually during 54 h presumably due
to the creation of preferential paths and dead zones. Therefore, the
chitosan particles-immobilized pectinase appears a good substitute
when applied in a fluidized-bed reactor for juice clarification on a large
scale.
4.2. Bioscouring of fibrous materials
The cotton fiber consists of structural and reserve polysaccharides.
Enzyme-mediated bioscouring is an eco-friendlier and attractive method
to eliminate non-cellulosic “impurities” from raw cotton making the
surface more hydrophilic (Li & Hardin, 1998). Pectins are associated
with the hydrophobic properties of raw cotton and its depolymerization
by pectinases facilitates the removal of waxes, leading to a significant
reduction in water and chemical consumption and thereby discharge of
effluent. Compared with the drastic alkaline conditions used conven­
tionally, catalytic treatment with pectin-depolymerizing biocatalysts do
not affect the cellulose backbone and thus prevent fiber damage (O’Neill
et al., 1999). By disrupting cell wall constituents such as pectin, enzymes
promote the liberation of the oil. Nevertheless, the requirement for
highly active, stable, and specific pectinase enzymes has gained prodi­
gious interest. Khan, Choi, Kim, and Yoo (2018) characterized the bio­
scouring efficiency of thermotolerant alkaline polygalacturonase by
comparing weight loss for a purified enzyme with commercial pectinase.
Results revealed a higher fabric weight loss treated with purified poly­
galacturonase compared to the scoured with commercial pectinases.
Notably, crude polygalacturonase, purified polygalacturonase BPN3,
and commercial pectinase showed 2.35%, 3.08%, and 2.63% weight loss
12
Food Research International 140 (2021) 109979
of bio-scoured cotton fabric, respectively, after 2 h treatment time. The
wax content in the grey organic cotton fabric has been stated to be
0.87%. After treatment with 2.0, 4.0, 6.0, and 8.0% of poly­
galacturonase at 60 ◦ C for 45 min, the wax context was reduced to 0.71,
0.59, 0.42, and 0.34%, respectively, because the polygalacturonase
catalyzed the hydrolysis of wax and pectin degradation in the organic
cotton fiber. In another report, bioscouring of cotton fabric was evalu­
ated with recombinant pectate lyase immobilized (0.05 to 0.5 mg/mL)
on MNPs (Chakraborty et al., 2017). The nanobiocatalyst exhibited
promising efficiency in eliminating pectin substances from the fabric
surface. Improved fabric wettability led to decreased water-absorbing
time from 3 min (by the free enzyme) to only 15 s by the MNPs-
immobilized PL1B treated fabric. MNPs-immobilized PL1B was
demonstrated to be more proficient in the removal of pectin compared
with free enzyme owing to its increased catalytic bioactivity and
improved specificity towards pectic substrates. Easy recovery using a
magnet was another important advantage of MNPs-coupled enzymes.
Therefore, the immobilized form of pectinases can potentially grasp new
opportunities in textile industrial sectors and offer an environmentally
responsive and economically viable alternative technique to the chem­
ical bio-scouring process.
Degumming of plant fibres by microbial-derived pectic enzymes is a
sustainable and eco-acceptable bioprocess, which involves the decom­
position of pectin of bark and releasing fibres (Shet, Desai, & Achappa,
2018). Pectinolytic enzymes play a principal role in the degumming of
natural fibres, which entails the removal of densely coated, non-
cellulosic gummy substances from the cellulose portion of the plant fi­
bres (Mukhopadhyay, Dutta, Chattopadhyay, & Chakrabarti, 2013).
Alkaline pectinases have been effectively used for degumming of sunn
jute, flax, hemp, and ramie and coconut fibers for textile exploitation
(Zhang et al., 2013; Chiliveri, Koti, & Linga, 2016). Degumming can be
carried out by incorporating a pectinolytic cocktail or by fiber fermen­
tation (dew retting) using pectinolytic-secreting microbial strains such
as xylanase, and mannanase-producing bacteria, and pectinolytic bac­
teria (Ruan, Raghavan, Gariepy, & Du, 2015; Liu et al., 2015). The
S. Zhang et al. Food Research International 140 (2021) 109979

Table 3 the surface of buel and banana fibres observed smoother and cleaner
Summary of application of immobilized pectinases. after enzyme treatment. Pectin lyase-mediated degumming of banana
Name of pectinase Immobilization Application Reference and buel showed the highest liberation of galacturonide after 15 and 24
matrix h, respectively. The inclusion of 250 and 200 U of pectin lyase resulted
Polygalacturonase, Chitosan Clarification of Irshad et al. in the maximum liberation of galacturonide for banana and buel fibres,
pectin methylesterase, microspheres apples, mango, (2017) respectively.
and pectin lyase peach, and
apricot juices
4.3. Antioxidant extraction from waste fruit peels
A significant
reduction in
color and Bioactive compounds have acquired a great deal of attention from
turbidity industrial and scientific communities due to sprouting market demand
reductions after and prospective applications in cosmetics, functional foods, and phar­
60 min
macological industries (Sun-Waterhouse, 2011; Acosta-Estrada, Gutiér­
treatment with
immobilized rez-Uribe, & Serna-Saldívar, 2014). Phenolic compounds, among the
pectinases vast number of phytochemicals, have been widely investigated owing to
Crude pectinase Sodium alginate Apple juice Hachemi- their numerous health-beneficial properties along with diverse biolog­
clarification Benmalek
ical effects such as antimicrobial, antithrombotic, anti-allergic, anti-in­
et al. (2019)
Endopolygalacturonase – Bioscouring of Khan et al.
flammatory, and vasodilatory activities. Therefore, much effort has been
cotton fabrics (2018) directed for the extraction of these naturally occurring biologically
Polygalacturonase Ca-alginate beads Pectin de Carvalho active secondary metabolites from plant sources (Oroian & Escriche,
hydrolysis in Silva et al. 2015; Dai & Mumper, 2010). Generally, phenolic compounds are intri­
cashew apple (2019)
cately conjugated with cell wall structural entities such as pectin, cel­
juice
Polygalacturonase Ca-alginate beads Apple juice Deng et al. lulose, and hemicellulose. These cell wall polysaccharides intensely
clarification (2019) diminish the extraction efficacy of traditional methods. Enzyme-driven
Pectin lyase Ca-alginate beads Degumming of Kohli and extraction is a regarded as lucrative approach that has gained
plant fibres Gupta (2019) intriguing interest as an eco-sustainable extraction technique for the
Pectinase Chitosan beads Fruit juice Mehmood
clearance and et al. (2019)
augmented liberation of bioactive substances from fruits and plant res­
detergent idues (Puri, Sharma, & Barrow, 2012; Joana Gil-Chávez et al., 2013).
