Received: 28 July 2020 Revised: 30 November 2020 Accepted: 7 December 2020

DOI: 10.1002/kin.21468

ARTICLE

Purification, characterization, immobilization and kinetic

studies of catalase from a novel source Sechium edule

Nene Takio Meera Yadav Mridusmita Barman Hardeo Singh Yadav

Department of Chemistry, North Eastern

Regional Institute of Science and
Technology, Nirjuli, Itanagar, India
Correspondence
Meera Yadav, Department of Chemistry,
North Eastern Regional Institute of Sci-
ence and Technology, Nirjuli, Itanagar 791
109, India.
Email: drmeerayadav@rediffmail.com
Abstract
A catalase from a novel source Sechium edule (squash) has been purified to
homogeneity using ammonium sulfate fractional precipitation, dialysis, and
anion exchange chromatography on Diethylaminoethyl (DEAE) cellulose in
sodium acetate buffer pH 6.0. The purity of the enzyme was analyzed by SDS-
PAGE. The molecular weight of the enzyme using the SDS-PAGE method was
found to be 55 kDa with specific activity 6.35 U/mg. The molecular weight of
the enzyme was further confirmed by the INTACT mass spectrometric technique
and found to be 52.5 kDa. The Reinheitzahl (Rz) value of the enzyme was 1.5. The
MALDI-TOF analysis of the purified enzyme showed that it contains 529 amino
acid residues. The Km , Vmax, optimum pH, and optimum temperature of the free
enzyme using H2 O2 as the substrate were found to be 0.03 mM, 200 μmol/min,
7.6, and 24◦ C, respectively. The purified enzyme was immobilized on chitosan
beads which was prepared by extracting the chitosan from Cornu aspersum (gar-
den snail) having highest degree of deacetylation%. The kinetic characteristics,
Km , Vmax, optimum pH, and optimum temperature of the immobilized cata-
lase were found to be 0.065 mM, 250 μmol/min, 7.6, and 41◦ C, respectively. The
immobilized catalase is more thermostable in comparison to free catalase.

KEYWORDS
catalase, chitosan, immobilization, purification, thermostability

1 INTRODUCTION The first stage involves oxidation of the heme iron by

H2 O2 substrate to form the oxyferryl group with a π-
Catalase (CAT) (oxidoreductase, EC 1.11.1.6) first isolated cationic porphyrin radical (Cpd I) with displacement of
from bovine liver is a metalloenzyme localized in peroxi- water. In the second stage, Cpd I reacts with another H2 O2
somes, and it degrades hydrogen peroxide into water and molecule to release the original hemenzyme, H2 O and O2 .5
oxygen.1-4 CAT is a tetrameric heme protein with molecu- CAT undergoes several reactions with compounds like
lar weight around 240 kDa and possesses one of the highest cyanides, sulfides, halogens and also participate in hydrox-
turnover rates of enzymes. Dismutation of H2 O2 occurs via ylation of aromatic compounds, desulfurization, epoxi-
a two-stage degradation mechanism: dation, and oxidation of nitric oxide and so on.6 CAT
( III
) has been isolated and studied from animals, bacterial,
H2 O2 + Enzyme Por-Fe
( +∙ IV ) and fungal sources whereas well-characterized CAT from
→ H2 O + 𝐂𝐩𝐝𝐈
( Por -Fe )= O plants sources are rare.7–9 The CATs are biotechnologi-
(1)
H2 O2 + 𝐂𝐩𝐝𝐈 Por+∙ -Fe
(
IV = O
) cally important enzymes used in numerous applications
→ H2 O + Enzyme Por-FeIII + O2 . in bioremediation of organic pollutants such as phenols,

596 © 2020 Wiley Periodicals LLC wileyonlinelibrary.com/journal/kin Int J Chem Kinet. 2021;53:596–610.
TAKIO et al. 597

removal of H2 O2 residues in textile industries, preservation

of food, drug delivery, and in analytical field as a biosensor,
micromotors etc. However, the use of soluble enzyme is
limited due to denaturation, instability, high cost, and non-
reusability. The immobilization of enzyme on solid sup-
ports and using it in a column, enhances its reusability,
and thermal stability and reduces the cost of the industrial
process.10 This is the reason why immobilized enzymes are
preferred in industries.
CAT was immobilized on various organic and inor-
ganic supporting materials like cellulose, dextran, chi-
tosan, agarose, collagen, polystyrene, carbon nanotubes,
magnesium silicate, gold, hybrid support, eggshell and
were characterized and studied its performance in terms
of applications.11 Immobilization of CAT has been studied
from few plants species, but so far no report has been dis-
cussed from chayote squash.
Keeping this point in view, the authors have isolated
CAT from a novel source Sechium edule (squash) to homo-
geneity and determined its enzymatic characteristics like
molecular weight, Km , Vmax , pH, and temperature optima.
The enzyme has been immobilized on chitosan beads, and
the enzymatic characteristics of the immobilized enzyme
were determined. It was observed that the activity and ther-
mal stability of the immobilized enzyme were enhanced
without changing the pH optima of the enzyme. The
results are reported in this communication. This character-
istic features provide advantages over soluble CAT which Deacetylation of obtained
makes it an important industrial tool to overcome chal-
lenges of biocatalyst applications.

