Journal of Food Science - 2022 - Patel - A Review On Pectinase Properties Application in Juice Clarification and

Received: 20 January 2022 Revised: 20 May 2022 Accepted: 1 June 2022
DOI: 10.1111/1750-3841.16233
CONCISE REVIEWS & H YPOTH ESES IN FOOD SCIENCE
A review on pectinase properties, application in juice

clarification, and membranes as immobilization support
Vashishtha B. Patel1 Somak Chatterjee1 Abhishek S. Dhoble2
1 Department of Chemical Engineering,

Birla Institute of Technology and Science, Abstract
Pilani, India Pectic substances cause haziness and high viscosity of fruit juices. Pectinase
2 Schoolof Biochemical Engineering, enzymes are biological compounds that degrade pectic compounds. Nontoxicity
Indian Institute of Technology (BHU),
Varanasi, India
and ecofriendly nature make pectinases excellent biocatalysts for juice clarifica-
tion. However, the poor stability and nonreusability of pectinases trim down the
Correspondence effectiveness of the operation. The immobilization techniques have gained the
Abhishek S. Dhoble, School of
Biochemical Engineering, Indian Institute attention of researchers as it augments the properties of the enzymes. Literature
of Technology (BHU), Varanasi, 221005 has reported the stability improvement of enzymes like lipase, laccase, hydro-
UP, India.
gen peroxidase, and cellulase upon immobilization on the membrane. However,
Email: asdhoble.bce@iitbhu.ac.in
only a few research articles divulge pectinase immobilization using a mem-
brane. The catalysis-separation synergy of membrane-reactor has put indelible
imprints in industrial applications. Immobilization of pectinase on the mem-
brane can enhance its performance in juice processing. This review delineates
the importance of physicochemical and kinematic properties of pectinases relat-
ing to the juice processing parameters. It also includes the influence of metal-ion
cofactors on enzymes’ activity. Considering the support and catalytic-separation
facets of the membrane, the prediction of the membrane as support for pectinase
immobilization has also been carried out.
KEYWORDS
enzyme immobilization, juice clarification, membrane, pectic substances, pectinases, reusabil-
ity, stability
1 INTRODUCTION final product. Therefore, the raw extracted juice is clari-

fied by centrifugation and filtration. But these operations
As a source of dietary fibers, minerals, and vitamins, fruits fall short as smaller particles remain suspended (Maktouf
are essential for the human diet. People also consume them et al., 2014).
in juice form. These have raised the importance of juice Pretreatment of the juice with pectinolytic enzymes
at a commercial level, and hence, they have become an breaks the polysaccharide chain of pectic substances and
essential part of the beverage industry. However, the high solubilizes them in the bulk liquid. It reduces the viscos-
viscosity and cloudiness in the extract due to solid sus- ity and the haziness of juice. High selectivity, sustainable
pended particles affect the market value of the juice. These process, and mild operating conditions are the benefits of
suspended solids are pectic substances extracted from the pectinase-based enzymatic treatment (Azimi et al., 2021).
fruits (Pagnonceli et al., 2019). Removal of pectic con- However, low stability, nonreusability, recovery issue, and
stituents improves the appearance, texture, and taste of the cost of biocatalyst limit its usage in juice processing
3338 © 2022 Institute of Food Technologists.