compatibility The use of enzymes promotes the release of biologically active com­
Pectinase Epoxy-activated Clarification of Rajdeo et al. pounds entangled inside the cell walls network by catalyzing its depo­
acrylate apple juice (2016)
lymerization (Chamorro, Viveros, Alvarez, Vega, & Brenes, 2012).
copolymer
DILBEAD-VWR Thitiratsakul and Anprung (2014) used a pectinase cocktail (257 poly­
Pectinase Glutaraldehyde- Grape juice Dal Magro galacturonase units/g fruit) to extract antioxidants from longan. The
activated clarification et al. (2018) enzyme-treated longan extract possessed 4-fold augmented release of
magnetic particles antioxidants than that to the extract without enzyme treatment. Margo
Pectinase Magnetic CLEAs Grape juice Dal Magro
clarification et al. (2018)
et al. determined the synergistic effect of cellulose and pectinase prep­
Pectinase Polyethylene Pineapple juice Kharazmi aration for extraction of grape juice from Vitis labrusca L. In comparison
glycol-grafted clarification et al. (2020) to individual preparation, the concerted action of two enzymes greatly
magnetic enhanced the bioactive compounds extraction as well as juice yield (Dal
nanoparticles
Magro et al., 2016, 2016). However, elevated cost, effective separation/
Recombinant pectate Magnetic Bioscouring of Chakraborty
lyase nanoparticles cotton fabric et al. (2017) recovery, and repeatability of the biocatalysts after each reaction cycle
Pectinase Magnetic Antioxidant Nadar and are the major problems in enzyme-based extraction. To confront these
nanoparticles extraction from Rathod inadequacies, modifying enzymes via immobilization onto appropriate
waste fruit (2019) pre-fabricated supports is a graceful approach, and offers a set of fasci­
peels
Pectinase Chitosan magnetic Apple juice Sojitra et al.,
nating advantages such as improved catalytic stability, repetitive utili­
nanoparticles clarification (2017) zation, and easy separation from complex reaction system (Talekar
Pectinase Activated Pineapple juice Mohammadi et al., 2017; Sojitra, Nadar, & Rathod, 2016). In a drive towards valo­
montmorillonite clarification et al. (2020) rization of fruit peel waste residues, Nadar and Rathod (2019) carried
support
out the extraction of antioxidants by simultaneous immobilization of
Commercial enzyme Chitosan beads Clarification of Dal Magro
preparation orange juice et al. (2020) two enzymes [Celluclast® (36 U/mL) and Pectinex® (117 U/mL)] onto
exo-polygalacturonase Sodium alginate Clarification of Amin et al. surface modified MNPs. This multi-enzyme immobilization approach
apple, peach (2017a; profoundly improved the yield in a shorter combined with the effectual
and grape) 2017b) mass transfer during the enzyme-catalyzed reactions leading to consid­
erable economic viability and environmental efficacy. It also presented
released plant fibres are significant sources of natural textile materials 2-folds increased free radical capturing activity than classical solvent-
and exhibit a range of advantageous features than synthetic counter­ based extraction procedure. The higher DPPH scavenging activity
parts including renewability, adequate stiffness, low density, high following enzyme-mediated treatment was attributed to the augmented
disposability, and better mechanical properties (Kashyap et al., 2001). extraction of phenolic substances from cell walls (Štambuk et al., 2016;
Immobilized pectinases provide numerous advantages compared with M’hiri, N., Ioannou, I., Boudhrioua, N. M., & Ghoul, M. , 2015). It can be
biotechnological processes where the free form of enzymes are utilized. concluded that the immobilized biocatalysts facilitate the liberation of a
Kohli and Gupta (2019) explored the application of Ca-alginate beads- higher number of natural antioxidants and polyphenolic substances
entrapped purified pectin lyase in the degumming of buel and banana from waste fruit peels with superior efficacy.
plant fibres. Characterization analysis by SEM and FTIR unveiled that
the untreated buel and banana fibres were heavily coated by non- 5. Conclusions and prospects
cellulosic substances like waxes, pectin, and hemicelluloses, whereas
Based on the extensive literature inspection presented herein, it can

13
S. Zhang et al. Food Research International 140 (2021) 109979

Fig. 8. Operational scheme of packed-bed (a) and fluidized-bed (b) reactors. Reprinted from Dal Magro, Pessoa, Klein, Fernandez-Lafuente, and Rodrigues (2020)
with permission from Elsevier. Copyright © 2020 Elsevier B.V. License Number: 4956920263934.

be demonstrated that the immobilized pectinolytic enzyme system may
represent an interesting strategy for application in the food industry, in
particular, for the clarification of fruit juices. Owing to a set of distinc­
tive properties including eco-responsive, inexpensive, biodegradability,
biocompatibility, and malleability to chemical modification, bio­
polymers have been found as desired materials for pectinases immobi­
lization. Likewise, the nanomaterials particularly magnetic
nanoparticles are regarded as ideal supports for enzyme immobilization
because of the easy separation of nano-sized conjugates from the reac­
tion system and their multiple recycling. A substantial enhancement in
biocatalyst stability together with a prodigious activity under extreme
pH and temperature regimens suggests broad-spectrum applications of
the immobilized pectinases in various industrial bioprocesses. The
design and establishment of immobilized pectinase containing bio­
reactors for continuous uses demonstrated a new technology with a high
perspective for industrial employment that contributes to a reduction in
production cost. Thus, this review offers new avenues to develop
enzyme-mediated promising and novel technology for application in the
food industry.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence

14
S. Zhang et al.
the work reported in this paper.
Acknowledgments
The listed author(s) are highly obliged to their institutes and uni­
versities for the literature services.
References
Acosta-Estrada, B. A., Gutiérrez-Uribe, J. A., & Serna-Saldívar, S. O. (2014). Bound
phenolics in foods, a review. Food chemistry, 152, 46–55.
Ahmed, A., & Sohail, M. (2020). Characterization of pectinase from Geotrichum
candidum AA15 and its potential application in orange juice clarification. Journal of
King Saud University-Science, 32(1), 955–961.
Alagöz, D., Tükel, S. S., & Yildirim, D. (2016). Immobilization of pectinase on silica-
based supports: Impacts of particle size and spacer arm on the activity. International
journal of biological macromolecules, 87, 426–432.