2 MATERIALS AND METHODS

2.1 Chemicals

Chemicals includes DEAE-cellulose from Sigma Aldrich,

and protein markers for SDS-PAGE analysis was purchased
from Bangalore Genei Pvt. Ltd. All other chemicals were F L O W C H A R T 1 Flowchart of preparation of chitosan from
either from Merck or SD fine Co. Ltd. animal wastes. RT: room temperature [Color figure can be viewed at
wileyonlinelibrary.com]

2.2 Enzyme purification
Screening was done for CAT activity in vegetable fruits col-
lected from a local market, and it was found that Sechium
edule (squash) possessed the highest CAT activity among
all fruits collected. A screening chart is not shown. The
process of purification proceeded with the isolation of
enzyme from the plant Sechium edule. The fruit was cut
and crushed into tiny pieces by mortar and pestle, and juice
was extracted by filtering crushed pieces into four layers
of cheese cloth. The extracted juice was centrifuged using
a Sigma model 3K-30 refrigerated centrifuge at 4000 g for
20 min at 4◦ C to remove cloudiness and obtain a clear solu-
tion. The steps involved in purification of enzyme is given
in Flowchart 1.
2.3 Ammonium sulfate precipitation
The supernatant collected after centrifugation was concen-
trated by ammonium sulfate precipitation method. One
milliliter of of the centrifuged enzyme was collected in
598
each of 10 Effendorf tubes marked from 10 to 100% ammo-
nium sulfate distribution with reference to Wood.12 After
addition of ammonium sulfate in each tube, the mixture
was kept for 1 h at constant stirring and checked for enzy-
matic activity in a UV spectrophotometer. The saturation
of proteins was observed at 80%, and finally the ammo-
nium sulfate mixture of 80%, that is, 568 g was added in
1000 mL enzyme solution and left overnight for complete
precipitation of enzymes.
2.4 Dialysis
The precipitated enzymatic solution (10 mL) obtained from
above method was loaded into a semipermeable dialyzing
membrane and was placed in a dialyzing 10 mM sodium
acetate buffer (pH 7.0) with constant stirring with three
changes of buffer at 24 h of interval. The final volume
of dialyzed solution and dialysate determine the equilib-
rium concentration of small molecules on both sides of the
membrane.
2.5 Ion exchange chromatography
The dialyzed crude extract (13 mL) was loaded into an
anion exchanger DEAE-cellulose previously equilibrated
with 10 mM sodium acetate buffer (pH 6.0) at a flow
rate of 16 mL/H and was washed with 100 mL of same
buffer to remove unbound proteins. The protein was eluted
with NaCl linear gradient (0.01–0.2 M) in the same buffer.
The collected fractions (30.0 mL) were analyzed for CAT
activity and checked for the protein concentration using
Lowry’s method.13 The active fractions were combined and
concentrated using an Amicon concentration cell model
8200 and ultrafiltration membrane PM-10. The purified
enzyme was stored at 4◦ C and used as source in following
experiments.
2.6 Protein content analysis
Using Lowry’s method, the protein content in a sample
was estimated. 0.1 mL enzyme was added to 1 mL of 1 N
NaOH at 100◦ C and allowed to stand for 5 min. Then added
5 mL of alkaline copper reagent and allowed the mixture
to stand for 10 min. 0.5 mL of Folin–Ciocalteu reagent was
added and mixed, and the sample was kept at room temper-
ature for 30 min. Absorbance at 750 nm was measured for
the mixture. The amount of the protein content in the mix-
ture was calculated with a standard curve prepared using
bovine serum albumin.
TAKIO et al.
2.7 SDS-PAGE analysis
The homogeneity and purity of the enzyme preparation
was checked by the SDS-PAGE method.14 The separating
gel was 30% acrylamide in a Tris-SDS buffer of pH 8.8, and
stacking gel was 30% acrylamide in tris-SDS buffer (pH
6.8). The electrophoresis was performed at a constant volt-
age and current at 100 V and 20 mA for 4 h and was visu-
alized by using ezee blue staining. The molecular weight
markers were bovine serum albumin (66 kDa), ovalalbu-
min (43 kDa), carbonic anhydrase (29 kDa), and lysozyme
(14 kDa).
2.8 INTACT mass analysis
For further confirmation of molecular weight of purified
protein, INTACT mass analysis was done through Q-TOF
SYAPT G2 mass spectrometry. To 50 μL of the sample,
0.1% Formic acid (FA) was added and it was analyzed on
the ESI-Q-TOF instrument for intact molecular weight by
direct infusion. The raw data were processed and deconvo-
luted by MassLynx 4.1 WATERS software.
2.9 MALDI-TOF analysis
Sample was analyzed by trypsin digestion of SDS-PAGE gel
cut containing pure enzyme through MALDI-TOF mass
spectrometry. It is a simple operation, a good mass accu-
racy with high resolution and sensitivity. In this approach,
peptides are generated by digesting proteins of interest
with a sequence-specific enzyme like trypsin. Then the
peptides are analyzed by MALDI-TOF mass spectrome-
try to get the peptide masses. The experimental masses
are compared against a database containing theoretical
peptide masses from a given organism with the same
sequence-specific protease.
The sample was ionized in a TOF mass spectrome-
ter, and a full postdecay source (PSD) spectra from pep-
tides were successfully recorded, and their amino acid
sequences were determined. Using the peak masses deter-
mined from the PSD ion spectrum, a peptide sequence
delivers a set of sequence propositions by combinatory
calculations. For each sequence proposition, a ranking
coefficient is calculated based on the level of correspon-
dence between the theoretical fragmentation pattern of
the sequence proposition (N-terminal, C-terminal, and
internal fragment ions) and the PSD spectrum. Each pro-
posed sequence is then searched in a protein database.15
The sample was analyzed through Mascot: http://www.
matrixscience.com.
TAKIO et al. 599

