Microsoft Word - 04-215-224-PJAEC-29102021-407-C-Ree Revised Galley


Cross Mark

ISSN-1996-918X

Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022) 215 – 224

http://doi.org/10.21743/pjaec/2022.12.04

Evaluation of Cotton Seeds as Environmentally Liable
Source for Neutral Protease

Asghar Ali Shaikh
*

and Asif Ali Bhatti
1

Department of Chemistry, Government College University, Hyderabad,71000, Pakistan.
*Corresponding Author Email: dr.asghar.ali@gcuh.edu.pk

Received 29 October 2021, Revised 27 October 2022, Accepted 11 November 2022
--------------------------------------------------------------------------------------------------------------------------------------------

Abstract
Proteases are considered one of the most imperative groups of enzymes and are used in
bioremediation processes, leather, detergents, pharmaceutical and the food industry. The foremost
aim of this study was to extract, purify, and characterize the protease enzyme from cotton seeds.
Purification of the neutral protease was achieved with ammonium sulphate, which gave the best
results at 60% concentration with a specific activity of 51 units/mg protein and 1.52 purification
fold with a percentage yield of 10.5%. The protease was active and stable at a wide range of pH
from 4.0–10.0 with an optimum pH of 7.0. The highest activity of the purified enzyme was found
at 20 °C. The enzyme was ther mally stable and retained 25% of its activity at 50o C. The activity
of cottonseeds protease Fraction-IV was enhanced by 20% with ZnCl2 and 15% with CoCl2 as
enzyme samples were heated for 10 minutes. The Casein and peptone assays were also perfor med
to check its catalytic activity. Further more, the maximum hydrolysis rate (Vmax) and apparent
Michaelis–Menten constant (Km) values of the purified protease were 19 μmol/min and 0.08
mol/L, respectively, while activation energy was found to be 12.47 KJ/mol.

Keywords: Neutral, Thermostable, Cotton seeds, Metallo Protease
--------------------------------------------------------------------------------------------------------------------------------------------

Introduction

Enzymes, due to their low production cost,
easy methods of production and
environmentally friendly behavior are broadly
used in several commercial fields [1]. in
proteins, these enzymes accelerate the
degradation of peptide bonds present between
amino acids [2]. Researchers are focusing to
isolate new enzymes which are suitable for
commercial applications [3]. In 2018, the
world market for industrial enzymes extended
by around $5.6 billion in 2018, which will
increase by an estimated $7.0 billion in 2023
[4]. Among these enzymes, proteases cover
65% of the total industrial enzymes market
[5]. Proteases may be classified as
endopeptidases, which degrade protein
substrate from the inner site and

exopeptidases, which degrade protein
substrate from the end but can still be
classified according to the substrate
specificity, source of isolation (vegetable,
animal or microbial), molecular size, catalytic
action, charge and active site [6].

Proteases, according to their optimum
pH reactions, are classified as neutral
protease, acidic protease and alkaline protease
[7]. Neutral proteases are broadly applied in
the food [8,], feed [9], pharmaceutical [10]
and leather [11] industries due to their
divergent benefits, including low pollution
level, a mild catalysis process and high yield.
Neutral protease is usually used for brewing
beer [12] and de-bittering soy sauce in the



Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022) 216

food industry [13]. For commercial
applications, several researchers have focused
on isolating new enzymes having satisfactory
properties [14]. Due to good solubility, high
stability in extreme conditions, substrate
specificity and activity over broad-ranging
temperatures and pH scales, plant proteases
had been used in numerous industries [15]. In
several food industry procedures, plant
sources proteases are mostly used, therefore,
the proteases are cost-effective for use in
industries [16]. Furthermore, researchers are
searching for novel proteases from plant
sources which have cost-effective in industries
and have important physiological roles [17].

At present, the important focus is to
find a neutral protease from economical
sources which have a high affinity to protein,
acid–alkali resistance and good thermal
stability. Because Pakistan is an agricultural
country and cotton is a major crop and cotton
seeds are frequently available and cheap
sources, therefore, in this study, a neutral
protease from cotton seeds was purified and
characterized.