Alkorta, I., Garbisu, C., Llama, M. J., & Serra, J. L. (1998). Industrial applications of
pectic enzymes: A review. Process Biochemistry, 33(1), 21–28.
Amin, F., Mohsin, A., Bhatti, H. N., & Bilal, M. (2020). Production, thermodynamic
characterization, and fruit juice quality improvement characteristics of an Exo-
polygalacturonase from Penicillium janczewskii. BBA - Proteins and Proteomics, 1868,
Article 140379.
Amin, F., Bhatti, H. N., & Bilal, M. (2019). Recent advances in the production strategies
of microbial pectinases—A review. International journal of biological macromolecules,
122, 1017–1026.
Amin, F., Bhatti, H. N., Bilal, M., & Asgher, M. (2017a). Improvement of activity, thermo-
stability and fruit juice clarification characteristics of fungal exo-polygalacturonase.
International journal of biological macromolecules, 95, 974–984.
Amin, F., Bhatti, H. N., Bilal, M., & Asgher, M. (2017b). Multiple parameter
optimizations for enhanced biosynthesis of exo-polygalacturonase enzyme and its
application in fruit juice clarification. International Journal of Food Engineering, 13(2).
Amin, F., Bhatti, H. N., Bilal, M., & Asgher, M. (2017c). Purification, kinetic, and
thermodynamic characteristics of an exo-polygalacturonase from Penicillium
notatum with industrial perspective. Appl. Biochem. Biotechnol., 183, 426–443.
Ansari, S. A., & Husain, Q. (2012). Potential applications of enzymes immobilized on/in
nano materials: A review. Biotechnology advances, 30(3), 512–523.
Arsenault, A., Cabana, H., & Jones, J. P. (2011). Laccase-based CLEAs: chitosan as a
novel cross-linking agent. Enzyme research, 2011.
Barbosa, O., Ortiz, C., Berenguer-Murcia, Á., Torres, R., Rodrigues, R. C., & Fernandez-
Lafuente, R. (2014). Glutaraldehyde in bio-catalysts design: A useful crosslinker and
a versatile tool in enzyme immobilization. Rsc Advances, 4(4), 1583–1600.
Betthausen, E., Drechsler, M., Förtsch, M., Schacher, F. H., & Müller, A. H. (2011). Dual
stimuli-responsive multicompartment micelles from triblock terpolymers with
tunable hydrophilicity. Soft Matter, 7(19), 8880–8891.
Bibi, F., Irshad, M., Anwar, Z., Bhatti, K. H., & Raza, A. (2017). Improved catalytic
functionalities of purified pristine and chitosan-immobilized polygalacturonase, and
pectin lyase. Chemical Engineering Research and Design, 128, 146–154.
Bilal, M., Asgher, M., Iqbal, H. M., Hu, H., & Zhang, X. (2016). Gelatin-immobilized
manganese peroxidase with novel catalytic characteristics and its industrial
exploitation for fruit juice clarification purposes. Catalysis Letters, 146(11),
2221–2228.
Bilal, M., & Iqbal, H. M. (2020a). Microbial Peroxidases and Their Unique Catalytic
Potentialities to Degrade Environmentally Related Pollutants. In Microbial
Technology for Health and Environment (pp. 1–24). Singapore: Springer.
Bilal, M., Asgher, M., Iqbal, H. M., Hu, H., & Zhang, X. (2017). Delignification and fruit
juice clarification properties of alginate-chitosan-immobilized ligninolytic cocktail.
LWT, 80, 348–354.
Bilal, M., Iqbal, H. M., Hu, H., Wang, W., & Zhang, X. (2017). Enhanced bio-catalytic
performance and dye degradation potential of chitosan-encapsulated horseradish
peroxidase in a packed bed reactor system. Science of the Total Environment, 575,
1352–1360.
Bilal, M., & Iqbal, H. M. (2020b). State-of-the-art strategies and applied perspectives of
enzyme biocatalysis in food sector—current status and future trends. Critical reviews
in food science and nutrition, 60(12), 2052–2066.
Bilal, M., & Iqbal, H. M. (2020c). Armoring bio-catalysis via structural and functional
coordination between nanostructured materials and lipases for tailored applications.
International Journal of Biological Macromolecules. https://doi.org/10.1016/j.
ijbiomac.2020.10.239.
Bilal, M., & Iqbal, H. M. (2019a). Chemical, physical, and biological coordination: An
interplay between materials and enzymes as potential platforms for immobilization.
Coordination Chemistry Reviews, 388, 1–23.
Bilal, M., & Iqbal, H. M. (2019b). Tailoring multipurpose biocatalysts via protein
engineering approaches: A review. Catalysis Letters, 149(8), 2204–2217.
Bilal, M., & Iqbal, H. M. (2019c). Naturally-derived biopolymers: Potential platforms for
enzyme immobilization. International journal of biological macromolecules, 130,
462–482.
Bilal, M., Nguyen, T. A., & Iqbal, H. M. (2020). Multifunctional carbon nanotubes and
their derived nano-constructs for enzyme immobilization–a paradigm shift in
biocatalyst design. Coordination Chemistry Reviews, 422, Article 213475.
Bilal, M., Ashraf, S. S., Cui, J., Lou, W. Y., Franco, M., Mulla, S. I., & Iqbal, H. M. (2020).
Harnessing the biocatalytic attributes and applied perspectives of nanoengineered
15
Food Research International 140 (2021) 109979
laccases—A review. International Journal of Biological Macromolecules. https://doi.
org/10.1016/j.ijbiomac.2020.10.195.
Bilal, M., Asgher, M., Shah, S. Z. H., & Iqbal, H. M. (2019). Engineering enzyme-coupled
hybrid nanoflowers: The quest for optimum performance to meet biocatalytic
challenges and opportunities. International journal of biological macromolecules, 135,
677–690.
Bilal, M., Cui, J., & Iqbal, H. M. (2019). Tailoring enzyme microenvironment: State-of-
the-art strategy to fulfill the quest for efficient bio-catalysis. International journal of
biological macromolecules, 130, 186–196.
Bilal, M., Zhao, Y., Noreen, S., Shah, S. Z. H., Bharagava, R. N., & Iqbal, H. M. (2019).
Modifying bio-catalytic properties of enzymes for efficient biocatalysis: A review
from immobilization strategies viewpoint. Biocatalysis and Biotransformation, 37(3),
159–182.