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MEMBRANES AS PECTINASES IMMOBILIZER 3339
(Jochems et al., 2011). Inactivation due to aggregation of tors and insights on possible changes that may observe
enzyme molecules is also a problem of solution-based in enzyme properties upon membrane confinement are
biocatalysis (Datta et al., 2013). Immobilization of pecti- also discussed. Moreover, the changes that observed in
nases can overcome these pitfalls. The carriers like beads, membrane properties after enzyme immobilization are
nanoparticles, capsules, gel matrices, microspheres, resin, examined. This review may help researchers and scien-
pulp fiber, pumice, or membrane have been used for tists to fabricate a membrane that can retain the maximum
enzyme localization (Ur Rehman et al., 2021; Zdarta et al., activity of the enzyme during immobilization. The dis-
2018). Further, immobilization facilitates continuous oper- cussed physicochemical and kinetic properties might help
ation, better process control, economic production, and design the experimental studies that give optimum results
reduced inhibition of the enzyme (Carrin et al., 2000; for juice clarification. Figure 1 depicts the idea of the
Datta et al., 2013). Anchoring of enzyme has eliminated review discussed here.
the enzyme inactivation step, and hence, the energy con-
sumption, making the process energy efficient. This has
also eradicated the adverse effects caused by the inac- 2 PECTIC SUBSTANCES
tivation step on product quality (Jochems et al., 2011).
Immobilization also enables easy and continuous product Pectic polymers occur in the middle lamella of plant cell
separation improving the system’s productivity by shift- walls, promoting the structural stability in plants and
ing the reaction equilibrium toward the product side (Luo safeguarding them against phytopathogens (Wang et al.,
et al., 2014). The continuous operation utilizes bioreactor 2018). Pectinaceous compounds are colloidal polysaccha-
configurations like fixed bed, packed bed, fluidized bed, rides of 1, 4 glycosidic linked α–D galacturonic acid
and membrane reactor (Azimi et al., 2021; Echavarría et al., monomers (Tan et al., 2020). The reactive groups like
2012; Zdarta et al., 2019). acetyl or methoxyl group substitute the hydrogen atom
Membrane reactors possess good mechanical strength of galacturonic acid, causing the esterification of polysac-
and require a low fabrication and energy cost compared charide backbone. Based on the degree of esterification or
to packed and fluidized bed reactors (Veismoradi et al., acetylation, the American Chemical Society has classified
2019). Furthermore, scaling up and functionality of mem- pectic compounds into four groups, that is, protopectin,
brane reactor is easier (Ye et al., 2019). Besides, membrane pectic acid, pectinic acid, and pectin. Protopectin is a
ultrafiltration follows the enzymatic treatment in juice water-insoluble, highly esterified parent compound, when
processing (Maktouf et al., 2014). Hence, immobilization hydrolyzed using an enzyme, produces a pectin molecule
using the membrane can reward the catalysis-separation (Jayani et al., 2005). In contrast, pectic acid is a polymer
synergy. Membranes also offer substantial specific sur- of galacturonic acid molecules, deprived of the methyl
face area, large void volumes, and well-controlled porosity ester group in central components. Pectinic acid, on the
(Deng et al., 2004; Wu et al., 2018). Membrane fabricated other hand, is a molecule that consists of the esterified
from materials having functional groups immobilizes the galacturonic acid monomers in the polysaccharide chain.
enzyme with a one-step reaction. Reactive functional Monosaccharides, such as arabinose, galactose, rhamnose,
groups also facilitate the modification of membrane sur- and xylose inclusion as a side-chain molecule in pectinic
face to improve the immobilization performance of the acid, result in the complex structure of pectin. Struc-
membrane (Dizge et al., 2018). The features like topogra- ture of pectin molecule is represented as homogalacturo-
phy, surface charge, hydrophilicity, resistance to mechan- nan, rhamnogalacturonan-I (RG I), rhamnogalacturonan-
ical, thermal, and chemical action, and biocompatibility II (RG II), xylogalacturonan, and apiogalacturonan (Yang
of membrane are also of great importance for enzyme et al., 2020). Citrus fruits and apple pomace contain
accommodation (Zdarta et al., 2019). These features can 0.5–4% pectin and are prominent sources of pectin extrac-
provide better accommodation to pectinases and reduce tion in industries (Guan et al., 2020). Apart from that,
the mass transfer resistance and the blockage of enzyme food waste streams like orange peels, apple cake and apple
active sites. Despite these benefits, there is a scarcity pomace, sugar beets, hop, sour cabbage, and sour cucum-
of research revealing the importance of membranes as ber are potential pectin sources. Pectin is widely utilized
pectinases immobilization support. in the food industry as a gelling agent, thickener, emulsi-
The article reviews the physicochemical properties fier, or stabilizer and can be used in the pharmaceutical
of pectinases in consideration of juice clarification and industry as a blood pressure stabilizer, cholesterol con-
throws light on juice treatment by pectinases. The chal- troller, and detoxifier (Picot-Allain et al., 2020). However,
lenges associated with the existing juice clarification the presence of pectin causes fruit juices to be hazy and
process are also summarized. Here, we have elucidated dense. The settlement of suspended solids during storage
the importance of membranes as enzyme accommoda- is also an issue for industries. To overcome this setback,
3340 MEMBRANES AS PECTINASES IMMOBILIZER
FIGURE 1 Brief graphical representation of review article structure. The concept of enzyme immobilization on membranes
most industries prefer the enzyme-based degradation of of cell walls. Protopectinases are classified as type-A and
pectin. type-B protopectinases (Jayani et al., 2005). Protopectinase
of type-A attacks on the inner region of protopectin con-
sisting of partially methylated galacturonic acid. While the
3 PECTINASES type-B protopectinase attacks the hairy and side-chain ter-
ritory of protopectin (J. Zhang et al., 2018). The enzymatic
Pectinases play an essential role in cell growth, ripen- extraction using protopectinase has replaced the high
ing of fruit, abscission, senescence, and pathogenesis of temperature and noneco-friendly acid-based extraction of
plants. Protopectinase, depolymerases, and esterases are pectin in industries. Protopectinase found its application
classes of pectinases that depend on the path of interac- in protoplast and single-cell protein production. However,
tions between enzymes and substrate, which is depicted it is not employed in the enzymatic treatment of extracted
in Figure 2. These enzymes are foremost in the enzyme- juice.
producing industries and are commercially produced via
solid-state or submerged fermentation from the strains
like fungi, bacteria, or yeast even though found in nature 3.2 Depolymerases
(Anand et al., 2017). Based on the optimum pH of pecti-
nases, they are bifurcated into alkaline or acidic groups. As the name suggests, these enzymes depolymerize the
Acidic pectinases are used in juice processing and wine- pectinaceous matters into lower chain compounds. The
making industries to degrade pectic substances. Whereas, viscosity of fruit juice depends on the chain length of pec-
alkaline pectinases find their application in papermaking, tic substances present, and hence, their performance is
vegetable oil extraction, textile processing, processing of crucial in juice clarification. Therefore, pectic enzymes
tea and coffee, and wastewater treatment (Saoudi et al., are extensively studied and industrially utilized enzymes.
2015). The following section discusses different types of Depending on the glycosidic bond cleaving mechanism,
pectinases in detail. depolymerases are grouped as hydrolases and lyases.
Hydrolases break the glycosidic bond of pectic com-
pounds by introducing the water molecule across the
3.1 Protopectinases oxygen bridge. Considering the substrate attacked by
enzymes, hydrolases are classified as polygalacturonases
Protopectinases (3.2.1.99) are hydrolase enzymes that cut (PGs) and polymethylgalacturonases (PMGs). PGs and
down water-insoluble protopectin’s complex structure into PMGs depolymerize the pectic acid and pectin, respec-
pectin. The maceration, a process of plant cell wall separa- tively. PGs follow a specific pattern to catalyze the sub-
tion, results from pectosinage activity on the protopectin strate. Enzymes targeting the terminal part of the pectic
F I G U R E 2 Hierarchical representation of enzymatic catalysis of pectic substances (i.e., from protopectin as primary substrate to
galacturonic acid as product) by pectinases