Material and Methods

Cotton seeds (BS-15) were purchased
from a local market. Ammonium sulphate,
Casein acid hydrolysate, peptones and other
reagents/chemicals were used in high
analytical grade and purchased from a local
vendor of Sigma (USA), Merck (USA), and
Fluka Company (Switzerland).

Preparation of Soluble Enzyme and
determination of Protein and Protease
activity

Cotton seeds were de-coated, crushed,
defatted and homogenized in chilled acetone.
On the other hand, 10% enzyme solution and
2% casein acid hydrolysate substrate were
prepared in chilled 0.2 M Tris-HCl buffer as
reported earlier [18,19]. Protein content was

calculated by the method of Lowry [20], with
bovine serum albumin as a standard.
Enzymatic solution protein absorbance was
monitored at 280 nm. The activity of Protease
was checked by the method of Anson [21]
with slight modification. In this method, 2%
casein acid hydrolysate was used as a
substrate. At the end of the reaction, protease
activity was checked by spectrophotometer at
625 nm, as reported earlier [18].

One unit of protease activity was
defined as the amount of enzyme that liberated
1ug of tyrosine under the standard assay
conditions.

Isolation, Dialysis and Purification of cotton
seeds Protease by using Sephadex G-100
Column Chromatography

Two-fold ethanol, acetone, methanol
and 40%, 60% and 80% saturated ammonium
sulphate was used to purify protease. All these
chemicals were retained at 4

o
C for 4-5 hours.

The collected precipitates from different
chemicals were dissolved separately in 10 ml
of Universal buffer of 7.0 pH and their
protease activity was determined by an earlier
reported method. On the basis of the highest
protease activity, precipitates were obtained
from 60% saturated ammonium sulphate
dialyzed for 24 h in a universal buffer of 7.0
pH. The collected precipitates and dialyzed
samples were centrifuged in a cooled
refrigerated centrifuge at 7000 rpm for 20
minutes and protease activity was determined
by the reported method.

Sephadex G–100 gel column (60 ×
2cm), previously packed and equilibrated with
Universal buffer pH 7.0. On this Sephadex G–
100 gel column, 5.0 ml of dialyzed sample
was loaded. The dialyzed sample was eluted
with the same buffer pH 7.0 at the flow rate of
40.0 mL/h. The fraction of 5.0 mL was
collected using fraction collector EYELA



Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022)217

UV–9900, UV-Visible detector. The protein
absorbance was monitored at 280 nm.

SDS-Gel Electrophoresis

SDS gel electrophoresis using buffer
system, as described by Peterson [22] and
Hames [23] was used to determine
homogeneity of the purified enzyme.

Kinetic Study and Characteristic Properties
of Protease Enzyme Effect of Substrate
Concentration and substrate specificity on
Protease Activity

Casein acid hydrolysate and peptone
animal were used as substrates of different
concentrations from 0.5-3% and checked their
effects on the rate of enzymatic reaction of
protease. 1.0 mL of purified fraction sample
and 1.0 mL substrate of different
concentrations were incubated at 20°C for one
hour.

The substrate specificity with various
substrates was also calculated by using
different substrates. The mixture of a fixed
amount of enzyme and substrate was
incubated for one hour at 20°C. After
incubation protease activity was checked as
previously described by the standard protease
assay method.

Effect of pH, Temperature, Thermal stability
and metal ions / reagents on Protease
Activity

To check the effect of pH on the
protease activity of purified Fraction-IV,
various pH ranges from 4 to 10 by using a
universal buffer pH 7.0 were used. The
enzyme was incubated with casein acid
hydrolysate as a substrate. The optimum
temperature of the purified protease enzyme
was determined by incubating the enzyme-
substrate mixture at various temperatures in
the range of 10°C to 50°C. On the other hand,

the thermal stability of protease was achieved
from purified fractions by measuring the
residue activity after incubation of the enzyme
at various temperatures ranging between 20°C
to 80°C for 10 min with and without the
addition of activators at 20

o
C. The effect of

time period on enzyme thermal stability was
also noted by heating enzyme samples at 5–20
min with and without the addition of an
activator at 40

o
C. The different concentrations

(mM) of several chemicals and metal ions
were incubated with purified Fraction-IV for
10 minutes at optimum temperature before
adding substrate. The remaining protease
activities of each parameter were determined
by the previously described method.

