Characterization of novel amylase enzyme from mango (Mangifera indica cv. Chokanan) peel

Abstract

Amylase is one of the important industrial enzymes used in different types of industries such as food, detergent, pharmaceutical, pulp and paper. Mango peel could be a potential source of amylase, which has been extracted and purified from mango (Mangifera indica cv. Chokanan) peel using alcohol/salt, aqueous two phase system. In this study, the effect of temperature, pH, metal ions, inhibitors and surfactant agents on amylase activity and stability were investigated. In addition, purity and molecular weight of amylase was determined using sodium dodecyl sulphate gel electrophoresis. Amylase showed the highest activity and stability at 50°C for 20 min after enzyme incubation at different temperatures (20 to 90°C) in interval time. Amylase from mango peel is thermostable because more than 85% of enzyme activity was retained at temperatures of 20-55°C for 20 min. The amylase was incubated at pH 3-10 and the highest enzyme activity was obtained at pH 7.0. The enzyme activity was significantly decreased at pH 3.0 and 10 because of protein denaturation. Molecular weight of amylase from Mangifera indica L. cv. Chokanan was 42 kDa. Activity of amylase was significantly (p < 0.05) increased in presence of Ca 2+ but Zn 2+ and Cu 2+ reduced the enzyme activity due to replacing of calcium cation from the binding site of amylase. In addition, the effect on amylase activity was investigated at a concentration of 5 mM. The enzyme was completely deactivated in presence of carbodimine and p-chloromercuribenzoic acid whereas iodoacetamide did not show any significant (p <0.05) effect on amylase activity. Thus, amylase from mango peel with this unique characteristic has potential application in various kind of industries such as food, detergent, pharmaceutical and biotechnological applications.

Full text

Journal of Food, Agriculture & Environment, Vol.11 (3&4), July-October 2013 47
www.world-food.net
Journal of Food, Agriculture & Environment Vol.11 (3&4): 47-50. 2013
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Science and Technology
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Characterization of novel amylase enzyme from mango (Mangifera indica cv.
Chokanan) peel
Amid Mehrnoush * and Abdul Manap Mohd Yazid
Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, Malaysia. *e-mail: mehrnoush_amid@yahoo.com, mehrnoush@food.upm.edu.my
Abstract
Amylase is one of the important industrial enzymes used in different types of industries such as food, detergent, pharmaceutical, pulp and paper.
Mango peel could be a potential source of amylase, which has been extracted and purified from mango (Mangifera indica cv. Chokanan) peel using
alcohol/salt, aqueous two phase system. In this study, the effect of temperature, pH, metal ions, inhibitors and surfactant agents on amylase activity
and stability were investigated. In addition, purity and molecular weight of amylase was determined using sodium dodecyl sulphate gel electrophoresis.
Amylase showed the highest activity and stability at 50°C for 20 min after enzyme incubation at different temperatures (20 to 90°C) in interval time.
Amylase from mango peel is thermostable because more than 85% of enzyme activity was retained at temperatures of 20-55°C for 20 min. The
