Indian Journal of Biotechnology
Vol 13, October 2014, pp. 551-554
Partial purification and characterization of
amylase and starch from Elephant foot yam
[Amorphophallus campanulatus
(Roxb.) Blume]
C Preethi* and T Veerabasappa Gowda
PG Department of Biotechnology, J S S College of Arts,
Commerce and Science, Mysore 570 025, India
Received 17 January 2013; revised 29 May 2013;
accepted 21 June 2013
Amylase (EC 3.2.1.2) and starch from Amorphophallus
campanulatus (Roxb.) Blume (Elephant foot yam) were partially
purified. Kinetic characterization of yam amylase at its optimum
conditions of activity was carried out. Its activity profile was
compared with that of salivary amylase upon yam and commercial
potato starch as substrates. A differential behaviour in the activity
of yam amylase compared to salivary amylase was observed.
Presence of isoforms of amylase in yam was detected. The
partially degraded yam starch appeared to be better substrate
compared to native starch. Ca2+ ions stimulated the yam amylase
activity upon endogenous starch than Na+ and K+ ions.
Keywords: Amylase, endogenous starch, isoforms, kinetic
characterization, Zymogram
Most of the carbohydrates found in nature are
polysaccharides of high mol wt. On hydrolysis with
acid or specific enzymes, these polysaccharides yield
monosaccharides
or
simple
monosaccharide
derivatives. Among the storage polysaccharides,
starch is most abundant in plants and is usually
deposited in the form of large granules in the
cytoplasm of cells. Starch is composed of amylose
and amylopectin1. Amylases, the starch degrading
enzymes, are widely distributed in microbial, plant
and animal kingdom. Historically, the term amylase
was used to designate enzymes hydrolyzing α-1,
4-glycosidic bonds of amylose, amylopectin and
glycogen1-3. They act by hydrolyzing bonds between
glucose units yielding characteristic products of the
particular enzyme involved. A number of enzymes
associated with degradation of starch and related
polysaccharide structures have been detected and
studied4. In the present study, partial purification and
——————
*Author for correspondence:
Tel: +91-821-2480117
Mobile: +91-9739482355
E-mail: [email protected]
kinetic characterization of amylase from yam
[Amorphophallus campanulatus (Roxb.) Blume)] was
carried out and it was compared with the activity of
salivary amylase to understand the differential
behaviour of yam amylase upon endogenous starch,
i.e., yam starch.
The yam was obtained from Mysore district of
Karnataka state (India). Sephadex G-75 from Sigma
Chemicals, USA; acrylamide, bis-acrylamide; Tris,
commercial starch, Glycine from Sisco Research
Laboratory, Pvt. Ltd., Mumbai; potassium iodide,
glacial acetic acid, ammonium sulphate, iodine,
copper(II) sulphate, sodium acetate, di-sodium
hydrogen phosphate, sodium di-hydrogen phosphate
were from commercial sources of analytical grade.
Amylase was extracted from the peel of yam; 100 g
peel in 50 mL of water was centrifuged at 5000 rpm
for 5 min at 4oC to remove debris. Supernatant was
used as crude source for amylase. The aqueous extract
of enzyme was subjected to ammonium sulphate
precipitation (25%) and was allowed to stand at 4oC
for a minimum of 6 h. The pellet was separated by
centrifugation at 4oC. Saturation of the supernatant
was raised to 30%. Again the pellet was obtained by
centrifugation and the supernatant obtained was
further raised up to 45% saturation with ammonium
sulphate. Pellets were then separately suspended in
0.1 M acetate buffer, pH 5. Thus, the crude enzyme
was separated into three fractions (0-25%-I, 25-30%-II
and 30-45%-III saturation of ammonium sulphate) in
0.1 M acetate buffer, pH 5. The starch from yam was
isolated by the method described earlier7. Amylase
was assayed according to the procedure of Bernfeld1.
Absorbance was read at 540 nm. Protein
concentration was estimated according to Lowry et al8
with bovine serum albumin as reference protein.