Results and Discussions
Purification of Protease by Chromatography

The solvent extraction of cotton seeds
enzymatic protein extract (20%) at pH 7.0 was
done. For this purpose, cotton seeds, and
enzymatic protein extract (20%) precipitated
with different solvents such as methanol,
acetone, ethanol and various concentrations of
ammonium sulphate (40, 60 and 80%). It was
observed that cotton seeds enzymatic protein
extract with 60% ammonium sulphate
precipitation produced the highest protease
activity in compression to solvents
precipitation, as illustrated in Table 1.
Therefore, ammonium sulphate (60%
saturated) concentration was selected for
precipitating enzymatic protein for further
investigation.

Table 1. Enzyme protei n and acti vi ty of preci pitates of sol vents.

Preci pitation
Wi th

Total
protei n

mg

Total
protease

acti vity uni ts

Specifi c Acti vity
uni ts/mg protei n

Control 253 8500 33.6

Ethanol 134 5000 37.3

Methanol 199 5750 28.9

Acetone 167 5500 33

Ammoni um sul phate

40% 51.9 1200 23.12

60% 115 6000 52.17

80% 83 1300 15.66



Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022) 218

Purification of cotton seeds protease
was done by Sephadex G-100 column
chromatography and the elution pattern is
shown in Fig.1. The precipitates obtained
from 60% ammonium sulphate were dissolved
in 20ml Universal buffer pH 7.0 and then
dialyzed overnight at 4

o
C. The dialyzed

sample was applied to Sephadex G-100
column (60 × 2 cm), which was packed and
equilibrated with the same buffer at a flow
rate of 40 mL/h with 10 mL of fraction
volume. The elution of enzymatic protein was
monitored at 280 nm. The protease activity
was checked in each tube and the active
fractions were pooled for further
characterization. These pooled fractions were
named Fraction-I, Fraction-II, Fraction-III,
Fraction-IV and Fraction-V. The Fraction-I,
Fraction-II, Fraction-III and Fraction-IV were
found homogeneous, showing a single protein
band by SDS gel electrophoresis, while
Fraction-V was not homogeneous as exhibited
in Fig. 2.

Figure 1. Puri fication profile of protease of cotton Seeds by

Sephadex G-100

Figure 2. SDS Pol yacryl ami de Gel Electrophoresis of Cotton
Seeds Protease (Fraction-IV)

All fractions were individually
characterized and found alkaline, acidic and
neutral. However, in terms of optimum
temperature, pH, substrate concentration,
thermos ability, substrate specificity and effect
of various reagents, Fraction-IV was neutral in
nature, further characterized and reported in
present study. The protease of Fraction-IV
was purified to 1.52 fold with the percent
yield of 10.5. While specific activity was
found to be 51 units /mg protein. The overall
purification profile of alkaline protease
fraction is presented in Table 2.

Table 2. Puri fication profile of protease from cotton seeds
(Fraction- IV).

Puri fication
Steps

Total
Protei n

mg

Total
Protease

Acti vity g

Speci fi c
Activity
g /mg

Purific-
ation
Fold

%
Yi eld

Enzyme
Crude

253 8500 33.6 1 100

After dialysis 190 6750 35.5 1.06 79.4

Sephadex
G-100

123.8 5800 46.8 1.39 68.2

Fraction-IV 17.5 895 51 1.52 10.5

Effect of Temperature on Protease Activity

From the industrial point of view, the
optimum temperature of an enzyme,
especially proteases is considered a significant
factor. Temperature affects the speed of
enzymatic reactions by disturbing the structure
of the enzyme and boosting the meeting of the
substrate with the active site [24]. Hence, as
the temperature is elevated, the speed of
enzymatic reaction also increases as long as
the natural structure of the protein is spoiled.
Higher temperatures denaturation the enzymes
mainly by breaking hydrogen bonds. To
determine the optimum temperature of
Fraction-IV protease, the enzyme-substrate
mixture was incubated at a different
temperatures ranging from 10 to 50

o
C. Fig. 3,

shows the maximum relative activity (100%)
for Fraction-IV protease at a temperature of



Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022)219

20°C. It is mentioned that the protease activity
with an optimum temperature 20

o
C or less

than 20
o
C is considered a cold protease [25].