residue was washed with distilled water until neutral pH

was obtained and oven dried.

Deproteinization
The dried residue was treated with 4% NaOH with a solid-
to-solvent ratio of 1:5 (w/v) for 20 h at room temperature
for the removal of protein and formation of chitin slurry.
The obtained residue was again washed until neutral pH
and oven dried.

Deacetylation
The chitosan was obtained by treating deproteinized chitin
with 4% NaOH at room temperature with solid-solvent
ratio of 1:10 (w/v) for 24 h. The final residue was washed
and oven dried for 2 h at 80◦ C. The chitosan product was
used for its characterization.

2.10.2 Chitosan verification

To verify presence of chitosan, the produced residue was

treated with iodine/KI solution and acidified with two
to three drops of 1% H2 SO4 .17 The residue was initially
changed to dark brown on I/KI addition but turned to dark
purple on H2 SO4 addition which indicated presence of chi-
tosan.

2.10.3 Chitosan characterization

F L O W C H A R T 2 CAT enzyme purification method from Yield
Sechium edule [Color figure can be viewed at wileyonlinelibrary.com] The yield percentage was obtained by comparing the
weight of the raw material to the chitosan obtained:
2.10 Immobilization study ( )
weight of chitosan produced
Yield% = × 100.
2.10.1 Extraction of chitosan weight of the raw material