Absorbance was read at 660 nm. The enzyme activity
(activity/mL) and specific activity (activity/mg of
protein) was calculated. The fraction III (6 mg) was
loaded on to a Sephadex G-75 column (length 75 cm,
radius 0.5 cm, with bed volume of 60 mL). The
protein was eluted with 0.1 M acetate buffer, pH 5.
The fractions were screened at 280 nm in a
spectrophotometer. Amylase activity was estimated
for peak tubes and the specific activities were
calculated. The protein in tubes with significant
activity was concentrated by lyophilization.
552
INDIAN J BIOTECHNOL, OCTOBER 2014
The time kinetics was followed for salivary
amylase and yam amylase (Table 1) on both yam and
commercial starch as substrates. Salivary amylase
sample was prepared by diluting 200 times the freshly
collected saliva from a healthy individual before
lunch time. Diluted saliva served as a source of
salivary amylase.
a) Colorimetric Method: The activity of salivary
amylase and yam amylase were determined
separately by incubating amylases with gelatinized
starch (1%), in sodium acetate buffer 0.1 M, pH 5
for Yam amylase and phosphate buffer 0. 1 M,
pH 7.0 for salivary amylase and were incubated
at their optimum temperature (50°C & 37°C,
respectively). The reaction was stopped at regular
time intervals.
b) Turbidometric Method: The activity of salivary
amylase and yam amylase on yam starch were
estimated separately by incubating amylases with
gelatinized yam starch (1%), in sodium acetate buffer,
0.1 M, pH 5 for yam amylase and phosphate buffer,
0.1 M, pH 7 for salivary amylase and were incubated
at their optimum temperature of activity. The turbidity
clearance at regular time intervals were recorded
using a turbidometer.
Effect of substrate concentration on yam amylase
was estimated by keeping the amount of enzyme
constant and substrate concentration was gradually
increased. The fraction III (200 µL) was incubated
with sodium acetate buffer, 0.1 M, pH 5 and with
gelatinized starch (0, 0.03, 0.06, 0.3, 0.6, 0.9, 1.21 ×
10-3 µmol) at the optimum temperature of activity for
30 min. To determine the effect of metal ions on yam
amylase activity, ammonium sulphate fraction III
(200 µL) was incubated with 500 µL of 5 mM
solution of the following metal chlorides: Ca2+, Ba2+,
K+, Na+, Fe3+, Hg2+ with gelatinized starch, 1%,
500 µL in 0.1 M sodium acetate buffer, pH 5 at its
optimum temperature for 60 min and the activity was
estimated as before. The enzyme activity without
metal ions was taken as 100%. The relative activities
were calculated for each metal ion.
Polyacrylamide gel electrophoresis (PAGE) with
7.5% gel under native basic conditions was carried
out. Samples (150 µg) of crude along with ammonium
sulphate fractions I, II, and III were run
simultaneously and stained for both protein and
enzyme activity. The gels were stained for protein
with CBB (Coomassive brilliant blue) staining9 and
enzyme activity staining10,11. For Zymogram, the gel
after electrophoresis was incubated in gelatinized
starch (2%) prepared in sodium acetate buffer 0.1 M,
pH 5 for 1 h. Subsequently, the gel was stained with
iodine reagent (3%).
Yam tubers are rich sources of starch. The starch
was isolated from yam according to the mentioned
procedure and its purity as per acid hydrolysis was
95%. The percentage of starch in wet tuber was 10%.
The peel water extract showed amylase activity. The
crude extract upon ammonium sulphate fractionation
yielded fractions I, II and III. Supernatant showed
diminished activity for amylase. The activity of fractions
along with the crude is represented in (Table 2).
Fraction III that exhibited highest activity yielded
several peaks upon gel filtration on Sephadex (Fig. 1).
The peaks 2, 3, 4 and 6 showed amylase activity,
which may represent the presence of isozymes. Time
kinetics graph (Fig. 2a) though showed similar type of
activity for both the amylases on commercial starch,
salivary amylase had more efficiency towards the
product release than yam amylase. Also, salivary
amylase had similar activity pattern on both
commercial and yam starch (Figs 2a & b). Yam
amylase showed difference in its time kinetics pattern
on yam starch (Fig. 2b) compared to potato starch.