amylase was incubated at pH 3-10 and the highest enzyme activity was obtained at pH 7.0. The enzyme activity was significantly decreased at pH
3.0 and 10 because of protein denaturation. Molecular weight of amylase from Mangifera indica L. cv. Chokanan was 42 kDa. Activity of amylase
was significantly (p < 0.05) increased in presence of Ca2+ but Zn2+ and Cu2+ reduced the enzyme activity due to replacing of calcium cation from the
binding site of amylase. In addition, the effect on amylase activity was investigated at a concentration of 5 mM. The enzyme was completely
deactivated in presence of carbodimine and p-chloromercuribenzoic acid whereas iodoacetamide did not show any significant (p <0.05) effect on
amylase activity. Thus, amylase from mango peel with this unique characteristic has potential application in various kind of industries such as food,
detergent, pharmaceutical and biotechnological applications.
Key words: Amylase, mango peel, temperature stability, optimum temperature, pH stability, optimum pH, protein concentration, molecular weight,
inhibitors, metal ions.
Received 30 May 2013, accepted 16 October 2013.
Introduction
Amylase is one of the groups of enzymes which can catalyze
glycosidic bonds in starch to make oligosaccharides and glucose 1.
The enzyme plays an important role in plants due to starch
degradation during seed germination 2. Amylase is the basis for
several industrial processes such as bread making, brewing, and
glucose syrups. The enzyme is also used in detergents,
pharmaceuticals, in sewage treatment and in animal feed 3. Plant
amylase has unique characteristics such as wide substrate
specificity, high activity, and stability in a wide range of pH and
temperatures and thus, the enzyme is suitable and also inexpensive
for use in industry 4. For these reasons, there is growing interest
in seeking more plant sources of amylase. The tropical mango is a
widely-popular tropical fruit 5 and, mango peel can be used as an
abundant and cost-effective source to produce and extract natural
enzyme 6. Mango peel, which contains several enzymes
demonstrates a high level of pectinase, peroxidase, protease,
polyphenol oxidase, amylase and xylanase activities 7. There has
been much work done to extract enzymes like amylase and protease
and many others from mango pulp but there are very few reports
available on the use of mango peel, although polyphenol oxidase 8
and peroxidase 9 have been reported to be present in mango peel.
Thus, this study was done for the main purpose of characterizing
amylase from mango peel and observing temperature and pH
effects on its activity and stability. Furthermore, the effects of
metal ions and inhibitors on amylase activity were also studied.
Materials and Methods
Materials: Mango fruits were purchased from local market
(Selangor, Malaysia) at slightly under ripens commercial maturity
stage with the brix of 14. All chemicals and reagents used were of
analytical grade. Soluble starch, bovine serum albumin (BSA), p-
chloromercuribenzoic acid (PCMB), Bradford reagent, acrylamid,
β-ME (mercaptoethanol) and Coomassie brilliant blue G-250 were
obtained from Sigma Chemical Co. (St. Louis, U.K.).
Ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate
(SDS), 3,5-dinitrosalicylic acid (DNS), di-sodium hydrogen
anhydrous, sodium hydrogen phosphate monohydrate were
purchased from Merck (Darmstadt, Germany).