After that optimum temperature, enzyme
activity starts to decrease as temperature
increases and is completely lost at a
temperature reached 40

o
C. After 20°C decline

in activity was observed and this is due to the
thermal denaturation of Fraction-IV protease.
Huston et al. have isolated a protease from
PsychrophileColwellia psychrerythraea Strain
34H, which has a low optimum temperature
(19oC) [26]. The activation energy for the
protease of Fraction-IV is calculated at 12.47
KJ/mol.

Figure 3. Effect of temperature on puri fied protease acti vity of
cotton seeds (Fraction- IV)

Effect of pH on protease activity

pH is an important factor for all
enzyme activity. The pH at which the enzyme
showed maximum activity, is called optimum
pH. The optimum pH is considered a key
factor for all enzymes in terms of their
activities and production, variation in pH leads
to enzyme inactivation [27]. Nevertheless,
some proteases extracted from plants are
exceptional, they are active in a wide range of
pH and temperature. In this study, the pH
activity of proteases isolated from cotton
seeds protease samples was calculated at
different pHs (5, 6, 7, 8, 9 and 10). The
enzyme activity increases as pH increases to 7
and then declines steadily up to pH 10. This

trend showed that the enzyme has optimum
activity at pH 7 as presented in Fig. 4. It is
documented that Metallo-proteases are in the
range of neutral to alkaline [28], and pH
studies reported an increased enzyme activity
at a pH range of 5-7. The functions of an
enzyme rely on its structural integrity, as the
structure of the enzyme is changed, the
enzyme loses its functions. The remarkable
ranges of pH activity of enzymes favor acidic
and neutral environments. Bijina isolated
protease inhibitor from Moringa oleifera with
potential application as therapeutic drug and
as seafood preservative at pH 7. [29],
Siritapetawee isolated and purified protease
from Artocarpus heterophyllus (jackfruit)
latex and found maximal activity between 55
and 60 °C at pH 8. [30].

Figure 4. Effect of pH on puri fied protease acti vity of cotton
seeds (Fraction-IV)

Effect of Metal ions / chemical reagents on
Protease activity

The effects of different metal ions on
protease activity are presented in Table 2. The
activity of the purified protease was enhanced
up to 15 and 20% by Ca2+ and Zn2+

respectively, while metal ions Ag+, Hg
2+

,
Co

2+
and Mn

2+
decrease enzyme activity with

the proportions of decrease 30, 35, 70 and
65%, respectively. On the other hand, tested
chemicals such as Tween 80, Triton X-100,
Sodium dodecylsulphate (SDC), Sodium
deoxycholate (SDS), Mercaptoethanol and
Cysteine also have strong inhibition effects
35%, 20%, 85%, 75%, 70% and 50%



Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022) 220

respectively on the enzyme activity in variable
degrees in comparison with the control as
exhibited in Fig.5. It was documented that
Plant metalloproteases are generally zinc and
calcium-dependent enzymes and calcium are
well-known to interfere with the three-
dimensional structure and catalytic properties
of enzymes [31]. These findings showed that
Fraction-IV protease belongs to Metallo
protease and Zn

2+
and Ca

2+
ions stabilize the

enzyme structure and protect the protease
against thermal denaturation [32]. Ca

2+
and

other metal ions dependent protease from
leaves of Moringa oleifera was also reported.
Ramakrishna has isolated Metallo-protease
from Dry Grass Pea Seeds [33].

Table 3. Effect of vari ous metal s / reagents on puri fied protease

acti vity of cotton seeds (Fraction-IV).

Reagents Activity % of
rel ati ve

% of

5mM Conc. units/
mL

protease
activity

activation/
[inhibition]

Control 8 100

Tween 80 5.2 65 35

Triton X- 100 6.4 80 20

SDC 1.2 15 85

SDS 2 25 75

AgNO 3 5.6 70 30

HgNo3 5.2 65 35

Mercaptoethanol 2.4 30 70

Cysteine 4 50 50

O-Phenanthroline 7.4 93 07

EDTA 7.6 95 5

CoCl2 2.4 30 70

MnCl2 2.8 35 65

CaCl2 9.2 115 15

ZnCl2 9.6 120 20

Effect of Thermostability on Protease
Activity

The stability of an enzyme depends on
the sum of numerous weak, non-covalent
interactions such as Van der Waal
interactions, hydrogen bonds and hydrophobic

effects. All of these non-covalent interactions
are upset by environmental conditions, such as
temperatures. The enzyme degradation
mechanisms are significantly speeded at high
temperature, and thus impart an important role
in the thermo-inactivation of enzymes.
Thermo-stability is considered a crucial
characteristic of protease enzymes required in
various industries. Those enzymes which are
thermostable are considered beneficial from
the industrial point of view. It was mentioned
in the literature that the thermal stability of an
enzyme is a key factor for its biotechnological
applications.