Initially (up to 60 min), the degradation of yam starch
Table 1—Physical characteristics of salivary and yam amylase
Characteristics
Yam amylase
Salivary amylase
5.0
50°C
6.9
37°C
Optimum pH
Optimum temperature
Table 2—Summary of purification of yam amylase and its activity
Steps
Enzyme
activity
(µmol/
min/mL)
Total
protein
(mg/mL)
Crude
0.28
1.325
Specific Enzyme
activity
unit*
(µmol/
min/mg)
0.21
4.8
Ammonium
sulphate
precipitates
0-25%
25-30%
30-45%
0.07
0.09
0.35
1.5
1.0
3.35
0.05
0.09
0.11
20
11.1
9.1
Sephadex G-75
fractions (Peaks)
2
0.05
0.25
0.20
5.0
3
0.027
0.1
0.27
3.7
4
0.049
0.5
0.098
10.2
6
0.060
0.125
0.48
2.08
*One unit of amylase activity was defined as the amount of
enzyme producing 1 µmol reducing sugar per min under standard
assay conditions
SHORT COMMUNICATIONS
was low. Then the velocity picked up slowly.
However, it was not comparable with its action on
potato starch. It may be due to the incompatibility
between the substrate (yam starch) and the enzyme.
Further, the partially degraded yam starch appeared to
be the better substrate compared to the native starch.
553
This fact was evident from the time kinetic curve. The
turbidometric measurement of time kinetics for yam
amylase was also similar to that of colorimetric
measurement of activity for its endogenous substrate
(Fig. 3). The results of PAGE and Zymogram (Fig. 4)
confirmed the presence of amylase activity and
presence of isozymes of amylase.
We propose to explain this as a requirement of the
sprouting yam. The rate of degradation should match
the rate of growth. Since, the degradation products are
expected to serve the supply of energy source and
precursor to the building structural blocks of the
growing plant. Hence, slow degradation of reserve
nutrients is probably desired to sustain slow growth
Fig. 1—Elution profile of fraction III on Sephadex G-75 column.
Fig. 3—Time kinetics for salivary and yam amylases on
endogenous (yam) starch by turbidometric method.
Fig. 2—Time kinetics (colorimetric method) for salivary and yam
amylases on: (a) Commercial starch, & (b) Yam starch.
Fig. 4—Gel profile for amylase fractions with crude sample
(150 µg): (a) Basic PAGE, & (b) Zymogram. [Lane 1, Crude
sample; Lane 2, Fraction I; Lane 3, Fraction II; Lane 4, Fraction
III; Arrows indicate the corresponding protein bands on native gel
as well as on Zymogram]
INDIAN J BIOTECHNOL, OCTOBER 2014
554
Table 3—To compare km of amylase from different origin
Amylase
km
(µmol)
Ref.
α-Amylase from Bacillus
licheniformis mutant
α-Amylase from Penicillium
citrinum HBF62
β-Amylase from Oryza sativa
β-Amylase from Curculigo pilosa
Yam amylase
46.11
12
1.11
13.
16.67
8.33
0.11
14
15
Present
precursors for the building blocks of large molecules
at a required rate for the plant.
Acknowledgement
Authors are grateful to Post Graduation
Department of Biotechnology, J S S College of Arts,
Commerce and Science, Mysore, India for providing
facilities and assistance to carry out this project.
References
1
Table 4—Effect of metal ions (5 mM) on Yam amylase
Metal ions
Enzyme activity
(µmoles/min/mL)
% activity
Control
0.074
100
Ca2+
0.148
200
Ba2+
0.120
162.1
K+
0.064
86.4
Na+
0.027
36.4
Fe3+
0.009
12.1
Hg2+
0.00
0
Values are average of 3 independent experiments, error less than 2%
pattern of the plant. On the other hand, salivary
amylase is a digestive enzyme it is required to
degrade the substrates immaterial of the source. The
estimated km value (0.11 µmol) for yam amylase
was lowest when compared to km values for
other amylases (Table 3)12-15. The size of km indicates
the tightness with which the enzyme binds to the
substrate. Lower the kmvalue, higher is the affinity of
the enzyme for the substrate. The process of
enzyme action for such enzymes is slow. Therefore,
the rate of release of products is slow for such
pairs. On the contrary higher the km value, faster
is the release of products. The enzyme substrate
combination is evolved to regulate metabolic
requirements.