The raw material exoskeleton wastes were collected Moisture content

from different animal sources Cornu aspersum (garden
snail), Periplaneta americana (cockroach), Dendrobranchi-
ata (prawn), Brachyura (crab), Labeo rohita (fish rohu) ,
and Colossoma brachypomum (fish red pomfret) collected
from rivers, households, and nearby local fish markets.
The extraction of chitosan involved three stage processes
of demineralization, deproteinization, and deacetylation.
Prior to demineralization, the exoskeletons were washed
with distilled water to remove organic residues and dried
in oven for 24 h.16 The steps involved in extraction of chi-
tosan is given are Flowchart 2.
Demineralization
The dried wastes were treated with 2% HCl with a solid-to-
solvent ratio of 1:5 (w/v) at room temperature for 22 h for
the removal of inorganic matter of CaCO3 upper layer. The
The moisture content of chitosan was determined by oven
drying chitosan at 110◦ C for 12 h.
( )
𝑤2 − 𝑤1
Moisture % = × 100,
𝑤1
Where w1 is the weight of chitosan and w2 is the weight
of oven dried chitosan.
Water binding capacity
Water binding capacity (WBC) was measured by weighing
0.5 g of chitosan in 10 mL distilled water in a centrifuge
tube. The mixture was shaken every 5 s for 10 min and cen-
trifuged at 3200 rpm for 25 min. The tube was weighed after
decantation of supernatant, and WBC was calculated.
WBC % = (waterbound∕sampleweight) × 100.
600
Solubility
The solubility was checked by weighing 0.1 g of chitosan
in a centrifuge tube was dissolved with 10 mL of 1% acetic
acid for 30 min in a magnetic stirrer at room tempera-
ture. The tube was then immersed in boiling H2 O bath for
10 min and centrifuged at 3500 rpm for 20 min. The residue
was isolated and poured distilled water in the tube and
was again centrifuged at 3500 rpm for 20 min. The pellet
obtained was oven dried at 60◦ C overnight.
(Initial weight of tube + Chitosan) − (Final weight of tube + Chitosan)
Solubility % =
(Intial weight of tube + Chitosan) − (Initial weight of tube)
Determination of degree of deacetylation (DD)
0.1 g of chitosan was dissolved in 25 mL of 0.06 M HCl for
1 h, and further it was diluted to 50 mL for titration with
0.1 N NaOH solution. The titration was continued until pH
3.75 under constant stirring. The volume was checked and
again continued until pH 8, and the total volume of NaOH
was recorded.17-18
Using given below formula, the degree of deacetylation
was calculated:
(𝐶1 𝑉1 − 𝐶2 𝑉2 ) × 0.016
NH2 % = × 100, (3)
𝐺 (100 − 𝑊)
where C1 is the HCl concentration, C2 is the NaOH concen-
tration, V1 is the volume of HCl solution, V2 is the volume
of NaOH solution, 0.016 is the molecular weight of NH2 in
0.06 M HCl solution, G is sample weight, and W is moisture
content of chitosan (%).
NH2 %
DD% = × 100, (4)
9.94%
where 9.94% is a theoretical NH2 % value
Fourier transform infrared spectrocopy
The infrared red spectra of chitosan was determined
using a Perkin-Elmer Fourier transform infrared
(FTIR) spectrophotometer at a scanning range of
400–4000 cm−1 with KBr pellet at room temperature.
The chitosan sample was mixed with KBr powder and
compressed into a thin pellet for identification of chitosan
formed.
2.10.4 Preparation of chitosan beads
Around 1 g of extracted chitosan was dissolved in 100 mL of
5% CH3 COOH under constant stirring for 1 h and was left
overnight for the removal of air bubbles. With the help of
TAKIO et al.
a microsyringe, the chitosan solution was slowly dropped
into 1% Tripolyphosphate (TPP) solution to form activated
chitosan beads. After immersing beads for 3–4 h in a TPP
solution, the beads were separated out and again dropped
in the NaOH solution (pH 9.0) for 1 h to neutralize as well
remove excess unreacted TPP. Cross-linked chitosan beads
were taken out and washed with distilled water and oven
dried at 100◦ C for 30 min.19
× 100. (2)
2.10.5 Catalase immobilization on
chitosan beads
One milliliter of CAT enzyme was mixed with activated
chitosan beads for 5 h under constant stirring and washed,
dried, and stored at 4◦ C in a fridge.
2.11 Enzyme assay of CAT
Spectrophotometric determination of CAT activity was
monitored by decreased in absorbance of H2 O2 substrate
at λ = 240 nm and molar extinction coefficient value of
39.4 M−1 cm−1 . One milliliter of reaction mixture consisted
of 59 mM H2 O2 in 50 mM phosphate buffer (pH 7.0) and
a suitable aliquot of the enzyme solution at room temper-
ature. One enzyme unit is defined as decomposition of 1
μM of H2 O2 per minute. To check activity of immobilized
enzyme, the CAT immobilized beads were immersed for
in 10 mL of 10 mM H2 O2 solution in 50 mM phosphate
buffer (pH 7.0) at room temperature for 5 min. The chi-
tosan beads were removed from the H2 O2 solution, and UV
absorbance of reaction mixture was monitored and calcu-
lated for immobilized CAT activity.10
2.12 UV/Vis spectral studies of pH,
temperature optima, and Km , Vmax
The optimum pH of both free and immobilized CAT
enzyme was determined by measuring steady-state veloc-
ity of enzymatic reaction in solution of pH ranging from
3.5 to 9.0 using 50 mM phosphate buffer, and a graph was
plotted of steady-state velocity versus various pH in 59 mM
H2 O2 solution at room temperature. The optimum tem-
perature of both free and immobilized enzyme was deter-
mined by change in the absorbance rate at varying tem-
peratures in the range of 10–70◦ C, and a graph was plotted
for comparison. The Km and Vmax of free and immobilized
TAKIO et al. 601

TA B L E 1 Purification chat for CAT from Sechium edule fruit juice

Total Specific Total Total
Sl volume Protein Activity activity protein activity Purification Percentage
no. Steps (mL) (mg/mL) (U/mL) (U/mg) (mg) (U) fold recovery
1 Crude enzyme 1010.00 0.33 0.23 0.69 333.30 232.30 1.00 100
2 Concentrated enzyme 10.00 1.85 4.30 2.32 18.50 43.00 1.60 18.50
3 Dialyzed enzyme 13.00 0.75 2.30 3.06 9.75 29.9 4.43 12.74
4 DEAE 30.00 0.20 0.96 4.8 6.00 28.80 6.95 12.38
5 Amicon concentrated 7.00 0.56 3.56 6.35 3.92 24.92 9.20 10.72