48 Journal of Food, Agriculture & Environment, Vol.11 (3&4), July-October 2013
Preparation of crude extract of amylase: Mango was peeled
with a stainless steel knife after washing with distilled water. The
mango peel was cut into the small cubes and blended with sodium
hydrogen phosphate (pH 6.9) for 2 min at high speed. The
homogenate was filtered through cheese cloth to remove the fiber.
Crude extract was kept at 4°C for future study 10.
Purification of crude extract from mango peel: Alcohol/salt
aqueous two phase system was employed for purification of
amylase from mango peel.
Amylase activity assay: Amylase activity in purified sample from
mango peel was assayed by the dinitrosalicylic acid method of
Miller 11 with some modifications. Purified enzyme was incubated
with 150 µl soluble starch (25% w/v) prepared in sodium phosphate
buffer (pH 6.9) containing 10 mM NaCl for 20 min at 37°C. The
reaction was stopped by the addition of 500 µl DNSA and heating
the tubes in a boiling water bath for 5 min. The absorbance was
determined at 540 nm by a spectrophotometer.
Protein concentration determination: Dye binding method as
described by Bradford 12 was used to determine the protein
concentration and the BSA was used as standard.
Determination of amylase optimum pH: Amylase was incubated
with 100 mM of different buffer (from pH 2 to 10) for determination
of optimum pH and residual amylase activity was measured. The
following system buffers were employed in this study: citrate–
phosphate (2.0–6.0), sodium phosphate (7.0–8.0) and sodium
carbonate (9.0–10.0) 13.
Determination of pH stability of amylase: The pH stability of
amylase was determined by measuring the residual activity
following maintenance for 24 h at 4°C in various pH from 3.0 to
9.014.
Determination of optimum temperature of amylase: Optimum
temperature of amylase was measured by residual activity of the
enzyme after incubation with substrate within a temperature range
of 20 to 90°C. Residual activity of amylase was determined by
using the method employed for amylase mentioned earlier 15.
Determination of thermal stability of amylase: Thermal stability
of amylase was determined by incubation of enzyme in a 50 mM
sodium phosphate buffer within temperatures of 10 to 90°C. At
intervals from 5 to 60 min, samples were removed and the residual
amylase activity was measured 16.
Determination of metal ion effect on amylase activity: Amylase
was incubated in the presence of 5 mM solution of salts of metal
ions Ca2+, Mg2+, Cu2+, Zn2+,Li+, Na+ and K+ and the residual enzyme
activity was determined. The samples with the presence of different
metal ions were incubated for 20 min at 50°C and the enzyme
activity was measured as previously described 17.
Substrate specificity assay: The effect of inhibitors such as PCMB,
iodoacetamide and carbodimide was investigated on amylase
activity at a concentration of 5mM. The enzyme with substrate
was incubated at 50°C for 20 min with the presence of various
chemical agents and the activity of the residual amylase was
evaluated as mentioned before 18.
Gel electrophoresis: Molecular weight and purity of amylase from
mango peel were determined by SDS-PAGE in electrophoresis
unit (Bio-Rad) using an acrylamide gel consisting of 12% of
resolving gel and 4.5% stacking gel 19. Protein bands were
observed using the silver staining method 20.
Statistical analysis: The data obtained from the measurements
were subjected to univariate analysis of variance (ANOVA) and
least significant difference tests (LSD) to determine the significant
differences among the samples. All measurements were carried
out in duplicate or triplicate for each sample. The experimental
data were reported as the mean ± SD of independent trials.
Significant differences among mean values were determined by
the Fisher’s test significance defined at (p < 0.05).
Results and Discussion
Optimum temperature and temperature stability of amylase:
Fig. 1 shows the effect of temperature on amylase activity and
stability from 10°C to 70°C in sodium phosphate buffer.
Enhancement of temperature from 10 to 50°C caused an increase
of amylase activity and the highest enzyme activity was obtained
at 50°C for 20 min (Fig. 1A). Denaturation of enzyme at higher
temperature could be the main reason for the decrease in amylase
activity at temperatures above 55°C. Temperature stability of
amylase was examined by measuring the residual enzyme activity
at different temperatures. Eighty-five percent of enzyme activity
was retained when it was incubated between 10°C and 60°C but
stability of enzyme markedly decreased at evaluated temperatures
above 65°C (Fig. 1B).
Optimum pH and pH stability of amylase: Amylase activity was
measured using starch soluble as substrate at different pH from 2
to 10 and the result is presented in Fig. 2. The 50% of amylase
0
20
40
60
80
100
0 20406080100
Residual amylase
activity (%)
Temperature (C)
A
0
20
40
60
80
100
0 20406080100
Residual amylase
activity (%)
Temperature (C)
B
Figure 1. Effects of temperature on optimum activity (A) and stability
(B) of amylase from mango peel at different temperatures. The data
plotted correspond to the mean ± SD of three replicates.