Benucci, I., Mazzocchi, C., Lombardelli, C., Cacciotti, I., & Esti, M. (2019). Multi-
enzymatic systems immobilized on chitosan beads for pomegranate juice treatment
in fluidized bed reactor: Effect on haze-active molecules and chromatic properties.
Food and Bioprocess Technology, 12(9), 1559–1572.
Bruce, I. J., & Sen, T. (2005). Surface modification of magnetic nanoparticles with
alkoxysilanes and their application in magnetic bioseparations. Langmuir, 21(15),
7029–7035.
Bustamante-Vargas, C. E., de Oliveira, D., Valduga, E., Venquiaruto, L. D., Paroul, N.,
Backes, G. T., & Dallago, R. M. (2016). Biomimetic mineralization of the alginate/
gelatin/calcium oxalate matrix for immobilization of pectinase: Influence of matrix
on the pectinolytic activity. Applied biochemistry and biotechnology, 179(6),
1060–1072.
Cao, L., van Langen, L., & Sheldon, R. A. (2003). Immobilised enzymes: Carrier-bound or
carrier-free? Current opinion in Biotechnology, 14(4), 387–394.
Cardoso, F. D. S. N., KOBLITZ, M. G. B., ORTIZ, G. M. D., CARVALHO, J. L. V. D., &
CARVALHO, L. M. J. D. (2019). Study of the parameters used in the encapsulation of
commercial pectinase in calcium alginate and its effect on its catalytic activity. Food
Science and Technology, 39(1), 247-252.
Cerreti, M., Liburdi, K., Benucci, I., & Esti, M. (2016). The effect of pectinase and
protease treatment on turbidity and on haze active molecules in pomegranate juice.
LWT, 73, 326–333.
Chakraborty, S., Jagan Mohan Rao, T., & Goyal, A. (2017). Immobilization of
recombinant pectate lyase from Clostridium thermocellum ATCC-27405 on magnetic
nanoparticles for bioscouring of cotton fabric. Biotechnology progress, 33(1), 236-
244.
Chamorro, S., Viveros, A., Alvarez, I., Vega, E., & Brenes, A. (2012). Changes in
polyphenol and polysaccharide content of grape seed extract and grape pomace after
enzymatic treatment. Food Chemistry, 133(2), 308–314.
Chiliveri, S. R., Koti, S., & Linga, V. R. (2016). Retting and degumming of natural fibers
by pectinolytic enzymes produced from Bacillus tequilensis SV11-UV37 using solid-
state fermentation. SpringerPlus, 5(1), 559.
Dai, J., & Mumper, R. J. (2010). Plant phenolics: Extraction, analysis, and their
antioxidant and anticancer properties. Molecules, 15(10), 7313–7352.
Dal Magro, L., Dalagnol, L. M., Manfroi, V., Hertz, P. F., Klein, M. P., & Rodrigues, R. C.
(2016). Synergistic effects of Pectinex Ultra Clear and Lallzyme Beta on yield and
bioactive compounds extraction of Concord grape juice. LWT-Food Science and
Technology, 72, 157–165.
Dal Magro, L., Hertz, P. F., Fernandez-Lafuente, R., Klein, M. P., & Rodrigues, R. C.
(2016). Preparation and characterization of a Combi-CLEAs from pectinases and
cellulases: A potential biocatalyst for grape juice clarification. RSC advances, 6(32),
27242–27251.
Dal Magro, L., Pessoa, J. P. S., Klein, M. P., Fernandez-Lafuente, R., & Rodrigues, R. C.
(2020). Enzymatic clarification of orange juice in continuous bed reactors: Fluidized-
bed versus packed-bed reactor. Catalysis Today. https://doi.org/10.1016/j.
cattod.2020.02.003.
Dal Magro, L., Silveira, V. C., de Menezes, E. W., Benvenutti, E. V., Nicolodi, S.,
Hertz, P. F., … Rodrigues, R. C. (2018). Magnetic biocatalysts of pectinase and
cellulase: Synthesis and characterization of two preparations for application in grape
juice clarification. International journal of biological macromolecules, 115, 35–44.
de Barros Nunes, C. A., Melo, P. G., Malaquias, S. G., Amaral, K. V.Á., Alves, G. R.,
Meira, A. A., … Bachion, M. M. (2019). Effectiveness of two bundles in venous leg
ulcer healing: A randomized controlled trial. Journal of Vascular Nursing, 37(4),
232–245.
de Carvalho Silva, J., de França, P. R. L., Converti, A., & Porto, T. S. (2019). Pectin
hydrolysis in cashew apple juice by Aspergillus aculeatus URM4953
polygalacturonase covalently-immobilized on calcium alginate beads: A kinetic and
thermodynamic study. International journal of biological macromolecules, 126,
820–827.
de Carvalho Silva, J., de França, P. R. L., Converti, A., & Porto, T. S. (2018). Kinetic and
thermodynamic characterization of a novel Aspergillus aculeatus URM4953
polygalacturonase. Comparison of free and calcium alginate-immobilized enzyme.
Process Biochemistry, 74, 61–70.
de Oliveira, R. L., da Silva, O. S., Converti, A., & Porto, T. S. (2018). Thermodynamic and
kinetic studies on pectinase extracted from Aspergillus aculeatus: Free and
immobilized enzyme entrapped in alginate beads. International journal of biological
macromolecules, 115, 1088–1093.
de Oliveira, R. L., Dias, J. L., da Silva, O. S., & Porto, T. S. (2018). Immobilization of
pectinase from Aspergillus aculeatus in alginate beads and clarification of apple and
umbu juices in a packed bed reactor. Food and bioproducts processing, 109, 9–18.
Defaei, M., Taheri-Kafrani, A., Miroliaei, M., & Yaghmaei, P. (2018). Improvement of
stability and reusability of α-amylase immobilized on naringin functionalized
magnetic nanoparticles: A robust nanobiocatalyst. International journal of biological
macromolecules, 113, 354–360.
S. Zhang et al.
Deng, Z., Wang, F., Zhou, B., Li, J., Li, B., & Liang, H. (2019). Immobilization of
pectinases into calcium alginate microspheres for fruit juice application. Food
hydrocolloids, 89, 691–699.
Esawy, M. A., Gamal, A. A., Kamel, Z., Ismail, A. M. S., & Abdel-Fattah, A. F. (2013).
Evaluation of free and immobilized Aspergillus niger NRC1ami pectinase applicable
in industrial processes. Carbohydrate polymers, 92(2), 1463–1469.