enzyme were calculated at different H2 O2 concentrations
and compared by linear regression analysis of double recip-
rocal plots, and a graph was plotted.
The Km (Michelis–Menten constant) and Vmax of CAT
was determined by steady-state velocities of enzyme
reaction by varying concentrations of H2 O2 in the
range of 0.001–0.070 mM in phosphate buffer (pH 7.6,
0.05 mM) at room temperature with a fixed CAT saturating
concentration.20 The value of Km and Vmax was established
by plotting a double reciprocal graph of the data calculated
using the Lineweaver–Burk formula:
( )( )
1 𝐾𝑚 1 1
= + , (5)
𝑉 𝑉max 𝑆 𝑉max
where V is the rate of reaction, S is the substrate concen-
tration, Km is the Michelis–Menten constant, and Vmax is
the maximum reaction rate of enzyme.
edule. The process here involved crushing, extracting,
ultrafiltration and anion-exchange chromatography on
DEAE-cellulose, which is summarized in Table 1. The
extracted enzyme was treated with different concentra-
tions of (NH4 )2 SO4 , which showed complete precipitation
at 80% as saturation point of CAT activity. CAT purified
at ninefold with 10.72% yield was achieved as shown in
Table 1. The enzyme elution profile is shown in Figure 1.
The SDS-PAGE analysis of purified enzyme concluded
that a single protein band in lane 2 with molecular mass
of 55 kDa was obtained as shown in Figure 2. The relative
migration distance (Rf ) graph plotted in Figure 2 validates
that molecular weight of enzyme is around 55 kDa. Molec-
ular weight was further confirmed from INTACT mass
analysis as shown in Figure 3, and molecular weight was
found to be 52.55 kDa. The number of amino acid residues
found in the purified CAT was determined by MALDI anal-
ysis and was found to be 529 residues as given below;

1 MASFHKGAAG DSMGSSKMSF DQRRQLVLKL SKESEREFKE VLKDWSCNEI

51 RELLRAESKK DIKYTGLTKD EIITRLFNIV SKKNTRDHEV EEIIPSPKRQ
101 KRDLVTPLAK AKGKGKMYCQ NLACQNKLRE EATFCKRCSC CICFKYDDNK
151 DPSLWLTCNS DSQFDGESCG LSCHLNCAFD SEKSGLKEDT PSSDIDGCFN
201 CVSCGKTNSK IECLKKQLII ANEERRVGVF CYRILLAHKL LKGTKKYIIV
251 SEEVEKAVMH LKNEFGVPIS CLPSKMSRGL VNRLCCAEKV KKHCSSALKE
301 LDGLPLPSTI QGSMKIRIES VLATSVTFDI EAEESFSWGD TNHYRMVYRK
351 VSEKHSSKDL TRELFSTSSH QRFTVMELTP ATEYWFKIVS FSGVEELSVD
401 EFIVSTKTLQ DEEVAAVLLN MSNCNNANKM EKSGSCSFGF EECVNLIRQL
451 ECSGQVKSDF RKKFLTWYCL KATDKEKHVV EIFVDTFKDD KEALAKQLID
501 TFSDCITRKH PEIGGGSESA GVSLILLQD