acid chain are called exopolygalacturonase. Exopolygalac- (Hamdy, 2005). Similarly, to PGs and PMGs, PNL and
turonase of two types has been reported. Exopolygalac- PL are classified as endo and exoenzymes. EndoPNL
trunase I (3.2.1.67) breaks the pectic acid from the (4.2.2.10) and endoPL (4.2.2.2) arbitrarily split the pectin
nonreducing end, producing monogalacturonates (Kant and pectic acid molecules, reducing the chain length of
et al., 2013). Whereas, exopolygalactrunase II (3.2.1.82) per- molecules. In contrast, exoPNL and PL (4.2.2.9) remove
forms the penultimate cleavage of polygalacturonic acid the terminal units of galacturonic acid in substrates dur-
(PGA), removing the digalacturonate molecule from the ing the reaction. ExoPNL scarcely exists in the crude of
polymer chain terminal (Anand et al., 2017). In contrast, lyases irrespective of the source of isolation, and hence,
the PG that strikes randomly on the chain is termed barely any research regarding exoPLs is found. Lyases
endopolygalacturonase (3.2.1.15). PGs are extracted from also include endorhamnogalacturonan I (4.2.2.23) and
microbial strains, especially Aspergillus niger (Jayani et al., exorhamnogalacturonan I (4.2.2.24), acting on RG moieties
2005). Similarly, PMGs are parted as endoPMGs and exoP- of pectinaceous compounds (Yadav et al., 2009).
MGs. No extensive studies regarding the production, char-
acterization, and application of PMGs have been reported
so far. Other accessory hydrolases, such as endoarabinase 3.3 Esterases
(3.2.1.99), α-arabinofuranosidase (3.2.1.55), β-galactosidase
(3.2.1.23), endoglucanase (3.2.1.89), EndoRG-I hydrolase Esterases do not alter the viscosity or turbidity of the juice;
(3.2.1.171), RG-I galacturonohydrolases (3.2.1.173), RG-I instead, they transform pectin into pectic acid, a substrate
rhamnohydrolases (3.2.1.174), and unsaturated rhamno- depolymerized by PLs and PGs. Esterase found in plants
galacturonyl hydrolases (3.2.1.172), also help in pectic works in the single-chain mechanism. They act on the
polymer degradation, especially in the removal of hairy esterified unit adjacent to the nonesterified galacturonic
moieties of pectin (Yadav et al., 2009). acid unit in the polymer chain and then proceed lin-
Lyases cleave the glycosidic bond at C4 (fourth num- early. In contrast, microbially isolated esterases eliminate
ber carbon in galacturonic acid unit) and remove the the esterified group randomly by following a multichain
hydrogen atom from C5, forming an unsaturated prod- mechanism (Kotnala et al., 2018). Esterases are classi-
uct (Yadav et al., 2013). Lyases that catalyze the pectin fied as pectin methylesterase (PME) (3.1.1.11) and pectin
molecule are known as pectin lyases (PNLs). In compar- acetylesterase (PAE) (3.1.1.6). PME removes the methyl
ison, pectate lyase (PL) trans-eliminates the pectic acid group from pectin’s esterified galacturonic acid unit,
forming PGA and methanol as a byproduct. Conversely, Precise maintenance of temperature throughout the
PAE deacetylates the pectin by eliminating the acetyl operation optimizes the yield of the process. However,
group from the side chains of pectin. The action of PAE exposure of proteins to temperature imposes the dena-
improves the functioning of other pectinases by reducing turing effect, depriving the biocatalytic performance. Sus-
the steric hindrance of acetyl groups present in the side tained stability with maintained catalytic ability over a
chains. RG acetyl esterase (3.1.1.86), an enzyme that per- processing period is an essential factor for the selection
forms the deacetylation of the RG I in pectin, has also been and design of enzymes. Pectic enzymes are mesophilic in
reported (Ahmed et al., 2021). this aspect. Pectinases show temperature optima (OT) in a
span of 40–60◦ C with appreciable stability. To determine
the thermal stability, an enzyme is exposed to a certain
4 PHYSICOCHEMICAL AND temperature for a varying period and the residual activity
BIOCHEMICAL PROPERTIES OF PECTIC of the enzyme is estimated at standard assay conditions at
ENZYMES the completion of incubation time. Aspergillus luchuensis
endoPG (OT-40◦ C), Datura stramonium PME (OT-50◦ C),
An acquaintance with the enzyme’s properties is vital PL from Bacillus subtilis PB1 (OT-50◦ C), Escherichia coli
for its commercial production and application. Enzymes’ (OT-50◦ C), PNL from A. niger (OT-50◦ C), and Fusarium
physicochemical and biochemical properties are signifi- oxysporum MTCC 1755 (OT-50◦ C) showed substantial sta-
cant constraints in designing and developing a biocatalytic bility at optimum temperature for 2 h of incubation time
process. The performance of pectinases selected based on (Dixit et al., 2013; Guan et al., 2020; He et al., 2018; Tan
these constraints is anticipated in industries. Hence, this et al., 2020; Yadav et al., 2017; Zhou et al., 2017). Ther-
section has given attention to the physicochemical and bio- mophile isolates can be a vital tool in high-temperature
logical characterization of pectinolytic enzymes embodied biochemical processes. Thermophiles obtained from var-
in Table 1. ious sources were stable at optimum temperature (Y. Chen
Enzymes must be purified to homogeneity before their et al., 2014; Cheng et al., 2016; Lu et al., 2016; Saoudi et al.,
utilization in food-processing applications. Purification 2015). However, S. purpureum GH28 endoPG reported 60%
of the enzyme enhances the catalytic efficiency elimi- residual activity after 30 min of incubation at OT (Carli
nating the chances of reaction mixture contamination. et al., 2019). Mesophiles from Aspergillus flavus MTCC
As molecular mass is assessed using purification tech- 10938 and A. niger ZJ5 also lost 50% of initial activity for an
niques, the macromolecule’s purity can easily be estimated hour of incubation at OT (Yadav et al., 2013; Z. Zhang et al.,
from the molecular mass statistics of enzymes. Pectina- 2018). Whereas, Kant et al. (2013) observed that exoPG
ceous enzymes, except exoPG, reported a molecular weight almost inactivated at 45◦ C after 75 min of operation. Tem-
between 30 and 50 kDa; however, SDS-PAGE analysis of perature optimum is also altered with substrate attacked
endoPG from Bispora sp. MEY-1 and Stereum purpureum by enzymes. Penicillium oxalicum endoPG exhibited tem-
GH28 and Aspergillus repens isolated PME expressed perature optimum of 65, 50, and 60◦ C for PGA, apple
molecular mass of 71, 60, and 141 kDa, respectively pectin, and citrus pectin, respectively (Cheng et al., 2016).
(Arotupin et al., 2008; Carli et al., 2019; Yang et al., 2011). Almost all researchers performed temperature optima test
Whereas, ExoPG from various sources showed molecu- for constant reaction time. Poturcu et al. (2017) studied
lar weight between 50 and 106 kDa (Anand et al., 2017; the effect of reaction time on maximum yield temperature.
Kant et al., 2013; Lu et al., 2016; Pagnonceli et al., 2019). PNL from A. niger WHAK1 recorded a peak performance
The higher mass of macromolecules is attributed to gly- at 40, 50, 60, and 70◦ C for 60, 30, 40, and 10 min of
cosylation. The addition of carbohydrates alters enzyme process time, respectively. However, the maximum con-
structure and molecular mass, affecting catalytic activity version of the substrate was reported at 40◦ C for 60 min
and stability, protein interaction, pathogenesis, and immo- (Poturcu et al., 2017). Simultaneous raise in reaction rate
bilization of enzymes (H. Xu et al., 2018; Yang et al., 2011). and enzyme deactivation with temperature can explain
PG with glycosylation showed lower substrate affinity than this phenomenon.
nonglycosylated enzymes (H. Xu et al., 2018). Due to strong The enzyme–substrate complexation is liable to the
protein–protein interactions, sometimes, the partition of enzyme’s ionic charge. The pH of the processing mix-
macromolecules becomes difficult during purification. In ture puts excellent emphasis on enzyme structure and
this case, enzyme exists as homo or heteromers, result- the electric state of amino acid residues in active sites.
ing in greater molecular weight. Kant et al. (2013) found Hence, it is crucial to consider the impression of the
106 kDa weight of exo-PG on native PAGE, which upon processing mixture’s pH on biocatalysts. Like optimum
SDS-PAGE analysis showed the existence of two subunits temperature, biocatalysts show optimal reactivity at a par-
of 69 and 34 kDa. ticular hydrogen potential called optimum pH. Pectinases
TA B L E 1 Physicochemical and kinetic properties of pectinases
Michaelis
Molecular constant Optimum Optimum Temperature Effect of ions on enzyme activity
Enzyme weight (kDa) (mg/ml) temperature (◦ C) pH stability (◦ C) pH stability Enhanced Reduced References
Endo PG 38 1.27 65 5 50 2.2–7 Na, K, Ca, Mg, Fe Mn, Co, Zn, Cu Cheng et al. (2016)
40 0.19 40 4.5 40 3.5–5.5 K Ca, Mn, Mg, Cu Tan et al. (2020)
42 – 35 6 30–40 4–9 Mg Na, Ca, Fe, Ba, Zn, Cu Sassi et al. (2016)
MEMBRANES AS PECTINASES IMMOBILIZER
71 1.25 55 3.5 55 2–7 Mn, Mg Ca, K, Na, Ni Yang et al. (2011)