Etienne, M., & Walcarius, A. (2003). Analytical investigation of the chemical reactivity
and stability of aminopropyl-grafted silica in aqueous medium. Talanta, 59(6),
1173–1188.
Fang, G., Chen, H., Zhang, Y., & Chen, A. (2016). Immobilization of pectinase onto
Fe3O4@ SiO2–NH2 and its activity and stability. International journal of biological
macromolecules, 88, 189–195.
Flores-Maltos, A., Rodríguez-Durán, L. V., Renovato, J., Contreras, J. C., Rodríguez, R., &
Aguilar, C. N. (2011). Catalytical properties of free and immobilized Aspergillus
niger tannase. Enzyme research, 2011.
Garcia-Galan, C., Berenguer-Murcia, Á., Fernandez-Lafuente, R., & Rodrigues, R. C.
(2011). Potential of different enzyme immobilization strategies to improve enzyme
performance. Advanced Synthesis & Catalysis, 353(16), 2885–2904.
Garg, G., Singh, A., Kaur, A., Singh, R., Kaur, J., & Mahajan, R. (2016). Microbial
pectinases: An ecofriendly tool of nature for industries. 3. Biotech, 6(1), 47.
Ge, J., Lei, J., & Zare, R. N. (2012). Protein–inorganic hybrid nanoflowers. Nature
nanotechnology, 7(7), 428–432.
Global Pectinase Market Research Report, http://www.marketresearchstore.com/
report/global-pectinase-marketresearch-report 2017 (2017-190713).
Gür, S. D., İdil, N., & Aksöz, N. (2018). Optimization of enzyme co-immobilization with
sodium alginate and glutaraldehyde-activated chitosan beads. Applied biochemistry
and biotechnology, 184(2), 538–552.
Hachemi-Benmalek, N., Nouani, A., & Benchabane, A. (2019). Valorization of brown
algae (Cystoseira caespitosa) from local region in Algeria for sodium alginate
extraction and their application in the immobilization of microbial pectinases.
Algerian Journal of Environmental Science and Technology, 5(4).
Hanefeld, U., Gardossi, L., & Magner, E. (2009). Understanding enzyme immobilisation.
Chemical Society Reviews, 38(2), 453–468.
Henriksson, G., Akin, D. E., Slomczynski, D., & Eriksson, K. E. L. (1999). Production of
highly efficient enzymes for flax retting by Rhizomucor pusillus. Journal of
Biotechnology, 68(2–3), 115–123.
Homaei, A. A., Sariri, R., Vianello, F., & Stevanato, R. (2013). Enzyme immobilization:
An update. Journal of chemical biology, 6(4), 185–205.
Hoondal, G., Tiwari, R., Tewari, R., Dahiya, N. B. Q. K., & Beg, Q. (2002). Microbial
alkaline pectinases and their industrial applications: A review. Applied microbiology
and biotechnology, 59(4–5), 409–418.
Irshad, M., Murtza, A., Zafar, M., Bhatti, K. H., Rehman, A., & Anwar, Z. (2017).
Chitosan-immobilized pectinolytics with novel catalytic features and fruit juice
clarification potentialities. International journal of biological macromolecules, 104,
242–250.
Jayani, R. S., Saxena, S., & Gupta, R. (2005). Microbial pectinolytic enzymes: A review.
Process Biochemistry, 40(9), 2931–2944.
Joana Gil-Chávez, G., Villa, J. A., Fernando Ayala-Zavala, J., Basilio Heredia, J.,
Sepulveda, D., Yahia, E. M., & González-Aguilar, G. A. (2013). Technologies for
extraction and production of bioactive compounds to be used as nutraceuticals and
food ingredients: An overview. Comprehensive Reviews in Food Science and Food
Safety, 12(1), 5–23.
Kashyap, D. R., Vohra, P. K., Chopra, S., & Tewari, R. (2001). Applications of pectinases
in the commercial sector: A review. Bioresource technology, 77(3), 215–227.
Khan, M. M., Choi, Y. S., Kim, Y. K., & Yoo, J. C. (2018). Immobilization of an alkaline
endopolygalacturonase purified from Bacillus paralicheniformis exhibits bioscouring
of cotton fabrics. Bioprocess and biosystems engineering, 41(10), 1425–1436.
Kharazmi, S., Taheri-Kafrani, A., & Soozanipour, A. (2020). Efficient immobilization of
pectinase on trichlorotriazine-functionalized polyethylene glycol-grafted magnetic
nanoparticles: A stable and robust nanobiocatalyst for fruit juice clarification. Food
Chemistry, 325, Article 126890.
Klein, M. P., Nunes, M. R., Rodrigues, R. C., Benvenutti, E. V., Costa, T. M., Hertz, P. F., &
Ninow, J. L. (2012). Effect of the support size on the properties of β-galactosidase
immobilized on chitosan: Advantages and disadvantages of macro and nanoparticles.
Biomacromolecules, 13(8), 2456–2464.
Kohli, P., & Gupta, R. (2019). Application of calcium alginate immobilized and crude
pectin lyase from Bacillus cereus in degumming of plant fibres. Biocatalysis and
Biotransformation, 37(5), 341–348.
Kumari, A., Kaur, B., Srivastava, R., & Sangwan, R. S. (2015). Isolation and
immobilization of alkaline protease on mesoporous silica and mesoporous ZSM-5
zeolite materials for improved catalytic properties. Biochemistry and biophysics
reports, 2, 108–114.
Laurent, S., Forge, D., Port, M., Roch, A., Robic, C., Vander Elst, L., & Muller, R. N.
(2008). Magnetic iron oxide nanoparticles: Synthesis, stabilization, vectorization,
physicochemical characterizations, and biological applications. Chemical reviews,
108(6), 2064–2110.
Lei, C., Shin, Y., Magnuson, J. K., Fryxell, G., Lasure, L. L., Elliott, D. C., …
Ackerman, E. J. (2006). Characterization of functionalized nanoporous supports for
protein confinement. Nanotechnology, 17(22), 5531.
Lei, L., Liu, J., Ma, X., Yang, H., & Lei, Z. (2019). A novel strategy to synthesize dual-
responsive polymeric nanocarriers for investigating the activity and stability of
immobilized pectinase. Biotechnology and applied biochemistry, 66(3), 376–388.
Li, C., Zhao, J., Zhang, Z., Jiang, Y., Bilal, M., Jiang, Y., … Cui, J. (2020). Self-assembly
of activated lipase hybrid nanoflowers with superior activity and enhanced stability.