Molecular weight is comparable with other known

species as summarized in Table 2. The molecular weight
3 RESULTS AND DISCUSSION of CAT varies from the range 53–72 kDa for its monomeric
unit. UV spectra of the pure enzyme shows its peak
3.1 Catalase purification at 420 nm shifted towards longer wavelength instead
of 405 nm for the presence of hemeprotein (figure not
The purification of CAT from plant sources has been stud- shown). However, the Soret band displayed by the enzyme
ied extensively but not yet been purified from Sechium was slightly shifted to longer wavelength (420 vs. 405 nm),
602
F I G U R E 1 Elution profile of CAT enzyme from DEAE column where (•) represent protein and (•) of CAT activity [Color figure can be
viewed at wileyonlinelibrary.com]
F I G U R E 2 SDS-PAGE analysis. Lane 1: protein marker, lane 2:
purified CAT, and lane 3: crude enzyme [Color figure can be viewed
at wileyonlinelibrary.com]
and the Reinheitzahl value (Rz index = (A405 /A280 ) was 1.5,
which is higher than in the case of the Ipomea batatas (Rz
index = 0.67)21 and Solanum tuberosum (Rz index = 0.88)22
CAT and equal to Lens culinaris CAT.23 The peaks
around 420 nm and above 475 nm are only due to heme
absorption.
TAKIO et al.
3.2 Chitosan characterization
3.2.1 Yield, moisture content, solubility,
degree of deacetylation, and water-binding
capacity
The yield, moisture content, solubility, water-binding
capacity, and degree of deacetylation percentage of
extracted chitosan from different sources are summa-
rized in Table 3. The yield % is obtained by calculat-
ing the dry weight of the extracted chitosan from the
dried raw material, where from Cornu aspersum shells,
the obtained yield was 10.3% compared to reported yield
of 12.73%.34 Colossoma brachypomum fish scales yield was
10.1%, Labio rohita fish scales yield obtained was 9.1%. A
similar study16,35 reported yield of 22.4% and 7.72%. Peri-
planata americana obtained yield was 5.05%, whereas from
Dendrobranchiate and Brachyura shells, the relative yield
percentage was 6.6 and 8.2. The variation in the yield
percentage can be explained by different species being
used and effect of acid and alkali reaction time on the
exoskeletons. However, the yield is also affected by loss
of sample weight during the deacetylation process lead-
ing to excessive removal of the polymeric group from the
shells.
The moisture content of the extracted chitosan of all
species showed average percentage range from 3 to 12%
compared to the report obtained by Lertsuttiwong et al.,36
where moisture content measured was 6–12% and also
Alishahi et al.37 acknowledged moisture content of chi-
tosan to be lesser than 10%. Being hygroscopic in nature,
the rate of moisture absorption differed in different species
TAKIO et al.
FIGURE 3 INTACT mass analysis of purified CAT [Color figure can be viewed at wileyonlinelibrary.com]
where lower moisture content suggests enhance quality
and stability.38
Chitosan are a semicrystalline rigid biopolymer, which
is not soluble in most aqueous solution but at certain
pH gets soluble in few acids like HCl, formic acid, acetic
acid, lactic acid, and so on.39 In the present work, the sol-
ubility of the extracted chitosan ranges from 20 to 53%
where lower solubility indicates incomplete removal of
proteins.17
There are several factors that affect degree of deacety-
lation of extracted chitosan from sample sources, instru-
ments used to purification procedure applied.40 They are
important parameters that affects various physiochemical
properties of chitosan. The duration and concentration of
alkali treatment greatly affect the deacetylation process
because an increase in the concentration promotes more
alkaline hydrolysis thus higher DD.41 The DD obtained
here ranges from 40 to 69.8% by the acid-alkali titration
method suggesting that the present extraction method is
more favorable for the extraction of species Colossoma
aspersum than Perilanata americana. Higher DD means
high degree of N-deacetylation of chitin, and better is the
chitosan formation.
In the present work, the WBC of the extracted chi-
tosan ranges from 105.91% to 121%, which is quite lower
compared to the studies done on Metapenaeus sstebbingi
shells reported WBC of 712.99%,42 reported from craw-
603
fish of about 660.6%43 and from shrimps of reported WBC
358%.39 This may be due to particle sizes of the extracted
chitosan, where a finer size promote increased absorption
or the procedure applied. Another possible explanation
could be water uptake capacity of polymeric groups, dif-
ferences in salt formation, or extraction process.41
3.2.2 FTIR studies of chitosan
The FTIR spectral studies of extracted chitosan from var-
ious sources were analyzed and compared to character-
ize and identify the functional group present (figure not
shown). Formation of different stretching vibrational large
bands between 3444.7 and 3400 cm−1 indicate overlap-
ping of OH and NH2 groups.44-46 The peak observed
at 2926.86 and 2888.05 cm−1 of snail and crab assigned
to the stretching vibration of alkane group (−CH, CH2 ,
CH3 ) indicating the presence of single bond.39,44,47,48 The
band observed at 1793.28 cm−1 denotes formation of the
acyl halide group stretching band (C=O). The NH2 bend-
ing vibration of amide II and C=O (amide I) group was
observed at 1648.28–1646.2 cm−1 .48 Further bands formed
between 1551.1 and 1556 cm−1 corresponds to N−H bend-
ing vibrations of amide II band of the −CONH− group.
The band formed between 1500 and 1700 cm−1 with
observed decreased intensity at 1656 cm-1 while increased
604 TAKIO et al.

TA B L E 2 Comparative chart of CAT characterization purified from different sources

S. Molecular
no Name of species weight (kDa) pH Km Vmax pI Temperature References

7
1 Pumpkin (Cucurbita sp. 59 6.5–10 124 mM 114 mmol/min 6.6 25
Amakuri Nankin)
8
2 Black gram (Vigna 56 7 16.2 mM 2.5 μmol/min – –
mungo)
9
3 Parsley (Petroselinum 45.8 7 84 mM 5000 U/mL – 35
hortense)
21
4 Sweet potato root 60 6–8 – – – –
(Ipomoea batatas)
22
5 Potato (Solanum 56 6–8 – – – –
tuberosum)
23
4 Lentil (Lens culinaris) 54 7.5–9.5 – – 5.5 –
24
5 Maize (Zea mays) 60 – – – – –
25
6 Papaya (Carica papaya) 40 61 – – – –
26
7 Pea leaves (Pisum 57 – – – – –
sativum)
27
8 Sunflower cotyledons 55–59 6–8 – – – –
(Helianthus)
28
10 Spinach leaf (Spinacia 72 5.3–8.9 – – – –
oleracea)
29
11 Castor bean (Ricinus 54–56 – – – – –
communis)
30
12 Tobacco (Nicotiana 53–55 – 0.057 M – – –
sylvestris)
31
13 Cotton seed 57 – – – – –
(Gossypium
hirsutum)
32
14 loblolly pine (Pinus 59 – – – 6.8 –
taeda)
33
15 Calla lily (Zantedeschia 54 7 5.2 40
aethiopica)
16 Squash (Sechium edule) 55 7.6 0.03 mM 200 μmol/min 8.2 24

