Accepted Manuscript

Characteristics of pectinase treated with ultrasound both during and after the
immobilization process
Xiaobin Ma, Danli Wang, Michelle Yin, Juliet Lucente, Wenjun Wang, Tian
Ding, Xingqian Ye, Donghong Liu
PII:
DOI:
Reference:

S1350-4177(16)30365-0
http://dx.doi.org/10.1016/j.ultsonch.2016.10.026
ULTSON 3410

To appear in:

Ultrasonics Sonochemistry

Received Date:
Revised Date:
Accepted Date:

13 July 2016
28 October 2016
28 October 2016

Please cite this article as: X. Ma, D. Wang, M. Yin, J. Lucente, W. Wang, T. Ding, X. Ye, D. Liu, Characteristics
of pectinase treated with ultrasound both during and after the immobilization process, Ultrasonics Sonochemistry
(2016), doi: http://dx.doi.org/10.1016/j.ultsonch.2016.10.026

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Characteristics of pectinase treated with ultrasound both during and

after the immobilization process

Xiaobin Ma a, Danli Wang a, Michelle Yin b, Juliet Lucente b, Wenjun Wang a, Tian
Ding a,d, Xingqian Ye a,c,d, Donghong Liu a,c,d,*

College of Biosystems Engineering and Food Science, Zhejiang University,

Hangzhou 310058, China

b

Department of Food Science, University of Illinois at Urbana-Chanpaign, Urbana

61801, America
c

Fuli Institute of Food Science, Zhejiang University, Hangzhou 310058, China

Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang R&D Center for Food

Technology and Equipment, Hangzhou 310058, China

* Corresponding author
Address: College of Biosystems Engineering and Food Science, Zhejiang University,
866 Yuhangtang Rd., Hangzhou 310058, China.
Tel.: +86 057188982169; Fax: +86 057188982144; E-mail: dhliu@zju.edu.cn

Abstract
In this study, ultrasound was applied both during and after the immobilization
process and characteristics of different immobilized pectinase samples were studied.
When introduced during the immobilization process, ultrasound at an intensity of 9 W
mL-1 for 20 min increased the immobilization yield 92.28% more than the control.
When introduced to the already immobilized pectinase, ultrasound at an intensity of
4.5 W mL-1 for 10 min increased the pectinase activity by 30.05%. Results of
scanning electron microscope demonstrated that ultrasound increased surface area and
loosened structures of immobilized enzymes. Higher Vmax and lower Km were
obtained after ultrasound treatment, indicating the increased catalytic efficiency and
enhanced affinity of immobilized pectinase. Furthermore, the optimum temperature
and pH for free and immobilized pectinase remained unchanged at 50 C and pH 4.
Thermostability, reaction stability and reusability of two ultrasound-treated pectinase
enzymes slightly decreased due to structural matrix changes.
Keywords: immobilization yield; catalytic activity; structure; enzymatic kinetics;
stability

1. Introduction
Pectin, a colloidal acid polysaccharide, is found in many waste products (such as
fruit peels and pomaces) [14] originating from fruit juice manufacturing and sugar
production. The presence of pectin increases the difficulty of waste treatments given
its degradation-resistance and high viscosity. Recently, enzymatic degradation is
starting to be applied in industrial processes, and pectinase is one of the most
commonly used enzymes [5]. Pectinases are a group of enzymes that catalyze the
breakdown of pectin and are useful in industrial terms such as extraction, waste
treatment, fermentation, and quite commonly used in fruit juice clarification [6]. They
have the potential to make an efficient conversion of waste pectin into smaller
molecules with high bioactivity, which is called the modified pectin [7]. Pectinase
naturally occurs as a free enzyme. Although free enzymes produce high catalytic
activity, they have low stability and cannot be reused. This increases the treatment
cost and restricts their applications [8,9]. For example, in certain industrial processes,
extreme values of pH, temperature, or reaction time can result in enzyme denaturation
or decreased output. This is why immobilized enzymes are introduced as another
aspect that those same industry operators use [10].
Immobilizing enzymes essentially allows the enzymes to locally stay in place;
therefore, removing its mobility region, while still maintaining its catalytic functions.
This certain confinement in turn results in another advantage of immobilization,
which is the increased, continuous reusability. As supported by Bashari et al. [11],
3

other advantages of immobilized enzymes include increased enzyme activity,

enantioselectivity, and ease of enzyme recovery after centrifugation or filtration.
Furthermore, it promotes multi-enzyme reactions, decreases industrial wastes, and
allows for more enzymatic processes to occur [12]. Immobilization of enzymes can be
brought out from many different methods such as entrapment, adsorption, or carrier
binding [9]. Amongst these, entrapment using calcium alginate beads as support is one
of the more effective approaches in biocompatibility, cost, and microbial
contamination resistance; especially when its cross-linked with glutaraldehyde, it
makes use of as much enzyme activity as possible [8].
Ultrasound is an environmentally-friendly technique and has been widely applied
in the modifications of food compositions [13]. Recently it has been used in the
improvement of several free enzymes, such as dextranase [14], alliinase [15],
cellulase [16,17], pectinase [18], Alcalase [19] and lipase [20], but its applications in
the immobilized enzymes has just started and very few studies [11,12,2124] have
been done into the effect of ultrasound on immobilization. In a study by Sharma et
al.[24], the immobilization time of horseradish peroxidase significantly decreased
from 4 h by conventional methods to 10 min by ultrasound treatment (40 kHz, 120 W).
In addition, ultrasound could also modify already immobilized enzymes. In a study by
Wang et al. [12], immobilized cellulase was researched under ultrasound treatment;
the use of ultrasound (60 W, 20 min) increased its catalytic activity by 24.67%.
Low-frequency and mild intensity ultrasound waves work by creating transient
cavitation effects, which generate mechanical and free radical effects. These
4