Thermal stability of purified cotton
seeds Fraction-IV protease was accomplished
by heating the enzyme samples at various
temperatures ranging from 20

o
C to 70

o
C for

10 minutes with and without the addition of
activators (ZnCl2 and CaCl2). After heating,
the enzyme samples were cooled, and the
remaining activities were determined by
standard method. Moreover, thermostability
was also determined at various times from
every interval of 5 min (5 to 20 min) at fixed
incubated temperature with and without the
addition of activators. The protease activity of
cotton seeds Fraction-IV was retained at 25%
at 50

o
C. Fraction-IV protease activity was

increased by 25% at 50oC by the addition of
5mM ZnCl2 in the enzyme samples heating for
10 min, and results are shown in Fig. 5.
Furthermore, the data depicted in Fig. 6
showed the effect of heat treatment on
protease activity at a variable time (5-20 min)
and fixed temperature (40

o
C) with and without

the addition of activators. However, Fraction-
IV protease activity was retained at 60%
without and 72% with ZnCl2 for 20 minutes of
incubation. These results are comparable with
other sources of proteases reported in the
literature. Protease from Salvadorapersica
was Stable up to 50oC [34], protease from
Baby Kiwi was stable up to 45

o
C [35] and

protease from Citrullus colocynthis was stable



Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022)221

up to 40oC. Some other researchers, such as
Gonçalves et al. have isolated thermostable
proteases from the leaves, seeds, roots, and
stem of Canavalia. Ensiformis [24].

Figure 5. Thermostability of Purified protease acti vi ty of cotton
seeds (Fraction-IV) wi th and wi thout ZnCl 2 for 10 mi n

Figure 6 . Effect of heat treatment at (40oC) with and without
ZnCl 2 at various time period on puri fied protease of cotton seeds
(Fraction-IV)

Effect of Substrate Concentration on
Protease activity

It is well-known consideration that
substrate concentration is a key factor for
determining the enzyme’s activity beside pH
and temperature. Furthermore, it is also used
for the calculation of enzyme kinetics (Km and
Vmax) to elucidate the enzyme specificity
and affinity. In this connection, different
concentrations of Casein acid hydrolysate
from 0.5 to 3% concentration at 20oC were
used with respect to their optimum time
period. The activity of Fraction–IV protease
was examined, and results are signified in
Fig. 7. Moreover, it was noted that the

substrate concentration increased up to 2%
when both casein hydrolysates which was
used throughout the study and peptone of
Soymeal and then dropped. Praiwala et al.
[36] have used several amounts (0.2, 0.4, 0.6,
0.8 and 1.0ml) of Hemoglobin as a substrate
incubated with protease obtained from Green
gram seeds and Soybean seeds. N--tosylL-
arginine methyl ester (L-TAME) was used as
substrate with proteases isolated from leaves,
seeds, roots and stem of Canavalia ensiformis.

Figure 7. Effect of substrates on puri fi ed Protease acti vity of
cotton seeds (Fraction-IV)

Estimation of Km and Vmax of protease

The Km value is a tool to measure the
enzyme affinity to the substrate. It is fact that
smaller km values indicate greater Enzyme-
Substrate affinity [37]. The value of Km shows
the highest rate achieved when the enzyme
sites are saturated by the substrate of the
reacted enzyme. The Km value of Fraction-IV
protease was calculated at 0.08M (Table 4) by
double reciprocal plots according to
Lineweaver and Burk [38]. The Km value of
(0.08) of the present work is lower than
reported by other researchers who isolated
protease enzymes from different plants like
Yemeni bean seeds [28]. Km is 0.19M, field
bean seeds enzyme 0.0105M [39], Moringa
Oleifera leaves is 0.107M [40] and from the
seeds of Cucumis melovaragresticis 0.02M
[41] and seeds of ash groud Benincasa hispida
(thumb) Cogn is 0.01M [42]. It is clear from



Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022) 222

the results obtained in this work, that the Km
value of Fraction-IV protease is lower than
other proteases hence greater affinity towards
substrate.