Biochemical Engineering Journal, 107582.
16
Food Research International 140 (2021) 109979
Li, T., Li, S., Wang, N., & Tain, L. (2008). Immobilization and stabilization of pectinase by
multipoint attachment onto an activated agar-gel support. Food chemistry, 109(4),
703–708.
Li, Y., & Hardin, I. R. (1998). Enzymatic Scouring of Cotton-Surfactants, Agitation, and
Selection of Enzymes. Textile Chemist & Colorist, 30(9).
Li, Z., Zhang, Y., Su, Y., Ouyang, P., Ge, J., & Liu, Z. (2014). Spatial co-localization of
multi-enzymes by inorganic nanocrystal–protein complexes. Chemical
Communications, 50(83), 12465–12468.
Lin, Z., Xiao, Y., Wang, L., Yin, Y., Zheng, J., Yang, H., & Chen, G. (2014). Facile
synthesis of enzyme–inorganic hybrid nanoflowers and their application as an
immobilized trypsin reactor for highly efficient protein digestion. RSC advances, 4
(27), 13888–13891.
Lin, Z., Xiao, Y., Yin, Y., Hu, W., Liu, W., & Yang, H. (2014). Facile synthesis of enzyme-
inorganic hybrid nanoflowers and its application as a colorimetric platform for
visual detection of hydrogen peroxide and phenol. ACS applied materials & interfaces,
6(13), 10775–10782.
Liu, K., Zhao, G., He, B., Chen, L., & Huang, L. (2012). Immobilization of pectinase and
lipase on macroporous resin coated with chitosan for treatment of whitewater from
papermaking. Bioresource technology, 123, 616–619.
Liu, M., Fernando, D., Daniel, G., Madsen, B., Meyer, A. S., Ale, M. T., & Thygesen, A.
(2015). Effect of harvest time and field retting duration on the chemical
composition, morphology and mechanical properties of hemp fibers. Industrial Crops
and Products, 69, 29–39.
López-Gallego, F., Betancor, L., Hidalgo, A., Alonso, N., Fernandez-Lorente, G.,
Guisan, J. M., & Fernandez-Lafuente, R. (2005). Preparation of a robust biocatalyst
of D-amino acid oxidase on sepabeads supports using the glutaraldehyde
crosslinking method. Enzyme and microbial technology, 37(7), 750–756.
Lü, F., Wang, J., Shao, L., & He, P. (2016). Enzyme disintegration with spatial resolution
reveals different distributions of sludge extracellular polymer substances.
Biotechnology for Biofuels, 9(1), 1–14.
Lu, Z., Zhang, J., Ma, Y., Song, S., & Gu, W. (2012). Biomimetic mineralization of calcium
carbonate/carboxymethylcellulose microspheres for lysozyme immobilization.
Materials Science and Engineering: C, 32(7), 1982–1987.
M’hiri, N., Ioannou, I., Boudhrioua, N. M., & Ghoul, M. (2015). Effect of different
operating conditions on the extraction of phenolic compounds in orange peel. Food
and bioproducts processing, 96, 161-170.
Ma, Z. Y., Jia, X., Zhang, G. X., Hu, J. M., Zhang, X. L., Liu, Z. Y., … Zhou, F. (2013). pH-
responsive controlled-release fertilizer with water retention via atom transfer radical
polymerization of acrylic acid on mussel-inspired initiator. Journal of agricultural and
food chemistry, 61(23), 5474–5482.
Mardani, T., Khiabani, M. S., Mokarram, R. R., & Hamishehkar, H. (2018).
Immobilization of α-amylase on chitosan-montmorillonite nanocomposite beads.
International journal of biological macromolecules, 120, 354–360.
Mateo, C., Palomo, J. M., Fernandez-Lorente, G., Guisan, J. M., & Fernandez-Lafuente, R.
(2007). Improvement of enzyme activity, stability and selectivity via immobilization
techniques. Enzyme and microbial technology, 40(6), 1451–1463.
Mateo, C., Palomo, J. M., Van Langen, L. M., Van Rantwijk, F., & Sheldon, R. A. (2004).
A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates.
Biotechnology and bioengineering, 86(3), 273–276.
Mehmood, T., Saman, T., Irfan, M., Anwar, F., Ikram, M. S., & Tabassam, Q. (2019).
Pectinase Production from Schizophyllum commune Through Central Composite
Design Using Citrus Waste and Its Immobilization for Industrial Exploitation. Waste
and Biomass Valorization, 10(9), 2527–2536.
Min, K., Kim, J., Park, K., & Yoo, Y. J. (2012). Enzyme immobilization on carbon
nanomaterials: Loading density investigation and zeta potential analysis. Journal of
Molecular Catalysis B: Enzymatic, 83, 87–93.
Mohammadi, M., Heshmati, M. K., Sarabandi, K., Fathi, M., Lim, L. T., &
Hamishehkar, H. (2019). Activated alginate-montmorillonite beads as an efficient
carrier for pectinase immobilization. International journal of biological
macromolecules, 137, 253–260.
Mohammadi, M., Mokarram, R. R., Shahvalizadeh, R., Sarabandi, K., Lim, L. T., &
Hamishehkar, H. (2020). Immobilization and stabilization of pectinase on an
activated montmorillonite support and its application in pineapple juice
clarification. Food. Bioscience, 100625.
Monier, M., Ayad, D. M., Wei, Y., & Sarhan, A. A. (2010). Immobilization of horseradish
peroxidase on modified chitosan beads. International Journal of Biological
Macromolecules, 46(3), 324–330.
Mukhopadhyay, A., Bhattacharyya, T., Dasgupta, A. K., & Chakrabarti, K. (2015).
Nanotechnology based activation-immobilization of psychrophilic pectate lyase: A
novel approach towards enzyme stabilization and enhanced activity. Journal of
Molecular Catalysis B: Enzymatic, 119, 54–63.
Mukhopadhyay, A., Dutta, N., Chattopadhyay, D., & Chakrabarti, K. (2013). Degumming
of ramie fiber and the production of reducing sugars from waste peels using
nanoparticle supplemented pectate lyase. Bioresource technology, 137, 202–208.
Nadar, S. S., & Rathod, V. K. (2016). Magnetic macromolecular cross linked enzyme
aggregates (CLEAs) of glucoamylase. Enzyme and microbial technology, 83, 78–87.
Nadar, S. S., & Rathod, V. K. (2019). A co-immobilization of pectinase and cellulase onto
magnetic nanoparticles for antioxidant extraction from waste fruit peels. Biocatalysis
and agricultural biotechnology, 17, 470–479.