60 2.45 70 4.5 50–60 6–8 – Ca, Fe, K, Mg Carli et al. (2019)
Exo PG 106 0.083 45 4.8 25 3.5–5.5 Mg, Cu Ca, K, Al, Zn Kant et al. (2013)
29 2.08 40 5 10–40 5–11 Co, K, Cu Ag, Ca, Hg, Anand et al. (2017)
102 2.56 50 5 50 3–5 Mg Ca, Na, K, Cu Pagnonceli et al. (2019)
75.28 5.44 60 5 30–50 3–11 Cu, Zn, Na, K, Mg Hg, Ba X. Lu et al. (2016)
50 0.31 72 5.2 70 – – Co, Ni, Ca, Mg Y. Chen et al. (2014)
Pectin lyase 31 3.87 50 7.5 50–60 7–9.5 Ca, K, Mg, Na Co, Cu, Mn, Zn Hamdy (2005)
23.3 5.2 40 8 – – Mg, Ca, Fe, Na K, Co Poturcu et al. (2017)
50 1.7 55 8 10–40 3–11 Mn, Ca Hg, Ag, Zn Yadav et al. (2013)
40 – 50 4.5 30–50 3–5 Na, Mg, Zn, Ni K, Ca, Cu He et al. (2018)
31 1.75 40 9 10–50 5–7 Ca, K, Na, Zn, Ag, – Yadav et al. (2017)
Pectate lyase 44 1.78 55 9.8 – 4–10 Ca, K Ba, Mn H. Wang et al. (2014)
38 1.64 50 8 40–60 5–10 Ca Ni, Mn, Zn, Cu Guan et al. (2020)
41 – 50 4 40 6–9 Al, Mg, Fe, Ba, Cu Yang et al. (2020)
43.1 0.312 50 9.5 30–60 5–11 Ca, Mg, Mn, Ba Cu, EDTA Zhou et al. (2017)
34 0.45 60 10.5 70–90 7.5–12 Ca, Mn, Ba, Mg, Hg, Cu, Sn, K Saoudi et al. (2015)
Pectin methyl 141 1.3 30 6.5 – – Ca, Na, Mg, K Pb, Hg Arotupin et al. (2008)
esterase
33 0.008 60 9 40–60 – Na – Dixit et al. (2013)
27 0.22 60 7 30–60 4–10 Na, K, Mg, Ca Zn, Ni, Cu, Fe Kotnala et al. (2018)
37 3.27 45 3.8 20–30 2–6 – – Z. Zhang et al. (2018)
3343
presented ambiguous pH optimum with considerable sta- substrate discussed in earlier sections. Still, the E. coli PL
bility between mild acidic to mild alkaline division. Endo showed greater affinity for 55–70% esterified pectin rather
and exoPGs showed optimum pH between 3 and 6 on the than PGA and pectin of esterification degree higher than
pH scale with palpable acidic activity. In contrast, PNL, 85% (Wang et al., 2014). Guan et al. (2020) observed simi-
PL, and PME expressed maximum efficiency in neutral lar behavior from PL, which expressed maximum activity
to the mild basic range, and hence, hardly utilized for with citrus pectin followed by apple pectin and PGA. PL
food-processing applications. Nevertheless, A. niger PNL, showed 80% and 55% relative activity with apple pectin
Aspergillus parasiticus PL, A. niger ZJ5, and A. repens PME and PGA considering 100% relative activity with orange
displayed optimal activity at 4.5, 4, 3.8, and 6.5 pH values pectin (Guan et al., 2020). This indicates that the sub-
(Arotupin et al., 2008; He et al., 2018; Yang et al., 2020; Z. strate’s source also plays a crucial part in biocatalytic
Zhang et al., 2018). Except for A. parasiticus PL, almost all kinesis. Michaelis’ constant values expressed in Table 1
pectic enzymes were stable at pH optima. PL from A. para- reveal the little lower affinity of pectinases toward its
siticus lost 60% initial activity for 24 h of incubation (Yang substrate.
et al., 2020). These studies were based on the individual The foreign compound present in biochemical reac-
effect of pH on enzyme stability, maintaining temperature tion media significantly alters the enzyme performance.
constant and vice versa in thermal stability analysis. How- Several enzymes require an entity, called a cofactor, to
ever, the simultaneous impact of pH and temperature may commence the biochemical reaction, whereas others get
alter the enzyme’s stability differently. Response surface stimulated by these compounds. The effect of metal ions
methodology showed that PMG, PL, and PG were max- like Na+ , K+ , Mg+2 , Ca+2 , Ni+2 , Mn+2 , Fe+2 , Zn+2 ,
imally stable at conditions other than the optima values Cu+2 , Hg+2 , Ba+2 , and Pb+2 on pectinases activity has
based on the individual effect studies (Sathya et al., 1998). widely examined. Pectinolytic enzymes, except PL, exhib-
The charge on substrate molecules also affects the pH opti- ited ambivalent responses to metal ions, suggesting no
mum of enzymes. P. oxalicum endoPG reported lower pH need for cations to express the activity. It is observed that
optima for pectin (OP-4) than pectic acid (OP-5) due to the PL needs calcium ions to display reactivity (Guan et al.,
negative charge on pectin (Cheng et al., 2016). 2020; Zhou et al., 2017). Wang et al. (2014) documented
Besides ionic charge, the substrate’s concentration also that Ca+2 helps PL recognize the substrate and stabilize
affects the enzyme’s catalytic capacity. The concentration the transient anion in the β-trans-elimination mechanism
of substrate required to attain half the maximum catalytic during catalysis. Saoudi et al. (2015) noticed the increase
efficiency of an enzyme is represented as Michaelis’ con- in optimal temperature of PL with better thermostabil-
stant (Km ). Pectinases reported Michaelis’ constant in the ity due to calcium ions. The optimum temperature of PL
range of 0.001–5.44 mg/ml. PME extracted from D. stramo- increased to 70◦ C with no loss in enzyme activity for
nium expressed a higher affection toward substrate with 10 h in the presence of Ca+2 ion (Saoudi et al., 2015).
Km of 0.004 mg/ml (Dixit et al., 2013). In comparison, Besides PL, Carica papaya PME required monovalent salts
exoPG from Zygoascus hellenicus reported a maximum to commence the reaction (Kotnala et al., 2018). How-
value of Michaelis’ constant, that is, 5.44 mg/ml (Lu et al., ever, PME from other sources reported no requirement
2016). Due to nonstandardize assay conditions, parame- for cations. EndoPG from P. oxalicum CZ1028, Penicillium
ters like pH, temperature, time, and buffer utilized for occitanis, and Pichia pastoris is stimulated in the pres-
assay may vary from article to article. Though temperature ence of magnesium ions (Cheng et al., 2016; Sassi et al.,
and pH are maintained at optimum working condition, 2016; Yang et al., 2011). However, A. luchuensis and S.
variation in time may change the overall output alter- purpureum endoPG activity degraded in the presence of
ing the ideal value of process data (Poturcu et al., 2017). magnesium ions (Carli et al., 2019; Tan et al., 2020). ExoPG
Different condition of temperature and pH for substrate also observed the same behavior in the presence of mag-
may alter the properties of substrate (Cheng et al., 2016). nesium ions (Anand et al., 2017; Chen et al., 2014; Kant
The source of origin of enzyme and substrate, enzyme’s et al., 2013; Lu et al., 2016; Pagnonceli et al., 2019). Cations
purity, type of substrate, and its esterification degree also of cadmium, copper, lead, tin, mercury, and silver showed
causes the deviation in Michaelis’ constant values (Cheng inhibitory effects on pectinases activity (Pagnonceli et al.,
et al., 2016; H. Xu et al., 2018). The lower affinity of exoPG 2019; Poturcu et al., 2017; Saoudi et al., 2015; Tan et al.,
could be a result of assay conditions other than ideal reac- 2020). The presence of Hg+2 oxidized the thiol group in
tion parameters. Besides, the poor parity of pectin with cysteine residue near the enzyme’s active site, hindering
PG in proportionate to PGA might be responsible for this the enzymatic catalysis (Lu et al., 2016). Pectinases under-
(Lu et al., 2016). Enzyme–substrate interaction is greatly went inhibition in the existence of chemicals like EDTA,
influenced by enzyme and substrate combinations. Most sodium dodecyl sulphate, potassium permanganate, and
pectinases showed better association with their respective sodium arsenate.
5 PECTINASES IN JUICE PROCESSING 6 IMMOBILIZATION OF ENZYMES ON