peak at 1597 cm−1 suggests existence of the effective
deacetylation process.49 Furthermore, the band observed
in the region 1466.4 cm−1 indicate deformed (symmet-
rical and asymmetrical) bending vibrations of methyl
groups whereas 1414.9–1414 cm−1 suggest C−H bending
vibrations of the CH2 group.39 The medium intensity
band formed between 1088.5,1030,1029.8, and 1010.14 cm−1
denotes the presence of secondary and primary OH group
and C−O−C stretching vibration, which are a contribut-
ing peaks of primary and cyclic alcohols,50 whereas 864.1
and 870.89 cm−1 signifies wagging stretching vibration of
NH2 (primary) and NH− (secondary) amine group, which
are characteristic peaks for β 1,4 glycosidic bonds.44, 49
Furthermore, the stretching vibrational peaks observed
between 697 and 555 cm−1 suggests the presence of
haloalkanes.
3.3 Enzymatic immobilization on
chitosan beads
In this study, the CAT enzyme purified from Sechium edule
was immobilized onto chitosan beads using TPP (sodium
tripolyphosphate) as cross-linking agent. The selection of
the chitosan used for immobilization was on the basis
of the highest degree of DD reported in Cornu aspersum
(garden snail) and discussed above. TPP-pretreated chi-
tosan beads were incubated with CAT for 5 h before study-
ing its spectral behavior. Chitosan are semipermeable lin-
ear polysaccharide comprising glucosamine and N-acetyl
glucosamine units linked by 1–4, glycosidic bonds offers
the combined benefits of the required properties of bio-
compatibility, biodegradability, and low toxicity. It con-
tains three types of reactive groups which are the primary
TAKIO et al.
TA B L E 3 Physiochemical characterization of extracted chitosan from exoskeletons of different sources
Sample Yield (%)
Cornua spersum (garden snail) 10.30
Periplaneta americana (cockroach) 5.05
Dendro branchiata (prawn) 6.60
Brachyura (Crab) 8.20
Labeo rohita (fish rohu) 9.10
Colossoma brachypomum (fish red pomfret) 10.10
*WBC: Water binding capacity.
*DD: Degree of deacetylation.
amine group and the primary and secondary hydroxyl
groups at C-2, C-3, and C-6 positions. Among the three
reactive groups, the primary amine at C-2 position of the
glucosamine residues is the most considerable functional
group for biological activities of chitosan.51 The amino
groups of chitosan can assist the TPP cross-linker bind-
ing to form covalent bonds with an enzyme. Cross-linking
with TPP increases the active side of the chitosan that bind
with enzyme through hydrogen bond. Most protein con-
tains amino acid residues located on its surface. The cross-
linking process began with the addition of an acid (here
acetic acid), chitosan’s −NH2 gets protonated to −NH3 + ,
then −NH3 + would bind ionotropically with the negative
ion (−PO4 3− ) from TPP.52 The amino acid residues present
in the enzyme binds with the chitosan through TPP cross-
linkers as shown in Figure 4.
3.4 Determination of optimum pH,
temperature, Km and Vmax of CAT and its
effect on immobilization
To obtain pH optima, free and immobilized enzyme were
treated in various pH ranges from 3.5 to 9.00 in phos-
phate buffer at room temperature. The rate of change in
enzyme activity in different pH is given in Figure 5A.
Data were plotted in triplicate with a standard deviation
value for both enzyme was found to be 0.05, and the
coefficient of variance was less than 1. Figures revealed
that both free and immobilized CAT gave optimum pH
at 7.60 suggesting immobilization promotes broader pH
stability and lesser sensitivity towards changing pH of
enzymes.10,53
The enzymatic activity of free and immobilized enzyme
was calculated in different temperature conditions in the
range of 10–70◦ C, and it is concluded that optimum tem-
perature of free and immobilized was found to be 24
and 41◦ C as shown in Figure 5B. Data were plotted in
triplicate with a standard deviation value of 0.022 (free
enzyme) and 0.05 (immobilized enzyme) and a coeffi-
605
Moisture Solubility
Content (%) (%) WBC* (%) DD* (%)
3.50 30 119.57 69.80
12.00 20 105.91 40.00
9.10 39 108.14 49.00
16.00 121.00 64.30
9.00 53 109.00 54.30
10.00 45 120.00 67.00
cient of variance less than 1.0 in both cases. Immobi-
lized enzyme showed thermal stability around 39–45◦ C,
whereas exhibit less activity loss in high temperature
which may be due to increased enzymatic rigidity during
immobilization in chitosan beads which helped immobi-
lized enzyme against thermal denaturation.54 Synergistic
relation between immobilized enzyme and support also
led to stable enzymatic conformation promoting improved
activity at harsh conditions.55 Stabilization of enzyme
was observed after immobilization by entrapment, cova-
lent immobilization, and physical adsorption. Entrapped
enzyme contained hydrophobic as well as secondary inter-
actions which may take part in conformational flexibil-
ity changes in an enzyme molecule at higher temper-
atures. The structural rigidity of the protein molecule
due to immobilization decreased the extent of distor-
tion when the enzyme was exposed to elevated tem-
perature. Chitosan beads provided an external backbone
for enzyme molecules which resulted in protection of
the temperature-induced structural modifications in the
enzyme causing an improvement in thermostability of
the CAT.56
Using double reciprocal plots, the graph was plotted and
Km of free and immobilized enzyme was calculated. The
kinetic parameters of free and immobilized CAT is shown
in Table 4.
The Km values were found to be 0.03 and 0.065 mM for
free and immobilized CAT using H2 O2 as variable sub-
strate, and Vmax for free and immobilized were calculated
as 200 and 250 μmol/min established from the Figure 6A,B,
respectively. Data were plotted in triplicate with a standard
deviation value of 2.05 (free enzyme) and 1.95 (immobilized
enzyme), and the coefficient of variance was less than 1.0 in
both cases. Increased Km value of immobilized CAT indi-
cates lower affinity for its substrate H2 O2 which is likely to
be caused by structural, conformational, or environmental
changes of CAT and steric hindrance.10,53,57,58 But the Vmax
value increased indicating that the activity of the enzyme
is retained and is higher even if low concentration of sub-
strate H2 O2 is present.
606 TAKIO et al.