sono-effects directly change the structure of the enzymes to make them more
accessible for a reaction [11,25]. They can also reduce the resistance of the substrate
to diffuse in the matrix and the resistance of the enzyme to diffuse out, which increase
the mass transfer characteristics [26]. Thus, catalytic activity of the enzyme and the
immobilization process are enhanced with ultrasound treatment. However, intense
ultrasound conditions can lead to a decrease in the immobilization yield and enzyme
activity due to deactivation of the enzymes [12].
While some studies have researched the effects of ultrasound during or after the
immobilization process, to the best of our knowledge, this is the first study that has
compared these two effects together. As well, it is the first time, that we know of,
reporting the effects of ultrasound on the immobilization process for pectinases. In
this study, ultrasound was applied both during and after the immobilization of
pectinase. Microstructures, enzymatic kinetics and properties (including the optimum
temperature, optimum pH, thermostability, reaction stability and reusability) of the
immobilized pectinase with different treatments were studied. This study will
illuminate the mechanisms and provide the basis for the application of ultrasound in
the immobilization of enzymes.

2. Materials and Methods

2.1. Materials
Pectinase from Aspergillus niger and pectin from a citrus peel were purchased
from SigmaAldrich (Shanghai, China) and used without further purification. All
other reagents, including the sodium alginate, calcium chloride, glutaraldehyde and 3,
5

5-dinitrosalicylic acid (DNS), were of analytical grade.

2.2. Assay of pectinase activity
Pectinase activity was measured using the 3, 5-dinitrosalicylic acid (DNS)
method as described in detail in our previous studies [18,27]. One unit of pectinase
activity (U) is the amount of pectinase that releases 1 mol of galacturonic acid per
minute under the assay conditions.
2.3. Immobilization of pectinase without ultrasound treatment
Immobilization of pectinase was conducted using sodium alginate as a support
and glutaraldehyde as a cross-linking agent. Sodium alginate with a concentration of
2% was syringed dropwise into a 0.15 M CaCl2 solution and hardened for 20 min at
4 C. The cross-linked network was formed by immersing the beads into 3%
glutaraldehyde. Then, it was magnetic stirred for 2 h at room temperature. The
acquired beads were rinsed, dried and weighed. Next, 2 g of the cross-linked beads
were added to 20 mL of 0.5 mg mL-1 pectinase solution. Then, it was magnetic stirred
for 1 h at 30 C. To remove the unentrapped pectinase, the calcium alginate beads
were washed with deionized water and then preserved in water at 4 C. The resulting
immobilized pectinase was named immobilized pectinase A.
The immobilization yield of pectinase in the alginate supports was calculated by
the following equation according to Won et al. [28]:
(1)
where

and

represent the activity of the immobilized pectinase (U) and the

free pectinase (U), respectively.

6

During immobilization, different concentrations of sodium alginate (1%5%)

and CaCl2 (0.050.3 M) were applied to investigate the effects of sodium alginate and
CaCl2 on immobilization of pectinase. Also, pectinase was immobilized at different
times (560 min) to determine the optimal immobilization time.
2.4. Ultrasound treatment
A probe sonicator (JY92-IIDN, Ningbo Scientz Biotechnology Co., Ningbo,
China) with a maximum output power of 900 W and an operating frequency of 22
kHz was applied to treat the calcium alginate beads. During ultrasound, the cylindrical
glass reactor (inner diameter 2.77 cm) was placed in a low-temperature thermostatic
water bath (DC-1006, Safe Corporation, Ningbo, China) to control the temperature of
the samples at 30 C
2.4.1. Ultrasound treatment during the immobilization process of pectinase
The cross-linked beads were prepared as described in Section 2.3. Entrapment of
pectinase was carried out under ultrasound irradiation. Two grams of cross-linked
beads and 20 ml of 0.5 mg mL-1 pectinase solution were mixed in the reactor, and the
probe was immediately inserted (about 1 cm into the solution) to dissipate the
ultrasonic field at different intensities (2.2513.5 W mL-1) for different times (560
min). After treatments, the beads were immediately washed with deionized water to
remove the unentrapped pectinase, and the immobilization yield was measured to
determine the optimum operating conditions. The immobilized pectinase prepared
under the optimum ultrasound conditions was named immobilized pectinase B.
2.4.2. Ultrasound treatment after the immobilization process of pectinase
7

The immobilized pectinase A was prepared as described in Section 2.3 and

was then processed by ultrasound prior to enzymatic reactions. Two grams of the
Immobilized Pectinase A were transferred into 20 mL of deionized water in the
cylindrical glass reactor. The ultrasound probe was immediately inserted (about 1 cm
into the solution) to dissipate the ultrasonic field at different intensities (2.2513.5 W
mL-1) for different times (530 min). After treatments, the calcium alginate beads
were filtered out for enzyme activity determination. The immobilized pectinase
treated with the optimum ultrasound conditions was named immobilized pectinase
C.
2.5. Scanning electron microscope (SEM) analysis
The surface morphology of the calcium alginate beads was observed using the
SEM (SU-8010; HITACHI Corp., Japan) at an acceleration voltage of 15 kV. Four
samples (including the beads before the immobilization of pectinase and immobilized
pectinase A, B and C) were dried and sputter-coated with gold in order to be
conductive before collection of the micrographs.
2.6. Enzymatic kinetics study
Pectin was dissolved in 1 mol L-1 citric acid-phosphate buffer at a pH of 4 to
obtain the substrate solution with different initial concentrations (1.678.33 mg mL-1).
Enzymatic reactions were conducted with an equal amount of free pectinase and
immobilized pectinase A, B and C. Pectin was incubated with each enzyme sample in
a test tube at 30 C for 10 min, with the reaction rate measured. Kinetics parameters
(Km and Vmax) were obtained from LineweaverBurk plots.
8