On the other hand, the value of Vmax of
Fraction-IV protease was also calculated
which comes out 19 µmol/min as presented in
Table 4. This value of Vmax is higher than
proteases isolated from seeds of Green
gram(Vmax 2 µmol/min) and Soybean(Vmax
1.01 µmol/min) but lower than those obtained
from proteases isolated from seeds of
Citrullus colocynthis (557 µmol/min) [43].
Ibraheem and Malom have reported two
neutral proteases, Pll and Plll from Citrus
sinensis fruit peel, having a value of Vmax are
192.31 and 111.11 µmol/min, respectively
[44].

Table 4. Km, Vmax val ues, Arrheni us plot of the acti vation energy
of puri fi ed cotton seeds protease (Ea=slopxR, where
R=8.314KJ/mol).

Km val ues
mol/L

Vmax val ues
µmol/min

Activation energy
KJ/mol

Fraction-
IV

0.08 19 12.47

Effect of substrate specificity of protease

The substrate specificity has great
importance in biotechnological fields. The
substrate specificity of purified protease
Fraction-IV was determined by incubating the
enzyme with different protein substrates
prepared in universal buffer pH 7 and results
are depicted in Fig.8. The comparative activity
of Faction-IV protease for the degradation of
casein was taken as 100%. Experimental
results showed that Fraction-IV protease
strongly hydrolyzed different peptones
proteins such as Peptone soy meal 198%,
Peptone meat 169%, Peptone animal 159%,
Peptones protease 143%, Peptone casein
109% but less hydrolyzed Hemoglobin 72%,
Caseinn soluble 43%, Lactalbumin 40%,
Azocol 24%, Azocasien 15% and Albumin

10%. These findings reflect that peptones are
favorable substrates for Fraction –iv protease.
It is the obvious conclusion from the above
results that cotton seed Fraction-IV protease
strongly favors hydrolyzed peptones as
compared to other substrates. Some other
investigators have also demonstrated substrate
specificity. Raghunath has isolated protease
from the latex of the Euphoriba Prunifolia
Jacq, which shows proteolytic activity of
72.14% with keratin, 58.61% with egg
albumin, 53.18% with gelatin, 38.61% with
hemoglobin and 27.51% with bovine serum
albumin used as substrates [45]. It was
reported in the literature that Salvadora
persicaprotease hydrolyzed proteins as casein
100%, hemoglobin 95%, egg albumin 72%,
gelatin 68% and bovine serum albumin 53%.
Ademola Ibraheem and Malom have purified
two neutral proteases Pll and Plll from Citrus
Sinensis fruit peel. Both enzymes Pll and Plll
exhibited relative activities are 75% and 91%,
but for gelatin 125% and 109%, respectively
[44].

Figure 8. Substrate Specifici ty of puri fied protease acti vity of
cotton seeds (Fraction-IV)

Conclusion

It is concluded that the obtained
protease Fraction-IV from cotton seeds by
Sephadex G-100 column chromatography was
found neutral, having pH 7. On the basis of
kinetic parameters, protease activity of



Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022)223

Fraction-IV was found heat stable and
increased by 20% at 50°C in the presence of
an activator (ZnCl2). Both ZnCl2 and CaCl2
enhance activity of Fraction-IV protease. The
Fraction-IV was found Metallo protease. This
enzyme favors degraded peptones as
compared to other substrates. On the basis of
investigational results, it is concluded that the
neutral protease of Fraction-IV is successfully
used in food, pharmaceutical and leather
industries because of their diverse benefits,
including low pollution levels.

Acknowledgement

We are thankful to the Institute of
Biotechnology & Genetic Engineering,
University of Sindh, Jamshoro, for facilitating
to carry out work; special thanks to Dr. Syed
Habib Ahmed Naqvi for technical support.

Conflict of Interest

The authors declare that there is no
conflict of interest.