Nadar, S. S., Gawas, S. D., & Rathod, V. K. (2016). Self-assembled organic inorganic
hybrid glucoamylase nanoflowers with enhanced activity and stability. International
journal of biological macromolecules, 92, 660–669.
Nakkeeran, E., Umesh-Kumar, S., & Subramanian, R. (2011). Aspergillus carbonarius
polygalacturonases purified by integrated membrane process and affinity
precipitation for apple juice production. Bioresource technology, 102(3), 3293–3297.
S. Zhang et al.
O’Neill, C., Hawkes, F. R., Hawkes, D. L., Lourenço, N. D., Pinheiro, H. M., & Delée, W.
(1999). Colour in textile effluents–sources, measurement, discharge consents and
simulation: A review. Journal of Chemical Technology & Biotechnology: International
Research in Process, Environmental & Clean Technology, 74(11), 1009–1018.
Oroian, M., & Escriche, I. (2015). Antioxidants: Characterization, natural sources,
extraction and analysis. Food Research International, 74, 10–36.
Patidar, M. K., Nighojkar, S., Kumar, A., & Nighojkar, A (2018). Pectinolytic enzymes-
solid state fermentation, assay methods and applications in fruit juice industries: A
review. 3 Biotech, 8(4), Article 199.
Pedrolli, D. B., & Carmona, E. C. (2010). Purification and characterization of the
exopolygalacturonase produced by Aspergillus giganteus in submerged cultures.
Journal of industrial microbiology & biotechnology, 37(6), 567–573.
Puri, M., Sharma, D., & Barrow, C. J. (2012). Enzyme-assisted extraction of bioactives
from plants. Trends in biotechnology, 30(1), 37–44.
Qu, J. B., Chapman, R., Chen, F., Lu, H., & Stenzel, M. H. (2017). Swollen Micelles for the
Preparation of Gated, Squeezable, pH-Responsive Drug Carriers. ACS applied
materials & interfaces, 9(16), 13865–13874.
Rajdeo, K., Harini, T., Lavanya, K., & Fadnavis, N. W. (2016). Immobilization of
pectinase on reusable polymer support for clarification of apple juice. Food and
Bioproducts Processing, 99, 12–19.
Ramirez, H. L., Gómez Brizuela, L., Úbeda Iranzo, J., Arevalo-Villena, M., & Briones
Pérez, A. I. (2016). Pectinase immobilization on a chitosan-coated chitin support.
Journal of Food Process Engineering, 39(1), 97–104.
Rehman, H. U., Aman, A., Nawaz, M. A., Karim, A., Ghani, M., Baloch, A. H., &
Qader, S. A. U. (2016). Immobilization of pectin depolymerising polygalacturonase
using different polymers. International journal of biological macromolecules, 82,
127–133.
Rehman, H. U., Aman, A., Silipo, A., Qader, S. A. U., Molinaro, A., & Ansari, A. (2013).
Degradation of complex carbohydrate: Immobilization of pectinase from Bacillus
licheniformis KIBGE-IB21 using calcium alginate as a support. Food chemistry, 139
(1–4), 1081–1086.
Rehman, H. U., Aman, A., Zohra, R. R., & Qader, S. A. U. (2014). Immobilization of
pectin degrading enzyme from Bacillus licheniformis KIBGE IB-21 using agar-agar as
a support. Carbohydrate polymers, 102, 622–626.
Ren, S., Wang, Z., Bilal, M., Feng, Y., Jiang, Y., Jia, S., & Cui, J. (2020). Co-
immobilization multienzyme nanoreactor with co-factor regeneration for conversion
of CO2. International Journal of Biological Macromolecules., 155, 110–118.
Ridley, B. L., O’Neill, M. A., & Mohnen, D. (2001). Pectins: Structure, biosynthesis, and
oligogalacturonide-related signaling. Phytochemistry, 57(6), 929–967.
Rodrigues, R. C., Ortiz, C., Berenguer-Murcia, Á., Torres, R., & Fernández-Lafuente, R.
(2013). Modifying enzyme activity and selectivity by immobilization. Chemical
Society Reviews, 42(15), 6290–6307.
Ruan, P., Raghavan, V., Gariepy, Y., & Du, J. (2015). Characterization of flax water
retting of different durations in laboratory condition and evaluation of its fiber
properties. BioResources, 10(2), 3553–3563.
Seenuvasan, M., Malar, C. G., Preethi, S., Balaji, N., Iyyappan, J., Kumar, M. A., &
Kumar, K. S. (2013). Fabrication, characterization and application of pectin
degrading Fe3O4–SiO2 nanobiocatalyst. Materials science and engineering: C, 33(4),
2273–2279.
Sheldon, R. A. (2007). Enzyme immobilization: The quest for optimum performance.
Advanced Synthesis & Catalysis, 349(8–9), 1289–1307.
Sheldon, R. A. (2011). Characteristic features and biotechnological applications of cross-
linked enzyme aggregates (CLEAs). Applied microbiology and biotechnology, 92(3),
467–477.
Sheldon, R. A., & van Pelt, S. (2013). Enzyme immobilisation in biocatalysis: Why, what
and how. Chemical Society Reviews, 42(15), 6223–6235.
Shen, Q., Yang, R., Hua, X., Ye, F., Zhang, W., & Zhao, W. (2011). Gelatin-templated
biomimetic calcification for β-galactosidase immobilization. Process biochemistry, 46
(8), 1565–1571.
Shen, X. C., Fang, X. Z., Zhou, Y. H., & Liang, H. (2004). Synthesis and characterization of
3-aminopropyltriethoxysilane-modified superparamagnetic magnetite nanoparticles.
Chemistry Letters, 33(11), 1468–1469.
Shet, A. R., Desai, S. V., & Achappa, S. (2018). Pectinolytic enzymes: Classification,
production, purification and applications. Res J Life Sci Bioinform Pharm Chem Sci, 4,
337–348.
Shi, J., Zhang, S., Wang, X., Yang, C., & Jiang, Z. (2014). Preparation and enzymatic
application of flower-like hybrid microcapsules through a biomimetic mineralization
approach. Journal of Materials Chemistry B, 2(27), 4289–4296.
Soares, A. M., Gonçalves, L. M., Ferreira, R. D., de Souza, J. M., Fangueiro, R.,
Alves, M. M., … Cantanhêde, W. (2020). Immobilization of papain enzyme on a
hybrid support containing zinc oxide nanoparticles and chitosan for clinical
applications. Carbohydrate Polymers, 116498.