2.7. Optimum temperature and pH of pectinase samples

To determine the optimum temperature for each enzyme sample, 0.5 g of
immobilized pectinase A, B and C (immersed in 1 mL of deionized water) and an
equal amount of free enzyme (1 mL) were incubated with 5 mL of 5 mg mL-1 pectin
for 10 min at a pH of 4 within the temperature range of 30 C to 70 C (at 10 C
intervals). To determine the optimum pH for pectinase, pectin was dissolved into a
citric acid-phosphate buffer at different pH values (26) and reacted with four enzyme
samples for 10 min at 30 C. For both reactions, the maximum enzyme activity in
each experimental group was designated as 100%.
2.8. Thermostability of pectinase samples
The thermostability of pectinase was studied by preserving four pectinase
samples at different temperatures ranging from 40 C to 60 C for different times
(1060 min). After heat preservation, enzyme activity of each pectinase sample was
measured at 30 C. The enzyme activity of the unheated pectinase was designated as
100%.
2.9. Reaction stability of pectinase samples
Equal amount of four pectinase samples were incubated with the substrate at
30 C for 10 min to 60 min with their residual enzyme activity measured every 10
min. The initial enzyme activities were determined by conducting the enzymatic
reaction at 1 min and were all defined as 100%.
2.10. Reusability of immobilized pectinase samples
The repeated hydrolysis of pectin using immobilized pectinase A, B and C was
9

conducted to measure the reusability of immobilized pectinase samples. In each

reaction cycle, 0.5 g of the immobilized pectinase was incubated with 5 mL of 5 mg
mL-1 pectin at 30 C for 30 min. After each batch, the calcium alginate beads were
immediately filtered out and washed with deionized water and the reactants were
collected for reducing sugar measurement. The enzyme activities of each immobilized
pectinase sample in the first run designated a relative activity of 100%.
2.11. Statistical analysis
The experiments were all done equally three times, while the data was
represented with a mean standard deviation. ANOVA (p < 0.05) and Duncans
multiple range tests were used with the SPSS 17.0 (SPSS Inc., Chicago, IL, USA) to
analyse the collected experimental data. All figures were generated with the Origin
Softward 8.5 (OriginLab Corp., MA, USA).

3. Results and Discussion

3.1. Effect of sodium alginate and calcium chloride concentrations on pectinase
immobilization
Fig. 1 shows the immobilization yield of the immobilized pectinase under
different concentrations of sodium alginate and CaCl2. The best concentrations for
sodium alginate and calcium chloride were 2% and 0.15 M, respectively. Sodium
alginate and calcium chloride were effectively used to immobilize enzymes because
they could react to form an insoluble calcium alginate gel-like matrix. This structure
increases the stability of the enzyme through entrapment of pectinase by affecting the
pore size of the beads. By using different concentrations of sodium alginate, the best
10

pore size that results in an easily diffusible substrate yet still retains the enzyme can
be tested. By using different concentrations of calcium chloride, the immobilized
enzymes activity and density will vary. However, too high concentrations of a
sodium alginate and calcium chloride solution produce smaller pore sizes, which
lower the immobilization yield efficiency, and too low of a concentration will produce
greater pore sizes and more enzyme leakage. Thus, Fig. 1 represents the
concentrations that produce the strongest formation of cross-linked alginate gels,
along with the protected entrapment of the enzyme [8,29,30].
3.2. Effect of ultrasound conditions on pectinase immobilization
The immobilization yield of pectinase with ultrasound treatment at different
intensities of 013.5 W mL-1 for 20 min is shown in Fig. 2 (a). The optimal ultrasound
intensity for pectinase immobilization was observed at 9 W mL-1, where the
immobilization yield significantly increased by 92.28% compared with the pectinase
immobilized using the routine entrapment method for 20 min. The ultrasound at 9 W
mL-1 provided enough radiation for the cavitation effect to take place; thus, it changed
the enzyme structure in way that improved the pectinase activity [11]. Furthermore,
low-intensity ultrasound allowed for a homogeneous system to occur [11]. The strong
acoustic streaming produced by cavitation enhanced mass transfer, which in turn
promoted the collision frequencies between the pectinase molecules and the
cross-linked calcium alginate beads [24], and improved the entrapment of enzyme
molecules onto the support. However, looking at Fig. 2 (a), the immobilization yield
at an intensity of 13.5 W mL-1 is shown to be lower than the control, indicating that
11

after exceeding a certain level of ultrasound irradiation both the free and entrapped
enzymes were destroyed. This can be attributed to the intense mechanical stress and
large amounts of free radicals generated from extreme ultrasound conditions leading
to pectinase inactivation. Also, at this level of ultrasound, it may increase the
vulnerability of previously entrapped enzymes to leak out due to the increased pore
size or damaged matrix.
Fig. 2 (b) illustrates the immobilization yield of pectinase with and without
ultrasound treatment at 9 W mL-1 for different immobilization times (060 min). For
the routine immobilization process, the immobilization yield of pectinase increased
with the increase in the immobilization time, and achieved its maximum value of
36.69% at 60 min. For the ultrasound-treated immobilization, with an increase in
sonication time, the immobilization yield increased to its maximum value of 50.42%
at 20 min, and then decreased with the prolonged ultrasound time; it even reached
below the control at 60 min, demonstrating the negative effects of prolonged
ultrasound exposure. As stated before, low-frequency and mild intensity ultrasound at
short bursts of time could increase the activity of the immobilized enzyme because of
the transient cavitation, but excessive ultrasound energy may inactivate pectinase and
destroy the matrix of immobilization beads. It is also noteworthy to see that
immobilization under ultrasound irradiation for 20 min even achieved higher
immobilization yield than using the routine entrapment method for 60 min. The
ultrasound used in the right doses has thus been proven to be a very efficient method
in achieving high immobilization yield in a shorter amount of time.
12