References

1. R. Singh, M. Kumar, A. Mittal and P.
Mehta, 3 Biotech, 6 (2016) 1.
doi.org/10.1007/s13205-016-0485-8

2. H.-Y. Zhao and H. Feng, BMC
Biotechnol., 18 (2018) 1.
doi.org/10.1186/s12896-018-0451-0

3. A. Hirata, Y. Hori, Y. Koga, J. Okada,
A. Sakudo, K. Ikuta, S. Kanaya and K.
Takano, BMC Biotechnol., 13 (2013) 19.
doi.org/10.1186/1472-6750-13-19

4. M. Omrane Benmrad, S. Mechri, N.
Zarai Jaouadi, M. Ben Elhoul, H. Rekik,
S. Sayadi, S. Bejar, N. Kechaou and B.
Jaouadi, BMC Biotechnol., 19 (2019) 43.

doi.org/10.1186/s12896-019-0536-4
5. R. Gupta, Q. K. Beg and P. Lorenz,

Appl. Microbiol. Biotechnol., 59 (2002)
15. doi.org/10.1007/s00253-002-0975-y

6. J. G. S. Aguilar and H. H. Sato, Food
Res. Int., 103 (2018) 253.
doi.org/10.1016/j.foodres.2017.10.044

7. M. Chaudhuri, A. K. Paul and A. Pal, J.
Environ. Biol., 42 (2021) 955.
doi.org/10.22438/jeb/42/4/MRN-1645

8. O. L. Tavano, J. Mol. Catal. B Enzym,
90 (2013) 1.
doi.org/10.1016/j.molcatb.2013.01.011

9. M. Yuzuki, K. Matsushima and Y.
Koyama, J. Biosci. Bioeng, 119 (2015)
92.
doi.org/10.1016/j.jbiosc.2014.06.015

10. H. Zhang, B. Zhang, Y. Zheng, A. Shan
and B. Cheng, Biodegradation, 93
(2014) 235.
doi.org/10.1016/j.ibiod.2014.05.024

11. M. M. S. Asker, M. G. Mahmoud, K. El
Shebwy and M.S. Abd el Aziz, J. Genet.
Eng. Biotechnol., 11 (2013) 103.
doi.org/10.1016/j.jgeb.2013.08.001

12. J. Wang, A. Xu, Y. Wan and Q. Li, Appl
Biochem Biotechnol, 170 (2013) 2021.
doi: 10.1007/s12010-013-0350-8

13. M. Machida, K. Asai, M. Sano and T.
Tanaka, Nature, 438 (2005) 1157.
doi: 10.1038/nature04300

14. A. Hirata, Y. Hori, Y. Koga, J. Okada,
A. Sakudo, K. Ikuta, S. Kanaya and K.
Takano, BMC Biotechnol., 13 (2013) 19.
doi: 10.1186/1472-6750-13-19

15. F. M. Abbas and A. E. Abdelrahman,
Biochem. Mol. Biol., 7 (2021) 12.
doi: 10.36648/2471-8084.21.7.102

16. T. Popovic and V. P. J. Brzin, FEBS
Lett., 530 (2002) 163.
doi.org/10.1016/S0014-5793(02)03453-1

17. M. Amid, M.Y. Manap and N.K. Zohdi,
Biomed. Res. Int., 2014 (2014) 1.
doi: 10.1155/2014/259238

18. A. Ali and M. U. Dahot, Sindh Univ.
Res. J., 41 (2009) 7.

19. M. U. Dahot and A. A. Sheikh, Pak. J.
Biotechnol., 2 (2005) 49.

20. O. H. Lowry, N. J. Rosebrough, A. L.
Farr and R. J. Randall, J. Biol. Chem.,
193 (1951) 265.



Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022) 224

21. M. L. Anson, J. Gen, Physiol., 22
(1938) 79.
doi: 10.1085/jgp.22.1.79

22. G.L. Peterson, Anal. Biochem., 83
(1977) 346.
doi.org/10.1016/0003-2697(77)90043-4

23. B. D. Hames, 3
rd

Edition, Oxford
University Press, (1998) 53.