Journal of Food, Agriculture & Environment, Vol.11 (3&4), July-October 2013 49
activity was retained at pH 4 to 8 with the highest enzyme activity
at pH 7 (Fig. 2A). After incubation of the amylase for 24 h at 4°C in
the presence of sodium phosphate buffer, the enzyme activity
was stable at pH 5 to 9 with optimum amylase activity at pH 7.
Less than 25% of the initial enzyme activity remained when enzyme
was incubated at pH 3 and 9 for 24 h (Fig.2B), which could be due
to denaturation of amylase at extremes of pH. Similar observation
was reported by Najafi 21 who worked on amylase from Bacillus
subtilis.
Effect of metal ions on the activity of amylase: The effect of
various metal ions on activity of amylase was determined at pH 7
and 50°C by addition of the respective cations to the reaction
mixture (Table 1). The enzyme activity was significantly (p < 0.05)
increased in the presence of Ca2+ whereas Mg2+ had negligible
effect on amylase activity (Table 1). Amylase activity was
significantly (p <0.05) inactivated in the presence of Zn2+ and
Cu2+. The enzyme inactivation could be due to its binding to either
catalytic residues or by replacing of Ca2+ from the binding site of
amylase. The ability of Ca2+ ions to interact with negatively charged
amino acids (e.g. aspartic and glutamic acids) is the main reason
to enhance activity and stability of amylase as well as maintenance
of enzyme confirmation 22. It should be considered that, Li+, Na+
and K+ did not have any significant effect (p < 0.05) on amylase
0
20
40
60
80
100
120
024681012
Residual Amylase
activity (%)
pH
A
0
20
40
60
80
100
120
024681012
Residual Amylase
activity (%)
pH
B
Figure 2. Effects of pH on the activity (A) and stability (B) of amylase
from mango peel at different pH. The data plotted correspond to the
mean ± SD of three replicates.
Reagent Concentration (mM) Relative activity (%)
None _ 100±0.0
a
Ca
+2
5 110±0.6
b
Mg
2+
5 98±1.1
a
b
Li
+
5 100±0.3
c
Na
+
5 100±0.5
c
K
+
5 100±1.1
c
Cu
2+
5 72.3±0.2
d
Zn
2+
5 61.4±2.1
d
Table 1. Effect of metal ions on activity of amylase
from mango peel.
Means with the same letter are not statistically different (p > 0.05). The results
are expressed as a mean of three readings with an estimated error of ±10%.
Reagent Concentration (mM) Relative activity (%)
None _ 100±0.0
a
EDTA 5 120±0.2
b
iodoacetamide 5 97±1.2
a
b
carbodimine
5 52±0.3
c
p-chloromercuribenzoic
5 37±0.7
d
Table 2. Effect of inhibitors reagent on activity of amylase from
mango peel.
Means with the same letter are not statistically different (p > 0.05). The results are expressed as a mean
of three readings with an estimated error of ±10%.
Amylase
kDa
66.4
55.6
42.7
34.6
27.0
20.0
14.3
6.5
M 1 2 3
Figure 3. SDS-PAGE profile of amylase from mango peel.
activity. The result was in agreement with Shafiei et al. 23 who
worked on amylase from halophilic bacterium.
Effect of specific reagents on amylase activity: The amylase was
completely deactivated in the presence of PCMB and carbodimide.
Meanwhile, iodoacetamide only partially influenced (p < 0.05) the
activity of amylase as it had very insignificant effect (Table 2).
The inhibition effect of PCMB and carbodimide was due to the
presence of acidic amino acids such as aspartic at the active site.
It should be considered that amylase activity in the presence of 5
mM EDTA was increased by 20% due to structural rigidity of the
enzyme and tight bounding of Ca2+ and other divalent cations to
the enzyme. The removal by EDTA treatment of divalent cation
from enzymes is very difficult. There was the presumption that
increased conformation flexibility, due to the effect of chelators
on its native conformation, was responsible for the increase in
enzyme activity.
Molecular weight of amylase: As presented in Fig. 3, the
molecular weight of purified amylase was found to be 41 kDa on
12% SDS gels. It was eluted as a single peak of activity which
connotes the degree of homogeneity of the purified enzyme.
Conclusions
Characterization of amylase from mango peel (Mangifera indica
cv. Chokanan) was done in this study. Mango peel as one of the
major bio-products of the mango processing industry has been
found to be natural source of amylase with interesting
characteristics. The enzyme with high activity and stability in
high temperature and its stability toward different metal ions
showed potential for application in various industries. In addition,
the high stability of the enzyme against EDTA confirmed its
structural rigidity, which is one of the important parameters for
use in different types of industries such as the detergent industry.

50 Journal of Food, Agriculture & Environment, Vol.11 (3&4), July-October 2013
Thus, these properties make amylase from mango peel an enzyme
of great importance in industrial and biotechnological applications.
Acknowledgements
E-Science Fund (05-01-04-SF1036) from the Ministry of Science,
Technology and Innovation of Malaysia (MOSTI) supported this
study.
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