According to a previous study, the entrapment process of dextranase onto the

calcium alginate beads was improved under a milder ultrasonic field (40 W, 15 min)
[11]. In addition to the differences in the enzyme structures of dextranase and
pectinase, the more intense ultrasound conditions in this study may be due to the
existence of glutaraldehyde promoting the formation of the crosslinking network on
the surface of the beads, and thus increasing the mechanical strength of the supports,
which could provide more potent protection for pectinase.
3.3. Effect of ultrasound conditions on the activity of immobilized pectinase
Fig. 3 (a) depicts the activity of immobilized pectinase treated with ultrasound at
intensities of 013.5 W mL-1 for 15 min; and Fig. 3 (b) shows the pectinase activity at
4.5 W mL-1 of ultrasound intensity for different treatment times (030 min). As can be
seen, the optimum ultrasound condition for the highest pectinase activity was 4.5 W
mL-1 for 10 min, under which the enzyme activity increased by 30.05% compared
with the untreated immobilized pectinase. One reason for this was because ultrasound
opened up the matrix in a way that increased the surface area of the gel bead, allowing
more access to the enzymes active site, compared to the enzyme that was just
immobilized without any altering of its structure. Another reason was that mild
ultrasound could favorably unfold the pectinase structures and cause exposure of
more active sites, which leads to the enhanced activity of the embedded enzyme
molecules. However, too much ultrasound for too long of time destroyed the matrix
and active sites of the enzyme rather than helping to activate it [11,12]. Compared
with the ultrasound conditions used during the immobilization process (9 W mL-1, 20
13

min), both the intensity and the sonication time were milder. When ultrasound was
exerted during the immobilization process, it acted on the pectinase solution and the
cross-linked beads, and thus the attachment and leakage of pectinase molecules
co-existed in an equilibrium system. At the optimum ultrasound conditions (9 W mL-1,
20 min), the positive effects of ultrasound outweighed the negative effects more.
When ultrasound was exerted to the already immobilized pectinase, it acted on the
water and the cross-linked beads instead of the enzyme solution, therefore it lacked a
pectinase supplement in the system and thus the maximum ultrasonic effect was
achieved under milder conditions.
In a study by Wang et al. [12], ultrasound-treated free cellulase was compared to
that of immobilized cellulase with chitosan as a support. The free enzyme increased
dramatically in activity and peaked at 15 W and 10 min, while the immobilized
enzyme activity achieved its maximum value at 60 W and 20 min. It is noteworthy to
compare the two ultrasound conditions and to see the kind of improved effect
immobilization has by protecting the enzyme and improving the stability. However,
compared with our previous work [18], in which ultrasound was introduced to the free
pectinase and the maximum enzyme activity was obtained at 4.5 W mL-1 for 15 min,
the optimum ultrasound conditions in this study (4.5 W mL-1, 10 min) was slightly
milder. This can be attributed to the changes of network structures and the consequent
leakage of pectinase when ultrasound was exerted on the immobilized pectinase.
Longer ultrasound duration caused more leakage of pectinase, which offset parts of
the positive changes in pectinase activity, and thus led to the optimum conditions
14

occurring at a shorter ultrasound duration.

3.4. Scanning electron microscopy analysis of immobilized pectinase
Fig. 4 shows the SEM of the surface morphologies of the cross-linked calcium
alginate beads with and without immobilized pectinase. Microstructures of three
immobilized pectinases, including the pectinase immobilized using routine methods
for 1 h (immobilized pectinase A), pectinase immobilized with ultrasound treatment at
9 W mL-1 for 20 min (immobilized pectinase B) and immobilized pectinase A with
ultrasound treatment at 4.5 W mL-1 for 10 min (immobilized pectinase C) were tested
and illustrated in Fig. 4 (b), (c) and (d), respectively. The blank calcium alginate beads
can be seen to have a dense, tight, orderly network (Fig. 4 (a)). However, when
enzymes were added into the beads, they were densely dispersed onto the gel network
throughout the surface of the beads. In the immobilized pectinase A (Fig. 4 (b)), the
enzymes were obviously shown, but there was not too significant of a change
compared to the control calcium alginate beads, aside from the added enzyme
molecules and slightly ruffled surroundings around the enzyme. This was in
accordance with the previously reported SEM of immobilized pectinase onto the
calcium alginate beads [8].
When the immobilized enzymes were introduced to ultrasound in pectinase
enzyme B and C, shear forces and free radicals generated from the cavitation effect
altered the surface structures of the matrix and there were thus much more changes
than compared to enzyme A. Enzyme B was treated with more intense ultrasound
conditions than enzyme C so when compared to enzyme A, enzyme B showed greater
15

gaps, surface changes, and openings (Fig. 4 (c)), while enzyme C showed the
relatively same network, except with looser surface folds and less wide etchings (Fig.
4 (d)). Furthermore, there were obviously more enzyme molecules attached to enzyme
B than compared with enzyme A, which demonstrated the higher immobilization yield
under an ultrasonic field. In addition to the SEM images, diameters of four alginate
beads were also measured using a vernier caliper. The average diameters of the blank
bead, enzyme A, enzyme B and enzyme C were 3.1, 3.1, 3.3, 4.0 mm, respectively.
The enzyme C went through an extra water-swelling process and thus had a larger
size. In a study by Huang et al. [31], ultrasound was exerted to the entrapped casein
and proved to loosen the structures of the alginate beads by the increased effective
diffusion coefficient. These results were similar to the present study. In conclusion,
observations of the ultrasound-effected enzymes showed an expansion of the alginate
beads with the increased surface area and larger pores in the surface, which were very
helpful to the exposure of pectinase during enzymatic reactions and thus could be an
explanation of the enhanced immobilization yield and increased enzyme activity
under ultrasound irradiation [12].
3.5. Enzymatic kinetics of free and immobilized pectinase
The LineweaverBurk plots for free and immobilized enzymes are shown in Fig.
5. The correlation coefficients for the free pectinase, immobilized pectinase A, B and
C were 0.9837, 0.9778, 0.9989 and 0.9977, respectively. Table 1 lists the maximum
velocity of the reaction (Vmax), the Michalis constant (Km) and the catalytic efficiency
(Vmax / Km). As can be seen, the immobilized enzymes with and without ultrasound
16