AND THEIR LIMITATIONS MEMBRANE
Raw extracted juices are cloudy and dense. As discussed Immobilization of pectinases has been widely practiced
earlier, pectic substances available in fruits are responsible using different supports like beads, nanoparticles, micro-
for this. Consumer prefers juice, having low viscosity and spheres, matrix, resins, capsules, pulp fiber, and pumice,
crystal clarity with high nutrient content. Hence, the treat- which has been reviewed elsewhere (Zhang et al., 2021).
ment of pectinaceous compounds is an absolute necessity Membranes are used as a juice filtration medium in indus-
from a marketing viewpoint. Juice clarification by apply- tries, and hence, immobilization of pectic biocatalysts
ing pectinase has many advantages like increased pulp on the membrane can synergize two parallel operations
liquefication, and hence, increased juice yield, enhanced (i.e., enzymatic depectination and juice filtration), inten-
clarification and reduced sugar content, reduced turbidity sifying the clarification process (Maktouf et al., 2014).
and viscidness, and enhancement in total soluble solid However, research revealing membrane as immobiliza-
content (Ramadan, 2018). Further, mild processing con- tion support for pectinases, especially for juice treatment,
ditions preserve the natural flavor of the raw fruit. The is limited (Carrin et al., 2000; Echavarría et al., 2012;
depectination studies of juices were performed with pecti- Giorno et al., 1998). When enzymes are immobilized, the
nases’ amount ranging from 0.02 to 100 Units/ml for 1–24 vicinity of the membrane imposes certain changes in
h, maintaining temperature and pH between 20 and 60◦ C the catalytic characteristics of enzymes. The charge pos-
and 3–5, respectively (Anand et al., 2017; Carli et al., 2019; sessed by membrane material interacts with the enzyme
Cheng et al., 2016; Dixit et al., 2013; He et al., 2018; Lu et al., and adjusts the optimum pH of the enzyme immobilized
2016; Pagnonceli et al., 2019; Yang et al., 2011). Pectinases (Vaillant et al., 2000). Hydrophobicity or hydrophilicity of
reduced the viscosity of juice, whereas enhancement membrane also influences the enzyme–substrate interac-
in sugar content, light transmission, and yield were tion (He et al., 2006). Only H. Xu et al. (2018) have studied
observed. The light transmittance of orange, apple, and the influence of the membrane on properties of immo-
grape extract, respectively, increased by 19.2-, 7.3-, and bilized pectinases. Hence, to elucidate the changes that
3.8-fold after pectinase treatment (He et al., 2018). Simi- occurred in the enzyme’s properties upon immobilization,
larly, after enzymatic treatment, 71.43%, 57.5%, and 54.2% we have taken the reference of other enzymes, like lac-
reduction in pomegranate, apple, and orange juice viscos- case, lipase, cellulase, tyrosinase, chloroperoxidase (CP),
ity was reported (Poturcu et al., 2017). Depectination of β-galactosidase, acetylcholinesterase, horseradish peroxi-
pear and banana juice raised the reducing sugar content dase (HP), and urease. The comparison of various param-
by 22.5% and 28%. Also, the catalytic action of pectinolytic eters of free and immobilized enzymes is illustrated in
enzymes improved the papaya juice yield by 24% (Cheng Table 2.
et al., 2016; Sassi et al., 2016). The reported research reveals Enzymes are confined on the membrane by applying
the pectinases’ potential for juice clarification. Pectinases techniques like adsorption, covalent binding, and entrap-
showed excellent catalytic performance with considerable ment. Figure 3 depicts the methods used to immobilize
thermal and pH stability. However, the separation of the enzymes on the membrane. Adsorptive attachment
pectinase from the final product is difficult. To address and chemical bonding are widely employed for enzyme
this problem, thermal inactivation of the final product is attachment on/in membranes. The adsorption-based tech-
carried out. Thermal inactivation may degrade the prod- nique uses physical forces between enzyme molecules
uct quality by destroying essential natural compounds and support to immobilize the biocatalyst (Datta et al.,
(Echavarría et al., 2012). Also, the energy consumption 2013). In contrast, a covalent association is accomplished
of the inactivation step is high. The failure of expensive by performing the reaction of the enzyme and functional
pectinase recovery and reusability makes the process group of support. Sometimes, chemical connectors like
uneconomical (Ur Rehman et al., 2021). Also, the use of glutaraldehyde, glycidyl methacrylate, and carbondiimi-
PME for juice clarification generates methanol as a byprod- dazole are also employed on membranes to chemically
uct. Methanol causes a toxic effect on human health, and bind the enzymes (Liu et al., 2018a; Vasileva et al., 2016).
hence, it is favorable to replace PME with PNL or PMGL. The functional groups like α-amino of lysine, sulfhydryl
This indicates the need for reinforcement for the enzy- of cystine, carboxyl of aspartate and glutamate, phenolic
matic treatment step. Immobilization of the enzyme has of tyrosine, guanidino of arginine, imidazole of histi-
been practiced widely for stability reinforcement of bio- dine, disulfide of cystine, indole of tryptophan, thioether
catalyst. Immobilization also permits the easy separation of methionine, hydroxyl of serine and threonine, C-
and repeated use of enzymes. Hence, we have focused on terminus carboxyl group, or N-terminus amino group are
pectinases’ immobilization to surplus their performance. the reactive residues of enzyme proteins (Srere & Uyeda,
3346
TA B L E 2 Physicochemical and kinetic properties of enzymes immobilized on membranes