F I G U R E 4 (A) Interaction of chitosan with TPP, (B) cross-linking and activation of chitosan by TPP, and (C) immobilization of CAT
enzyme on activated chitosan support
TAKIO et al. 607

F I G U R E 5 Effect of (A) pH and (B) temperature on activity of free and immobilized enzyme was detected via UV–Vis spectrophotometer.
Conditions: (A) Reaction mixture of 1 mL solution consisted of 50 mM phosphate buffer, 59 mM H2 O2 and 40 μL enzyme at RT. The pH of
buffer was varied from 3.0 to 9.0. (B) Reaction mixture remains the same except as above but the buffer solution was prepared at pH 7.6. The
temperature was varied from 10 to 70◦ C and absorbance was monitored at 240 nm [Color figure can be viewed at wileyonlinelibrary.com]

TA B L E 4 Kinetics parameters of free and immobilized CAT enzyme from Sechium edule

Vmax Temperature
Enzyme Km (mM) (µmol/min) pH optima optima (◦ C)
Free CAT 0.030 200 7.60 24
Immobilized CAT 0.065 250 7.60 41

4 CONCLUSION
Sechium edule (squash) is a rich source of CAT with spe-
cific activity 6.35 U/mg. The CAT enzyme was purified
to homogeneity by adopting different steps of extraction
and purification and purified to ninefold from Sechium
edule (squash). Molecular weight determined by SDS-
PAGE analysis was 55 kDa, which was further confirmed
by INTACT mass analysis and found to be 52.5 kDa. The Rz
value (A405 /A280 ) found to be 1.5. Spectral analysis showed
that the purified CAT consists of heme and is a heme-
protein and showed its Soret band shifted to longer wave-
length at 420 nm. Sequences of purified enzyme have
been identified by MALDI-TOF analysis and found to
have 529 amino acid residues. Chitosan extracted from
from different animal sources Cornu aspersum (garden
snail), Periplaneta americana (Cockroach) Dendrobranchi-
ate (prawn), Brachyura (crab), Labeo rohita (fish rohu)
were characterized in terms of yield percentage, moisture
content percentage, solubility percentage, WBC percent-
age, and DD percentage. The DD percentage was high for
Cornu aspersum (garden snail). FTIR analysis of chitosan
608 TAKIO et al.

F I G U R E 6 Michelis–Menten and double reciprocal plots of (A) free and (B) immobilized enzyme using H2 O2 as variable substrates. In
both (A) and (B), the 1- mL reaction mixture consisted of 50 mM phosphate buffer of pH 7.6, distilled water, 40 μL enzyme, and concentration
of H2 O2 was varied at room temperature [Color figure can be viewed at wileyonlinelibrary.com]

extracted from different sources was done and concluded
from characterization based on different parameters that
chitosan extracted from Cornu aspersum (garden snail)
was better for the chitosan preparation. The CAT enzyme
was immobilized on chitosan beads prepared from chi-
tosan extracted. The enzymatic characteristic parameters
like Km , Vmax, optimum pH, and optimum temperature of
free and immobilized enzyme were calculated using H2 O2
as the substrate and found to be 0.03 mM, 200 μmol/min,
7.6, and 24◦ C for free CAT and 0.065 mM, 250 μmol/min,
7.6 and 41◦ C. Immobilized CAT is more thermostable to
free CAT and has lower affinity for H2 O2 . Thus CAT iso-
lated can be immobilized with better catalytic activity was
retained.
AC K N OW L E D G M E N T S
Department of Chemistry and Central Research Facility
(CRF), NERIST, Nirjuli, Itanagar, are highly acknowledged
for providing necessary facilities. This research work was
supported by UGC, New Delhi, India, under ‘National
Fellowship for ST’[award number: 201718-NFST-ARU-
01696].
TAKIO et al.
D A T A AVA I L A B I L I T Y S T A T E M E N T
The data that support the findings of this study are avail-
able on request from the corresponding author. The data
are not publicly available due to privacy or ethical restric-
tions.
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How to cite this article: Takio N, Yadav M
Barman M, Yadav HS. Purification,
characterization, immobilization and kinetic
studies of catalase from a novel source Sechium
edule. Int J Chem Kinet. 2021;53:596–610.
https://doi.org/10.1002/kin.21468