treatment had lower Vmax and Vmax / Km and higher Km compared with the free
pectinase, which demonstrated the decreased catalytic efficiency and lower affinity of
pectinase after immobilization. Similar variation trends of enzymatic parameters were
also observed for an alkaline pectinase immobilized onto the calcium alginate beads
[8]. In the enzymatic reactions catalyzed by the immobilized pectinase, the substrate
required more time to infiltrate the enzyme because the gel matrix protects the
enzyme compared with the free enzyme without the coating. The slower diffusion rate
led to high product concentrations surrounding the gel beads accounting for the high
Km value [11].
The two ultrasound-treated samples (enzymes B and C) had higher Vmax and
Vmax / Km and lower Km compared with the untreated immobilized enzyme sample
(enzyme A). This can be explained by the alteration in the structures of gel matrix as
mentioned above. The calcium alginate beads puffed up with larger pores in its
surface under an ultrasonic field, which allowed for substrate to transport more easily
into the matrix and reach the active sites of the enzymes. Furthermore, ultrasound
could also favorably change the structures of pectinase and in turn cause the enzyme
to change to a structure with more exposed active sites [18,27]. There were little
differences in the kinetics parameters of enzyme B and C, which suggested a similar
effect of ultrasound whether being exerted during or after the immobilization process.
3.6. Optimum temperature of free and immobilized pectinase
The enzyme activity of free and immobilized pectinase A, B and C tested in the
temperature range of 30 to 70 C is shown in Fig. 6 (a). Although some references
17

[8,11,12,32] reported a shift in optimum temperature after immobilization of the

enzymes, there were also others that reported no change happened [33,34]. For
example one study found that the optimum temperatures for free pectinase and its
immobilized form onto amphiphilic PS-b-PAA diblock copolymers were both at
60 C [33]. In this study, the optimum temperature for free and immobilized pectinase
remained unchanged at 50C, however aside from the optimum levels, the relative
activity of the three immobilized enzymes were higher throughout than the free
enzyme. For example, at 70 C free pectinase only maintained 12.59% of its
maximum activity, while the relative activities of immobilized pectinase A, B and C
were 51.92%, 57.25% and 52.04%, respectively. The gel matrix entrapped the enzyme
preventing it from leaking out and being exposed to temperature stresses; the matrix
itself can withhold thermal stability, which protected the enzyme from denaturation.
Furthermore, enzymes B and C showed higher catalytic activity at each temperature
compared to enzyme A, which demonstrated that mild ultrasound conditions yielded
more readily available enzymes.
3.7. Optimum pH of free and immobilized pectinase
The enzyme activity of free and immobilized pectinase A, B and C tested in the
pH range of 2 to 6 is shown in Fig. 6 (b). The optimum pH for free and immobilized
pectinase remained unchanged at pH 4. Similar to the optimum temperature, both the
changed [11,12] and unchanged [8,33] optimum pH for enzymes after immobilization
were reported before. Rehman et al. [8] found that the maximum activity for
immobilized and free pectinase enzymes were both achieved at a pH of 10. Looking
18

at the graph, relative activity of three immobilized pectinase obviously increased more
than that of the free enzyme in the tested pH range, especially in higher pH values.
Furthermore, the two ultrasound-treated enzyme samples even showed higher relative
activity than the untreated immobilized pectinase. The relative activity of enzyme B
can be seen at pH 5 achieved at 99.88%, which was almost the same as its optimum
pH activity. In another study by Bashiri et al. (Bashari et al., 2013), the maximum
activity of the free dextranase was found at pH 5, while the immobilized enzymes
shifted to pH 6. Paralleling to our findings, enzyme B had highest activity at pH 4 and
5, meaning the stability of the calcium alginate gel network along with the ultrasound
immobilization influences allowed the enzyme to be more tolerable to high pH
environments [8,11,12].
3.8. Thermostability of free and immobilized pectinase
Residual activities of free and immobilized pectinase samples at temperatures of
40 C to 60 C for 10 min to 60 min are shown in Fig. 7. It has been observed that the
thermostability of the three immobilized pectinase enzymes significantly increased
compared to the free. For example, the residual activity for free pectinase preserved
for 60 min at 40 C, 50 C and 60 C were 31.98%, 19.90% and 6.57%, respectively,
while for immobilized pectinase A, they were 67.42%, 35.04% and 19.71%,
respectively. This confirmed the improvement that immobilizing enzymes can bring
through the protection given by the calcium alginate network. With the enzyme being
molecularly confined within this entrapment gel, the enzyme location was
immobilized to generally fend off changes from the microenvironment up to a certain
19

level [12]. Furthermore, the thermostability of pectinase enzymes B and C were a bit
lower than pectinase enzyme A. Ultrasound changed the surface morphologies of the
calcium alginate beads, as can be observed in the SEM, causing more potential areas
of leakage and increased alterations infiltrated from the microenvironment. Therefore,
this suggests that ultrasound power still cannot be used in high amounts to yield
higher enzyme activity because it may destroy the cross-linked enzymes and lead to
low stability. However, it still benefits the immobilization enzymes by increasing the
immobilization yield and enzyme activity.
3.9. Reaction stability of free and immobilized pectinase
Fig. 8 shows the reaction stability of free and immobilized pectinase samples at
30 C for 10 min to 60 min. The reaction stability of pectinase was remarkably
improved after immobilization. For example, the residual activity of the free enzyme
and immobilized pectinase A at a reaction time of 60 min retained 28.73% and
42.60% of the initial activity, respectively. This increase in stability can be accounted
for by the calcium alginate beads protecting the enzyme molecule, making it less
susceptible to external environment and thus was more stable during reaction. On the
other hand, the two ultrasound-treated samples showed a slight decrease in reaction
stability compared to the immobilized pectinase without ultrasound treatment. This
can be explained by the structural alterations of gel matrix under ultrasound
irradiation. As stated before, the cross-linkages of enzymes B and C were partly
destroyed by ultrasound and the appearance of wide gaps and larger pores in the
surface of alginate beads caused the enzymes to be more prone to exposure. However,
20