Various metal ions, such as, Ca2+, Ba2+, K+, Na+,
3+
Fe and Hg2+ at 5 mM concentration influenced
the enzyme activity (Table 4). Ca2+and Ba2+ were
found to have both activating and stabilizing
effect as indicated by increased activity. The metal
ions Hg2+, Fe3+, Na+ inhibited the activity of
amylase perhaps indicating the involvement of
SH-group/cysteine in the catalytic function of the
enzyme.
The present investigation reveals the regulation of
enzyme activity by evolving enzyme substrate
combination to meet the metabolic requirement of the
growing plant. Stored nutrient starch is degraded by
amylase to release the energy providing products and
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Bernfeld P, Amylase α and β, in Methods in enzymology,
edited by S P Colowick & N O Kaplan (Academic Press Inc.,
New York, USA) 1955, 149-158.
Fisher E H & Stein E A, α-Amylase, in The enzyme, 2nd edn
(Academic Press Inc., New York, USA) 1960, 133-143.
Myrback K & Neumuller G, Amylases and the hydrolysis of
starch and glycogen, in The enzyme: Chemistry
and mechanisms of action, vol 1, 1st edn, edited by
J B Sumner, K Myrback (Academic Press Inc., New York,
USA) 1950.
Buonocore V, Caporale C, De Rosa M & Gambacorta A,
Stable, inducible thermoacidophilic α amylase from Bacillus
acidocaldarius, J Bacteriol, 128 (1976) 515-521.
Fogarty W M & Kelly C T, Developments in microbial
extracellular enzymes, in Enzyme and fermentation
biotechnology, edited by A Wiseman, (John Wiley and Sons,
New York, USA) 1979, 45-108.
Aiyer P, Amylases and their applications, Afr J Biotechnol,
4 (2005) 1525-1529.
Hodge J E & Hofreier B T, Determination of reducing sugars
and carbohydrates, in Methods in carbohydrate chemistry,
edited by R L Whistler & J N Be Miller (Academic Press
Inc., New York, USA) 1962, 17.
Lowry O H, Rosebrough N J, Farr A L & Randall R J,
Protein measurement with the Folin phenol reagent, J Biol
Chem, 193 (1951) 265-275.
Walker J M, Non denaturing polyacrylamide gel
electrophoresis of proteins, in Protein protocols handbook,
edited by J M Walker (Humana Press Inc., Totowa, NJ,
USA) 1996, 51-55.
Acevedo E & Cardemil L, Regulation of α-amylase
isoenzymes expression in Araucaria araucona by gibberellic
and abscisic acids, Phytochemistry, 44 (1997) 1401-1405.
Manchenko G P, Handbook of detection of enzymes on
electrophoretic gels (CRC Press, New York) 1994, 187.
Ikram-ul-Haq, Javed M M, Hameed U & Adnan F, Kinetics
and thermodynamic studies of alpha amylase from Bacillus
licheniformis mutant, Pak J Bot, 42 (2010) 3507-3516.
Metin K, Koc O, Ateslier Z B B, & Biyik H H,
Purification and characterization of α-amylase produced
by Penicillium citrinum HBF62, Afr J Biotechnol, 9 ( 2010)
7692-7701.
Matsui H, Chiba S & Shimomura T, Purification and some
properties of active ß-amylase in rice, Agric Biol Chem,
41 (1977) 841-847.
Dicko M H, Searle-van Leeuwen M J F, Beldman O G,
Hilhorst R et al, Purification and characterization of betaamylase from Curculigo pilosa, Appl Microbiol Biotechnol,
52 (1999) 802-805.