Optimum Temperature Michaelis’
Types of Immobilization temperature (°C) Optimum pH stability(°C) pH stability constant
Enzyme membrane method Free Immo. Free Immo. Free Immo. Free Immo. Free Immo. References
EE Tubular Adsorption 45 45 4 4 – – – – 0.027 0.442 de Cazes et al. (2016)
Covalent 45 45 4 4 – – – – 0.027 0.638
Laccase Nanofiber Adsorption 50 50 4.5 4.5 75 75 – – 0.78 1.84 Wu et al. (2018)
β-Galactosidase Spiral wound Covalent 37 40 7.1 6.8 – – – – – – Vasileva et al. (2016)
CP Thin film Adsorption – – – – 80 80 2–5 2–7 – – J. Lu et al. (2017)
CGTase Nanofiber Covalent 60 70 – – – – – – – – Sulaiman et al. (2017)
Pectinase Nanofiber Covalent – 55 – 5 55 55 2–6 2–7 8.17 9.26 H. Xu et al. (2018)
55 55 2–6 2–7 6.29 7.23
Cellulase Flat-sheet Adsorption 30 50 4 8 – – – – 0.0127 0.0143 W. Xu et al. (2018)
Lipase Flat-sheet Covalent – – 6.5 6.5 60 60 – – 0.213 0.111 Aghababaie et al.
(2016)
Lipase Nanofibrous Covalent 30 35 8 6 70 70 – – 0.169 0.19 X. Liu et al. (2018a)
Lipase Nanofibrous Covalent 30 35 8 7 70 70 – – 0.169 0.218 X. Liu et al. (2018b)
ACE Nanofibrous Covalent 30 35 7.4 8 – – – – 0.4733 0.5008 Çakıroğlu et al. (2018)
HP Nanofibrous Covalent 45 50 7 7 55 55 – – 1.86 2.42 Temoçin et al. (2018)
Tyrosinase Flat-sheet Covalent 30 30 6.5 6.5 – – – – – – Veismoradi et al.
(2019)
Laccase Flat-sheet Covalent 55 50 3.5 2.5 55 55 – – 0.12 0.91 Li et al. (2017)
WE Fibrous Adsorption 30 40 8 7 40 40 – – 0.85 6.73 L. Ye et al. (2019)
Lipase Hollow fiber Covalent 37 45 7.5 7 DN 50 4–6 04–07 0.45 1.43 P. Ye et al. (2005)
Urease Hollow fiber Adsorption 45 55 7 6 50 50 – – 18 22 Akgöl et al. (2002)
Trypsin Flat-sheet Covalent 50 60 8 8 – – – – – – Dizge et al. (2018)
Lipase Hollow fiber Adsorption 35 45 7.7 8.5 DN 50 – – – – Deng et al. (2004)
Laccase Nanofiber Adsorption 30 30 5 3 – – – – – – Taheran et al. (2017)
CITase Hollow fiber Covalent 50 50 6.5 6.5 – – – – – – Kawakita et al. (2002)
Abbreviations: ACE, acetylcholinesterase; AD, alcohol dehydrogenase; CITase, cycloisomaltooligosaccharide glucanotransferase; CGTase, cyclodextrin glucanotransferase; CP, chloroperoxidase; DN, denatured; EE, ereB
esterase; HP, horseradish peroxidase; WE, wheat esterase.
MEMBRANES AS PECTINASES IMMOBILIZER
F I G U R E 3 Schematic depiction of techniques used to immobilize the enzyme on membrane. (a) Adsorptive attachment, (b) covalent
binding, and (c) entrapment
1976). Enzyme desorption attributed to weak binding lipase retained 15% of residual activity at 70◦ C after 3 h,
forces is a major setback of adsorption binding. Whereas, which upon immobilization showed 40% residual activity
deactivation of enzymes due to structural deformation for the same assay conditions (Liu et al., 2018b). The par-
caused by the chemical reaction is a common prob- tition effect also hindered the transportation of hydrogen
lem of the covalent bonding method (Ur Rehman et al., ions and a product varying the pH optima and Michaelis’
2021). However, the leaching rate of enzymes mounted constant of enzymes (Çakıroğlu et al., 2018; Taheran
by chemical bonding was lower than the adsorbed et al., 2017). The poor transportation of ammonia from
enzyme. Jochems et al. (2011) have explicitly discussed the the microenvironment of immobilized urease to bulk fluid
enzyme immobilization techniques concerning membrane reduced the pH optimum of an immobilized enzyme from
immobilization. 7 to 6 (Akgöl et al., 2002). Moreover, the charge carried by
Enzyme confinement on the membrane raised the ther- the carrier too imposes changes in pH optima of immobil-
mal stability and temperature optima of the enzyme. The isate. Polar and ionic interaction as well as weak bonding of
immobilization limited the structural reforms of enzymes, biomolecules with charged carriers altered the pH optima
resulting in higher activation energy of biocatalysis (Dizge of immobilized enzymes (Li et al., 2017; Ye et al., 2019).
et al., 2018; Ye et al., 2019). Akgöl et al. (2002) observed The negatively charged support shifted the pH optimum of
a two-fold hike in urease’s activation energy upon bind- enzyme in the alkaline region, whereas enzymes attached
ing due to energy distribution in covalent bonds. This to positively charged support showed acidic displacement
increased the optimum temperature of urease from 45 to in pH optima after attachment (Vaillant et al., 2000). As
55◦ C. Besides, limited transport of substrate in the case discussed earlier, immobilization provides the structural
of a pore diffusion-controlled regime results in reduced rigidity due to which pH stability of enzyme also improved
enzyme–substrate interaction leading to reduced reaction upon support binding. The CP in free mode showed pH
velocity. An increase in temperature lowers the diffu- stability below the neutral pH. The localization of CP on
sion resistance enhancing the overall reaction velocity and the TiO2 membrane enhanced the stability to pH value 9
yield (Ye et al., 2005). However, heat transfer gradient (Lu et al., 2017). However, support binding of the enzyme
between porous microenvironment of macromolecule and diminished the Michaelis’ constant resulting in a reduced
bulk fluid as well structural rigidity due to multipoint sup- rate of reaction. Except for Aghababaie et al. (2016), all the
port attachment enhanced the thermal stability of enzyme articles reported a reduction in enzyme–substrate affinity.
(Liu et al., 2018b; Temoçin et al., 2018). Solution-based The reduction in enzyme–substrate affinity is attributed to
limited pour diffusion, steric hindrance imposed by sup- and lifespan (Girard & Fukumoto, 2000). The membranes
port, different microenvironmental conditions, concealed fabricated from reported materials were also used to immo-
active sites of enzymes, and conformational restrictions to bilize the enzymes and enhanced the performance of
enzyme mobility (Temoçin et al., 2018; Wu et al., 2018; H. enzymes without deteriorating the integrity of macro-
Xu et al., 2018; Ye et al., 2019). Aghababaie et al. (2016) molecules (Akgöl et al., 2002; Deng et al., 2004; Dizge et al.,
observed enhancement in enzyme–substrate complexa- 2018; Vasileva et al., 2016; Ye et al., 2005). Till date, poly-
tion upon immobilization of lipase on support. This was meric membranes of polysulphone, PVDF, and polyamide
attributed to balanced hydrophobicity and hydrophilicity have been used as an immobilization support for pecti-
of membrane surface due to surface modification. The nases for juice clarification application (Carrin et al., 2000;
Km value of lipase reduced to 0.111 from 0.213 mg/ml Echavarría et al., 2012; Giorno et al., 1998).
(Aghababaie et al., 2016). Enzyme incorporation on the membrane significantly
To further rectify the catalytic performance of enzymes, altered the membrane properties, and hence, the perfor-
researchers incorporated or grafted the additional com- mance of the membrane filtration. The immobilization
pounds on membranes. The surface modification by these of the enzyme improved the permeate flux reducing the
compounds enhanced the surface area for enzyme accom- membrane fouling compared to the free enzyme mem-