more activity decreased its stability at the same time because it was not as protected.
Previous studies [35,36] also reported the physical damages of immobilization
supports under ultrasound irradiation. Furthermore, ultrasound could also change the
internal bonds of enzyme molecules and result in the decreased stability of the
embedded enzymes [22,37]. This explains why the ultrasound-treated enzymes were
slightly less stable compared to the immobilized enzyme without ultrasound treatment.
But this decline in the enzyme stability was slight and the ultrasound-treated enzymes
still had a stable structure overall.
3.10. Reusability of immobilized pectinase
The reaction activities of immobilized pectinase A, B and C for 6 cycles of batch
experiments are shown in Fig. 9. On the second cycle, the residual activity of
enzymes A, B and C all maintained above 80% of their initial activity, but the relative
activity of these enzyme samples dropped to just 32.28%, 25.77% and 22.02%,
respectively, on their sixth cycle. There were not many differences in the relative
activity of the three immobilized enzymes during the first four cycles, but enzymes B
and C showed an obviously lower activity than enzyme A in the last two cycles. These
coincide with the same reasons as discussed in Section 3.8 and 3.9. Ultrasound
treatment destabilized the structures of the matrix and caused easier leakage of
enzyme molecules, and thus slightly decreased the reusability of the immobilized
enzyme. Nevertheless, immobilized enzymes can be re-used and have overall
long-term stability when compared to free enzymes, which makes them more
applicable to industrial processes.
21

3.11. Prospective of ultrasound immobilization applied in the industry

Ultrasound proves an effective method in enzyme immobilization; it significantly
shortens the immobilization duration while increasing the immobilization yield.
Although ultrasound-promoted immobilization of enzymes still stays in the
experimental stage, it has the potential to be industrialized given the decreased
processing cost and the enhanced production efficiency. In fact, there have been
several instrument corporations designing large-scale ultrasound devices that can be
used in industrial scales. According to Chemat et al. [38], Hielscher Company
(www.hielscher.com, Germany) has devised ultrasound apparatuses with the output
power ranging from 500 to 16,000 W, which could perfectly meet the requirement of
the ultrasound conditions used in this work. There are also some ultrasonic bath
apparatuses developed for industrial uses [39]. However, recent studies on enzyme
immobilization mainly use an ultrasonic probe and therefore, applications of
ultrasonic bath still need more research basis.
On the other hand, in this study ultrasound was also applied to the already
immobilized enzymes to produce higher catalytic activity. When compared with the
ultrasound-assisted immobilization, conditions used in this process are milder and
thus more conducive to saving energy; however, the method adds an extra step and
therefore is time-costing and less efficient. Overall, both methods could be applied in
large scales and choices mainly depend on the species of enzymes and the specific
operating conditions.

22

4. Conclusions
In this study, ultrasound was applied both during and after pectinase
immobilization. The immobilization yield and enzyme activity peaked at 9 W mL-1
intensity for 20 min and 4.5 W mL-1 intensity for 10 min, respectively. Results of
SEM showed an expanded and loosened matrix structure after ultrasound.
Furthermore, ultrasound increased the Vmax but decreased the Km of immobilized
pectinase, indicating that higher catalytic efficiency and enhanced affinity were
achieved. Immobilization and ultrasound did not change the optimum temperature and
pH for pectinase. Thermostability, reaction stability and reusability of the
ultrasound-treated pectinase slightly decreased due to structural matrix changes.

Acknowledgements
This work was financially supported by National Natural Science Foundation of
China (Project 31371872).

References
[1] J. Li, P. Zhou, H. Liu, C. Xiong, J. Lin, W. Xiao, Y. Gong, Z. Liu, Synergism of
cellulase, xylanase, and pectinase on hydrolyzing sugarcane bagasse resulting
from different pretreatment technologies, Bioresource Technol. 155 (2014)
258265.
[2] M. Boukroufa, C. Boutekedjiret, L. Petigny, N. Rakotomanomana, F. Chemat,
Bio-refinery of orange peels waste: A new concept based on integrated green and
solvent free extraction processes using ultrasound and microwave techniques to
obtain essential oil, polyphenols and pectin, Ultrason Sonochem. 24 (2015) 7279.
23

[3] L. Zhang, X. Ye, T. Ding, X. Sun, Y. Xu, D. Liu, Ultrasound effects on the
degradation kinetics, structure and rheological properties of apple pectin, Ultrason
Sonochem. 20 (2013) 222231.
[4] I. G. Moorthya, J. P. Maranb, S. Ilakyaa, S. L. Anithaa, S. P. Sabarimaa, B. Priyac,
Ultrasound assisted extraction of pectin from waste Artocarpus heterophyllus fruit
peel, Ultrason Sonochem. 34 (2017) 525530.
[5] D. R. Kashyap, P. K. Vohra, S. Chopra, R. Tewari, Applications of pectinases in
the commercial sector: a review, Bioresource Technol. 77 (2001) 215227.
[6] B. R. Thakur, R. K. Singh, A. K. Handa, Chemistry and uses of pectin - A review,
Crit Rev Food Sci. 37 (1997) 4773.
[7] E. G. Maxwell, N. J. Belshaw, K. W. Waldron, V. J. Morris, Pectin - An emerging
new bioactive food polysaccharide, Trends Food Sci Tech. 24 (2012) 6473.
[8] H. U. Rehman, A. Arnan, A. Silipo, S. A. Ul Qader, A. Molinaro, A. Ansari,
Degradation of complex carbohydrate: Immobilization of pectinase from Bacillus
licheniformis KIBGE-IB21 using calcium alginate as a support, Food Chem. 139
(2013) 10811086.
[9] S. Chauhan, A. Vohra, A. Lakhanpal, R. Gupta, IMMOBILIZATION OF
COMMERCIAL PECTINASE (POLYGALACTURONASE) ON CELITE AND
ITS APPLICATION IN JUICE CLARIFICATION, J. Food Process Pres. 39
(2015) 21352141.
[10] M. N. Islam, M. Zhang, B. Adhikari, The Inactivation of Enzymes by
Ultrasound-A Review of Potential Mechanisms, Food Rev Int. 30 (2014) 121.
24