24. R. N. Gonçalves, S. D. G. Barbosa and
R. E. S.Lopez, Biotechnol. Res. Int.,
2016 (2016) 11.
doi.org/10.1155/2016/3427098

25. H. Aoki, M. N. Ahsan, K. Matsuo, T.

Hagiwara and S. Watabe, Int. J. Food
Sci. Technol., 39 (2004) 471.
doi.org/10.1111/j.1365-
2621.2004.00806.x

26. A. L. Huston, B. Methe and J. W.
Deming, Appl. Environ. Microbiol., 70
(2004) 3321.
doi.org/10.1128/AEM.70.6.3321-
3328.2004

27. J. E. Lawn, H. Blencowe, P. Waiswa, A.
Amouzou, C. Mathers, D. Hogan, V.
Flenady, J. F. Froen and Z. U. Qureshi,
The Lancet, 387 (2016) 587.
doi.org/10.1016/S0140-6736(15)00837-5

28. M. A. A. Maqtari, K. M. Naji and L. K.
Ali. J. Modern Sci. Eng., 5 (2017) 8.
doi.org/10.47372/uajnas.2019.n2.a09

29. B.Bijina, S Chellappan, J. G.Krishna, S.
M. Basheer, K.K. Elyasa, A. Bahkali,
and M. Chandrasekaran, Saudi J. Biol.
Sci.,18 (2011) 273.
doi.org/10.1016/j.sjbs.2011.04.002J.

30. Siritapetawee, K. Thumanu, P. Sojikul
and S. Thammasirirak, Biochim.
Biophys. Acta - Proteins Proteom., 1824
(2012) 907.
doi.org/10.1016/j.bbapap.2012.05.002.

31. J. Callis, Plant Cell, 7 (1995) 845.
doi: 10.1105/tpc.7.7.845

32. H. Ostolaza, A. Soloaga and F. M. Goni,
Eur. J. Biochem., 228 (1995) 39.
doi.org/10.1111/j.1432-
1033.1995.0039o.x

33. V. Ramakrishna, S. Rajasekhar and L. S.
Reddy, Appl. Biochem. Biotechnol., 160
(2010) 63.
doi: 10.1007/s12010-009-8523-1

34. W. A. Abdulaal, BMC Biochem., 19
(2018) 1.
doi: 10.1186/s12858-018-0100-1

35. S. Miyazaki-Katamura, M. Yoneta-
Wada, M. Kozuka, T. Sakaue, T.
Yamane, J. Suzuki, Y. Arakawa and I.
Ohkubo, Open Biochem. J., 13 (2019)
54.
doi: 10.2174/1874091X01913010054

36. B. Praiwala, S. Priyanka, N. Raghu, N.
Gopenath, A. Gnanasekaran, M.
Karthikeyan, R. Indumathi, N. Ebrahim,
B. Pugazhandhi and P. J. Pradeep,
J. Biomed. Sci., 5 (2018) 10.
doi.org/10.3126/jbs.v5i2.23633

37. I. A. Mohamed Ahmed, I. Morishima,
E. E. Babiker and N. Mori,
Phytochemistry, 70 (2009) 483.
doi: 10.1016/j.phytochem.2009.01.016

38. H. Lineweaver and D. Burk, J. Am.
Chem. Soc., 56 (1934) 658.
doi.org/10.1021/ja01318a036

39. B. Paul and L. R. Gowda, Agric. Food
Chem., 48 (2000) 3839.
doi.org/10.1021/jf000296s

40. S. Banik, S. Biswas and S. Karmakar,
F1000Res., 7 (2018) 1151.
doi: 10.12688/f1000research.15642.1

41. B. G. Devi and K. Hemalatha,
Technology, 3 (2014) 88.
doi.org/10.15623/ijret.2014.0306016

42. N. Das, S. Maity, J. Chakraborty, S. Pal,
S. Sardar and U. C. Halder, Indian J.
Biochem. Biophys., 55 (2018) 77.
nopr.niscpr.res.in/handle/123456789/44349

43. M. B. Khan, H. Khan, M. U. Shah and
S. Khan, Nat. Prod. Res., 30 (2016) 935.
doi: 10.1080/14786419.2015.1079909

44. A. S. Ibraheem and S. Malomo, World
Sci. News, 67 (2017) 250.

45. W. H. Abdulaal, BMC Biochem., 19
(2018) 10.
doi: 10.1186/s12858-018-0100-1