modation. Further, a microenvironment provided by these brane reactor. Giorno et al. (1998) observed the permeate
compounds raised the catalytic activity of the enzyme flux of 15 L/m2 .h.bar for free enzyme membrane reac-
(Aghababaie et al., 2016; Akgöl et al., 2002; Dizge et al., tor, which upon enzyme immobilization increased to
2018; Li et al., 2017; W. Xu et al., 2018). TiO2 inclusion 20 L/m2 .h.bar due to hydrolytic action of pectinases on sur-
on cellulose membrane lessened Michaelis’ constant value face deposited pectin. However, the general trend shows
of laccase by 50% due to enhanced enzyme loading and reduction in permeability upon enzyme immobilization
simultaneous photo- and biocatalysis phenomena (Li et al., due to blockage of the pore (Luo et al., 2014). Konoval-
2017). Lipase responded similarly when immobilized on ova et al. (2016) reported that the attachment of amylase
polypeptide feathers-grafted membrane. The multipoint reduced the concentration polarization in membrane fil-
attachment of lipase widely opened the enzyme structure tration due to the degradation of starch in the membrane
and provided stability (Liu et al., 2018a). However, in both vicinity. The hydrolysis led to an increase in diffusiv-
cases, enzyme–substrate affinity was relatively lower than ity of the substrate in the boundary layer, enhancing
solution-based enzymes. The inclusion of the interfacial mass transfer coefficient, compared to a membrane having
layer showed similar behavior for temperature and pH of no biocatalyst attachment (Konovalova et al., 2016). The
the enzyme (Deng et al., 2004; Ye et al., 2005). Tethering immobilization of the enzyme also alters the hydrophilic-
of phospholipid analogous polymer elevated the temper- ity of the membrane. Immobilization of transglutaminase
ature optima from 40 to 45◦ C. The decrease in substrate on the membrane increased the hydrophilicity of the mem-
diffusion due to pore blockage by phospholipid analogous brane, lowering the contact angle by 20◦ (Wen-qiong &
polymer caused the change in OT (Deng et al., 2004). Xiao-feng, 2018). However, trypsin immobilization on the
Hence, the selection of the membrane support layer is membrane increased the water contact angle by increasing
important. Apart from being able to enhance the enzyme the hydrophobicity of the enzyme (Liu et al., 2017).
activity, the carrier material should be inexpensive, stable, To characterize the occurred changes, various tech-
nontoxic, biodegradable and biocompatible, regenerable, niques are employed. The characterization techniques
of good mechanical, chemical, and thermal strength, easily like scanning electron microscopy/field emission scan-
moldable, and durable in the membrane form (Cen et al., ning electron microscopy (SEM/FESEM), transmission
2019; Jochems et al., 2011). The material used should also electron microscopy (TEM), atomic force microscopy
withstand the properties of juices. (AFM), Fourier transform infrared spectroscopy (FTIR),
Generally, membranes fabricated from polymeric com- attenuated total reflectance-Fourier transform infrared
pounds are used in juice clarification. Polysulphone, spectroscopy (ATR-FTIR), thermal gravimetry analysis
polyvinylidene fluoride (PVDF), polyamide, polypropy- (TGA), X-ray photoelectron spectroscopy (XPS), Raman
lene, polyacrylonitrile, cellulose acetate, and ceramic spectroscopy, confocal laser scanning microscopy (CLSM),
membranes have been applied for juice clarification due and inverted fluorescence microscopy (IFM) have been
to pH, pressure, and temperature resistance (Bhattachar- applied to characterize the enzyme immobilized mem-
jee et al., 2017; Carrin et al., 2000; Echavarría et al., brane (Sandu et al., 2015; Taheran et al., 2017; Veismoradi
2012; Giorno et al., 1998). Also, their cost of fabrication is et al., 2019; Wen-qiong & Xiao-feng, 2018; Xu et al., 2017).
very low. However, polymeric membranes possess a poor These techniques provide a comprehensive understanding
antifouling property with a lower lifespan. Conversely, of the enzyme confinement on a membrane and the possi-
ceramic membranes are expensive with better durability ble effect on the performance of heterogeneous biocatalyst.
TA B L E 3 Illustration of enzyme loading with recovered activity and recyclability of enzyme immobilized on membrane
Loading capacity Reusability
Types of Immobilization (mg/m2 Recovered Number of Residual
Enzyme membrane method membrane) activity (%) cycle activity (%) References
EE Tubular Adsorption 870 – 4 – de Cazes et al.
(2016)
Covalent 510 – 4 –
Laccase Nanofibrous Adsorption 6.524 – 10 50 Wu et al. (2018)
a
CGTase Nanofibrous Covalent 159.34 45 10 55 Sulaiman et al.
(2017)
Cellulase Flat-sheet Adsorption 825 – 7 42.8 W. Xu et al.
(2018)
Lipase Flat-sheet Covalent 600 – 8 45 Aghababaie
et al. (2016)
ACE Nanofibrous Covalent – – 10 87 Çakıroğlu et al.
(2018)
HP Nanofibrous Covalent – – 25 54 Temoçin et al.
(2018)
Tyrosinase Flat-sheet Covalent – – 10 80 Veismoradi
et al. (2019)
Laccase Flat-sheet Covalent – – 10 60 Li et al. (2017)
Lipase Hollow fiber Covalent 66.5 44.5 10 53 P. Ye et al.
(2005)
β-Galactosidase Spiral wound Covalent – – 20 69.7 Vasileva et al.
(2016)
Urease Hollow fiber Adsorption 4.87 – – – Akgöl et al.
(2002)
Lipase Hollow fiber Adsorption 39.5 83.2 – – Deng et al.
(2004)
Laccase Nanofibrous Adsorption – – 10 40 Taheran et al.
(2017)
CITase Hollow fiber Covalent 18.9 – – – Kawakita et al.
(2002)
Abbreviations: ACE, acetylcholinesterase; AD, alcohol dehydrogenase; CITase, cycloisomaltooligosaccharide glucanotransferase; CGTase, cyclodextrin glucan-
otransferase; CP, chloroperoxidase; EE, ereB esterase; HP, horseradish peroxidase; WE, wheat esterase.
a
Unit is in mg/gm of membrane.
AFM, TEM, and SEM/FESEM studies gave insight into a good life cycle upon immobilization. Temoçin et al.
morphological changes reported on support. Whereas, (2018) reported that immobilized HP catalyzed 25 batches
FTIR, ATR-FTIR, Raman spectroscopy, and XPS analysis and was active with 54% residual activity. Similarly,
assessed the enzyme immobilization as well determined β-galactosidase possessed almost 70% residual activity
surface chemical composition. Investigation of thermal after 20 cycles (Vasileva et al., 2016). The reduction
behavior of enzyme carrying membrane sample was in residual activity is attributed to the detachment or
carried out by TGA analysis (Sandu et al., 2015). Whereas, denaturation of enzyme molecules while processing.
protein distribution on membrane support is examined The constant exposure to processing parameters causes
by IFM and CLSM. Bolivar et al. (2016) have thoroughly permanent changes in enzyme functioning leading to
discussed the various characterization techniques applied the deactivation of enzymes resulting in reduced enzyme
to support bonded enzymes. activity (de Cazes et al., 2016). Whereas, weak attachment
The immobilization significantly increased the life between enzyme and substrate leads to leaching of the