[11] M. Bashari, P. Wang, A. Eibaid, Y. Tian, X. Xu, Z. Jin, Ultrasound-assisted

dextranase entrapment onto Ca-alginate gel beads, Ultrason Sonochem. 20 (2013)
10081016.
[12] Z. Wang, X. Lin, P. Li, J. Zhang, S. Wang, H. Ma, Effects of low intensity
ultrasound on cellulase pretreatment, Bioresource Technol. 117 (2012) 222227.
[13] D. Pingret, G. Durand, A. Fabiano-Tixier, A. Rockenbauer, C. Ginies, F. Chemat,
Degradation of Edible Oil during Food Processing by Ultrasound: Electron
Paramagnetic Resonance, Physicochemical, and Sensory Appreciation, J. Agr
Food Chem. 60 (2012) 77617768.
[14] M. Bashari, A. Eibaid, J. Wang, Y. Tian, X. Xu, Z. Jin, Influence of low
ultrasound intensity on the degradation of dextran catalyzed by dextranase,
Ultrason Sonochem. 20 (2013) 155161.
[15] J. Wang, Y. Cao, B. Sun, C. Wang, Y. Mo, Effect of ultrasound on the activity of
alliinase from fresh garlic, Ultrason Sonochem. 18 (2011) 534540.
[16] P. B. Subhedar, P. R. Gogate, Enhancing the activity of cellulase enzyme using
ultrasonic irradiations, J. Mol Catal B-Enzym. 101 (2014) 108114.
[17] A. L. Prajapat, P. B. Subhedar, P. R. Gogate, Ultrasound assisted enzymatic
depolymerization of aqueous guar gum
solution, Ultrason Sonochem. 29 (2016) 8492.
[18] X. Ma, W. Wang, M. Zou, T. Ding, X. Ye, D. Liu, Properties and Structures of
Commercial Polygalacturonase with Ultrasound Treatment: Role of Ultrasound in
Enzyme Activation, Rsc Adv. 5 (2015) 107591107600.
25

[19] H. Ma, L. Huang, J. Jia, R. He, L. Luo, W. Zhu, Effect of energy-gathered

ultrasound on Alcalase, Ultrason Sonochem. 18 (2011) 419424.
[20] S. H. Jadhav, P. R. Gogate, Intensification in the Activity of Lipase Enzyme
Using Ultrasonic Irradiation and Stability Studies, Ind Eng Chem Res. 53 (2014)
13771385.
[21] B. Wang, T. Meng, H. Ma, Y. Zhang, Y. Li, J. Jin, X. Ye, Mechanism study of
dual-frequency ultrasound assisted enzymolysis on rapeseed protein by
immobilized Alcalase, Ultrason Sonochem. 32 (2016) 307313.
[22] K. C. Badgujar, P. A. Pai, B. M. Bhanage, Enhanced biocatalytic activity of
immobilized Pseudomonas cepacia lipase under sonicated condition, Bioproc
Biosyst Eng. 39 (2016) 211221.
[23] Y. Wang, Y. Hasebe, Tyrosinase-modified carbon felt-based flow-biosensors:
The role of ultra-sonication in shortening the enzyme immobilization time and
improving the sensitivity for p-chlorophenol, JOURNAL OF ENVIRONMENTAL
SCIENCES. 23 (2011) 10381043.
[24] P. Sharma, D. K. Kannoujia, S. F. Basir, P. Nahar, Rapid Immobilization of
Enzymes onto Solid Supports by Ultrasound Waves, ARTIFICIAL CELLS
BLOOD SUBSTITUTES AND BIOTECHNOLOGY. 39 (2011) 289292.
[25] X. Ma, W. Wang, D. Wang, T. Ding, X. Ye, D. Liu, Degradation kinetics and
structural characteristics of pectin under simultaneous sonochemical-enzymatic
functions, Carbohyd Polym. 154 (2016) 176185.
[26] L. Batistella, M. K. Ustra, A. Richetti, S. B. C. Pergher, H. Treichel, J. V.
26

Oliveira, L. Lerin, D. de Oliveira, Assessment of two immobilized lipases activity