17
Food Research International 140 (2021) 109979
Sojitra, U. V., Nadar, S. S., & Rathod, V. K. (2016). A magnetic tri-enzyme
nanobiocatalyst for fruit juice clarification. Food chemistry, 213, 296–305.
Sojitra, U. V., Nadar, S. S., & Rathod, V. K. (2017). Immobilization of pectinase onto
chitosan magnetic nanoparticles by macromolecular cross-linker. Carbohydrate
polymers, 157, 677–685.
Soozanipour, A., Taheri-Kafrani, A., Barkhori, M., & Nasrollahzadeh, M. (2019).
Preparation of a stable and robust nanobiocatalyst by efficiently immobilizing of
pectinase onto cyanuric chloride-functionalized chitosan grafted magnetic
nanoparticles. Journal of colloid and interface science, 536, 261–270.
Štambuk, P., Tomašković, D., Tomaz, I., Maslov, L., Stupić, D., & Kontić, J. K. (2016).
Application of pectinases for recovery of grape seeds phenolics. 3. Biotech, 6(2), 224.
Sun-Waterhouse, D. (2011). The development of fruit-based functional foods targeting
the health and wellness market: A review. International Journal of Food Science &
Technology, 46(5), 899–920.
Talekar, S., & Chavare, S. (2012). Optimization of immobilization of α-amylase in
alginate gel and its comparative biochemical studies with free α-amylase. Recent
Research in Science and Technology, 4(2).
Talekar, S., Joshi, A., Kambale, S., Jadhav, S., Nadar, S., & Ladole, M. (2017). A tri-
enzyme magnetic nanobiocatalyst with one pot starch hydrolytic activity. Chemical
Engineering Journal, 325, 80–90.
Tallury, P., Payton, K., & Santra, S. (2008). Silica-based multimodal/multifunctional
nanoparticles for bioimaging and biosensing applications, 3, 579–592.
Thai, H., Nguyen, C. T., Thach, L. T., Tran, M. T., Mai, H. D., Nguyen, T. T. T., …
Ramadass, K. (2020). Characterization of chitosan/alginate/lovastatin nanoparticles
and investigation of their toxic effects in vitro and in vivo. Scientific Reports, 10(1),
1–15.
Thitiratsakul, B., & Anprung, P. (2014). Prebiotic activity score and bioactive compounds
in longan (Dimocarpus longan Lour.): Influence of pectinase in enzyme-assisted
extraction. Journal of food science and technology, 51(9), 1947–1955.
Tran, D. N., & Balkus, K. J., Jr (2011). Perspective of recent progress in immobilization of
enzymes. Acs Catalysis, 1(8), 956–968.
Wahab, W. A. A., Karam, E. A., Hassan, M. E., Kansoh, A. L., Esawy, M. A., & Awad, G. E.
(2018). Optimization of pectinase immobilization on grafted alginate-agar gel beads
by 24 full factorial CCD and thermodynamic profiling for evaluating of operational
covalent immobilization. International journal of biological macromolecules, 113,
159–170.
Wang, C., Zhang, G., Liu, G., Hu, J., & Liu, S. (2017). Photo-and thermo-responsive
multicompartment hydrogels for synergistic delivery of gemcitabine and
doxorubicin. Journal of Controlled Release, 259, 149–159.
Wang, L. B., Wang, Y. C., He, R., Zhuang, A., Wang, X., Zeng, J., & Hou, J. G. (2013).
A new nanobiocatalytic system based on allosteric effect with dramatically enhanced
enzymatic performance. Journal of the American Chemical Society, 135(4),
1272–1275.
White, L. D., & Tripp, C. P. (2000). Reaction of (3-aminopropyl) dimethylethoxysilane
with amine catalysts on silica surfaces. Journal of colloid and interface science, 232(2),
400–407.
Wu, P., Luo, F., Lu, Z., Zhan, Z., & Zhang, G. (2020). Improving the Catalytic
Performance of Pectate Lyase Through Pectate Lyase/Cu3 (PO4) 2 Hybrid
Nanoflowers as an Immobilized Enzyme. Frontiers in Bioengineering and Biotechnology,
8, 280.
Wu, W., He, Q., & Jiang, C. (2008). Magnetic iron oxide nanoparticles: Synthesis and
surface functionalization strategies. Nanoscale research letters, 3(11), 397.
Wu, X., Ge, J., Yang, C., Hou, M., & Liu, Z. (2015). Facile synthesis of multiple enzyme-
containing metal–organic frameworks in a biomolecule-friendly environment.
Chemical Communications, 51(69), 13408–13411.
Yin, Y., Xiao, Y., Lin, G., Xiao, Q., Lin, Z., & Cai, Z. (2015). An enzyme–inorganic hybrid
nanoflower based immobilized enzyme reactor with enhanced enzymatic activity.
Journal of Materials Chemistry B, 3(11), 2295–2300.
Zhang, B., Li, P., Zhang, H., Fan, L., Wang, H., Li, X., … Zhang, Q. (2016). Papain/Zn 3
(PO 4) 2 hybrid nanoflower: Preparation, characterization and its enhanced catalytic
activity as an immobilized enzyme. RSC Advances, 6(52), 46702–46710.
Zhang, B., Li, P., Zhang, H., Wang, H., Li, X., Tian, L., … Zhang, Q. (2016). Preparation of
lipase/Zn3 (PO4) 2 hybrid nanoflower and its catalytic performance as an
immobilized enzyme. Chemical Engineering Journal, 291, 287–297.
Zhang, S., Shang, W., Yang, X., Zhang, S., Zhang, X., & Chen, J. (2013). Immobilization of
lipase using alginate hydrogel beads and enzymatic evaluation in hydrolysis of p-
nitrophenol butyrate. Bulletin of the Korean Chemical Society, 34(9), 2741–2746.
Zhang, Y., Lyu, F., Ge, J., & Liu, Z. (2014). Ink-jet printing an optimal multi-enzyme
system. Chemical Communications, 50(85), 12919–12922.
Zhen, Q., Wang, M., Qi, W., Su, R., & He, Z. (2013). Preparation of β-mannanase CLEAs
using macromolecular cross-linkers. Catalysis Science & Technology, 3(8), 1937–1941.
Zhu, J., Yang, J., & Deng, B. (2009). Enhanced mercury ion adsorption by amine-
modified activated carbon. Journal of hazardous materials, 166(2–3), 866–872.