cycles of biocatalyst compared to free enzyme reactor. enzyme, resulting in 60% loss of enzyme activity (Taheran
Reusability data of various immobilisate, discussed above, et al., 2017). Here, a residual activity defines the percentage
are given in Table 3. Due to easier separation, enzymes retention of immobilized enzymes activity compared to
were repeatedly used for biocatalysis. Enzymes showed a freshly immobilized enzyme. Similarly, the activity
recovery represents the relative catalytic performance to reduced enzyme loading (Chen et al., 2012). Chen et al.
of immobilisate in consideration of free enzyme perfor- (2012) observed that the loading of lipase increased with
mance. Activity recovery is a percentage ratio of freshly increase in hydrophilicity of membrane due to easy access
immobilized enzyme activity to free enzyme activity at the of immobilization surface to hydrophilic enzyme media.
same assay conditions. Few articles have taken consider- Membrane type also plays a critical role in the enzyme’s
ation in this regard. CGTase showed 45% activity recovery loading capacity. Flat-sheet, hollow fiber, nanofiber, and
upon immobilization (Sulaiman et al., 2017). Similarly, tubular geometries are utilized as enzyme carriers. The
lipase immobilization on chitosan tethered membrane membrane geometry possessing high-specific surface area,
resulted in 44.5% activity recovery (Ye et al., 2005). The good mechanical strength, well-controlled porosity, and
low recovery activity can be the result of poor loading or low fouling tendency with a self-supporting structure
denaturation of the enzyme on the membrane. Loading of is preferred as a carrier (Sulaiman et al., 2017). Easier
biocatalyst depends on factors, such as binding technique, modulation and low resistive flow pattern through the
enzyme concentration in solution, and parameters, such as membrane can elevate the efficiency of the operation.
time, temperature, and pH maintained during the immo- Likewise, the selected membrane should also fulfill the
bilization process. Enzyme loading increased initially, requirements of the juice clarification step. Hollow fiber
became maximum, and then reduced with an increase membrane consists of high surface-to-volume ratio with
in enzyme concentration, immobilization time, and pH. well-controlled porosity and excellent mechanical strength
Whereas, an increase in temperature reduced enzyme and found its application in the juice clarification pro-
loading on the membrane (Aghababaie et al., 2016; Akgöl cess (Akgöl et al., 2002; Deng et al., 2004; Ye et al., 2005).
et al., 2002; Kawakita et al., 2002; Taheran et al., 2017). From the late 1990s to the late 2000s, hollow fiber was
The change in immobilization temperature from 5 to 25◦ C foremost as a biomolecule immobilization carrier. How-
reduced enzyme loading of laccase to half, whereas opti- ever, the increasing trend of nanostructures, comparable
mum immobilization loading is obtained at around 4 h. features, and inexpensive cost of fabrication of nanofi-
However, immobilization time higher than 4 h lowered the brous membrane has reduced the demand for hollow
macromolecule loading on support (Taheran et al., 2017). fiber membrane as support. Nowadays, the nanofibrous
The properties of the membrane also affect the enzyme membrane is widely adopted in the enzyme immobiliza-
loading capacity. The enhanced specific surface area of tion research field (Çakıroğlu et al., 2018; Sulaiman et al.,
the membrane improves the enzyme loading, avoiding 2017; Taheran et al., 2017). Membrane immobilization is
the overcrowding of enzyme molecules compared to other also carried out upon a flat-sheet membrane. Flat-sheet
accommodators discussed earlier (Ye et al., 2005). The yielded lower loading capacity than hollow fiber mem-
surface area of the membrane is inversely proportional brane because of low accommodation area (Ganapathi
to membrane pore size. Smaller the pore size, higher et al., 1995). The tubular membrane has not been investi-
the surface area. However, a membrane having a pore gated broadly as an immobilization carrier. However, the
size smaller than an enzyme molecule may report lower tubular membrane is used extensively in the commercial
loading capacity. It can be attributed to inaccessibility of juice clarification process due to tangential flow, stable
pore void volume for enzyme accommodation. The mem- flux, and cleaning comfortability. But energy consumption
brane structure is also of great importance in enzyme is relatively higher compared to other membrane mod-
immobilization. Nishizawa et al. (2000) studied the effect ules (Bhattacharjee et al., 2017). Bhattacharjee et al (2017)
of membrane structure on enzyme immobilization and have enlightened concerning membrane modules in juice
concluded that symmetric membrane structure accom- processing applications.
modates a greater mass of biocatalysts. However, this The study reveals that the membrane provides a
cannot be compared to the fouling-based enzyme immo- better accommodation to enzymes enhancing the sta-
bilization technique as this technique mostly utilizes the bility and performance of the enzyme compared to the
asymmetric structure membrane (Luo et al., 2014). The solution-based biocatalytic process. The above discussion
topography of the membrane also plays a crucial role encourages the immobilization of pectinase enzymes on
in enzyme immobilization. Qiao et al. (2021) observed the membrane to synergize the chemical reaction and
that higher surface roughness provides better enzyme product separation steps. However, the changes observed
accommodation with improved enzymolysis efficiency. in enzymes properties due to immobilization cannot be
The hydrophobic/hydrophilic nature of the membrane sur- the same for pectinases as every enzyme responds differ-
face is also a point of consideration while immobilizing ently to the immobilization as its shape, structure, and
the enzyme. The nature of enzyme solution media (i.e., properties are distinct compared to other enzymes. Hence,
hydrophilic or hydrophobic) contradictory to nature of the discussion carried out may not fulfill all the criteria
membrane surface may affect the binding process leading for pectinase upon immobilization on the membrane.
However, the above analysis can pave the route that Abhishek S. Dhoble https://orcid.org/0000-0003-4889-
may lead to the stabilized reusable membrane-confined 0885
pectinase heterogeneous catalyst for juice clarification
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CONCISE REVIEWS & H YPOTH ESES IN FOOD SCIENCE

A review on pectinase properties, application in juice

Vashishtha B. Patel1 Somak Chatterjee1 Abhishek S. Dhoble2

1 Department of Chemical Engineering,

1 INTRODUCTION final product. Therefore, the raw extracted juice is clari-

3338 © 2022 Institute of Food Technologists.

71 1.25 55 3.5 55 2–7 Mn, Mg Ca, K, Na, Ni Yang et al. (2011)

5 PECTINASES IN JUICE PROCESSING 6 IMMOBILIZATION OF ENZYMES ON

TA B L E 2 Physicochemical and kinetic properties of enzymes immobilized on membranes

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