and stability to low temperatures in organic solvents under ultrasound-assisted
irradiation, Bioproc Biosyst Eng. 35 (2012) 351358.
[27] X. Ma, L. Zhang, W. Wang, M. Zou, T. Ding, X. Ye, D. Liu, Synergistic Effect
and Mechanisms of Combining Ultrasound and Pectinase on Pectin Hydrolysis,
Food Bioprocess Tech. 9 (2016) 12491257.
[28] K. Won, S. Kim, K. J. Kima, H. W. Park, S. J. Moon, Optimization of lipase
entrapment in Ca-alginate gel heads, Process Biochem. 40 (2005) 21492154.
[29] Z. Bibi, S. A. Ul Qader, A. Aman, Calcium alginate matrix increases the stability
and recycling capability of immobilized endo-beta-1,4-xylanase from Geobacillus
stearothermophilus KIBGE-IB29, Extremophiles. 19 (2015) 819827.
[30] S. Geethanjali, A. Subash, Optimization and Immobilization of Purified Labeo
rohita Visceral Protease by Entrapment Method., Enzyme research. 2013 (2013)
874050.
[31] Z. Huang, Y. Cao, D. Xu, C. Wang, D. Zhang, Effect of ultrasound on the
diffusion properties of casein entrapped in alginate-chitosan gel, Ultrason
Sonochem. 26 (2015) 149156.
[32] M. A. Abdel-Naby, A. Ismail, A. M. Abdel-Fattah, A. F. Abdel-Fattah,
Preparation and some properties of immobilized Penicillium funiculosum 258
dextranase, Process Biochem. 34 (1999) 391398.
[33] Z. Lei, S. Bi, Preparation and properties of immobilized pectinase onto the
amphiphilic PS-b-PAA diblock copolymers, J. Biotechnol. 128 (2007) 112119.
27

[34] Z. Lei, Q. Jiang, Synthesis and Properties of Immobilized Pectinase onto the
Macroporous Polyacrylamide Microspheres, J. Agr Food Chem. 59 (2011)
25922599.
[35] N. Paludo, J. S. Alves, C. Altmann, M. A. Z. Ayub, R. Fernandez-Lafuente, R. C.
Rodrigues, The combined use of ultrasound and molecular sieves improves the
synthesis of ethyl butyrate catalyzed by immobilized Thermomyces lanuginosus
lipase, Ultrason Sonochem. 22 (2015) 8994.
[36] G. V. Waghmare, M. D. Vetal, V. K. Rathod, Ultrasound assisted enzyme
catalyzed synthesis of glycerol carbonate from glycerol and dimethyl carbonate,
Ultrason Sonochem. 22 (2015) 311316.
[37] M. Zheng, L. Wang, F. Huang, P. Guo, F. Wei, Q. Deng, C. Zheng, C. Wan,
Ultrasound irradiation promoted lipase-catalyzed synthesis of flavonoid esters with
unsaturated fatty acids, J. Mol Catal B-Enzym. 95 (2013) 8288.
[38] F. Chemat, N. Rombaut, A. Sicaire, A. Meullemiestre, A. Fabiano-Tixier, M.
Abert-Vian, M. Abert-Vian, Ultrasound assisted extraction of food and natural
products. Mechanisms, techniques, combinations, protocols and applications. A
review, Ultrason Sonochem. 34 (2017) 540560.
[39] S. Achat, V. Tomao, K. Madani, M. Chibane, M. Elmaataoui, O. Dangles, F.
Chemat, Direct enrichment of olive oil in oleuropein by ultrasound-assisted
maceration at laboratory and pilot plant scale, Ultrason Sonochem. 19 (2012)
777786.

28

Figure Captions

Fig. 1. (a) Effect of concentrations of sodium alginate on the immobilization yield of

immobilized pectinase (concentration of calcium chloride: 0.2 M; immobilization
time: 1 h). (b) Effect of concentrations of calcium chloride on the immobilization
yield of immobilized pectinase (concentration of sodium alginate: 2%; immobilization
time: 1 h).
Fig. 2. (a) Effect of ultrasound intensity on the immobilization yield of immobilized
pectinase (ultrasound time: 20 min). (b) Effect of treatment time on the
immobilization yield of immobilized pectinase (ultrasound intensity: 9 W mL-1).
Fig. 3. (a) Effect of ultrasound intensity on the enzyme activity of immobilized
pectinase (ultrasound time: 15 min). (b) Effect of treatment time on the enzyme
activity of immobilized pectinase (ultrasound intensity: 4.5 W mL-1).
Fig. 4. Scanning electron microscope (SEM) of the surface morphologies of (a)
calcium alginate beads before immobilization, (b) immobilized pectinase A, (c)
immobilized pectinase B and (d) immobilized pectinase C.
Fig. 5. Plots of the initial velocity values obtained as a function of the correspondent
substrate concentration values using Lineweaver-linearization for free and
immobilized pectinase samples with and without ultrasound treatments.
Fig. 6. Effect of (a) temperature and (b) pH and on the relative activity of free and
immobilized pectinase samples with and without ultrasound treatments.
Fig. 7. Thermostability of (a) free pectinase, (b) immobilized pectinase A, (c)
immobilized pectinase B and (d) immobilized pectinase C.
Fig. 8. Reaction stability of free and immobilized pectinase samples with and without
ultrasound treatments.
Fig. 9. Reusability of the immobilized pectinase samples with and without ultrasound
treatments.

29

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Table 1
Kinetics parameters of free and immobilized pectinase samples with and without
ultrasound treatments (mean SD).
Vmax
Km
Vmax / Km
Samples
-1
-1
-1
(mg mL min ) (mg mL )
(min-1)
Free pectinase
1.58 0.01 a
3.17 0.04 b
0.50 0.01 a
Immobilized pectinase A 1.08 0.01 c
3.42 0.09 a
0.31 0.00 c
b
a, b
Immobilized pectinase B 1.18 0.01
3.24 0.06
0.36 0.00 b
Immobilized pectinase C 1.14 0.00 b
3.28 0.02 a, b
0.35 0.00 b
Note: values with different superscript letters (ac) in the same column within each
pectin indicate significant differences as estimated by Duncans multiple range test (P
< 0.05).

30

Highlights
1.
2.
3.
4.
5.

Mild ultrasound enhanced the immobilization yield of immobilized pectinase.

Mild ultrasound improved the catalytic activity of immobilized pectinase.
Ultrasound increased surface area and loosened structure of immobilized
pectinase.
The Vmax increased but the Km decreased after ultrasound treatment.
Immobilization did not change the optimum temperature and pH for pectinase.

31