1
SUPPLEMENTARY MATERIAL
PURIFICATION
AND
CHARACTERIZATION
OF
ALPHA
AMYLASE
PRODUCED BY MUTANT STRAIN OF ASPERGILLUS ORYZAE EMS-18
*ROHEENA ABDULLAHa AND IKRAM-UL-HAQb
*aDepartment of Biotechnology Lahore College for women university, Lahore, Pakistan.
b
Institute of Industrial Biotechnology,GCU Lahore, Pakistan
*Corresponding author e.mail: roheena_abdullah@yahoo.com
Abstract: Alpha amylase produced by mutant strain of Aspergillus oryzae EMS-18 has been purified to
homogeneity as judged by SDS gel electrophoresis. The enzyme was purified by using 70% ammonium
sulphate precipitation followed by anion exchange chromatography on DEAE-Sephadex column and gel
filtration on Sphadex G-100. An enzyme purification factor of 9.5 fold was achieved with final specific
activity of 1987.7U/mg protein and overall yield of 23.8%. The molecular weight of purified alpha amylase
was estimated to be 48kDa by SDS-PAGE. The purified enzyme revealed an optimum assay temperature,
and pH 40○C and 5.0 respectively. Except Ca++ all other metal ions such as Mg, Mn, Na, Zn, Ni, Fe, Cu,
Co, Ba were found to be inhibitory for enzyme activity.
Table SI
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Table S1: Purification summary of alpha amylase produced by mutant strain of A. oryzae EMS-18 by using
ammonium sulfate
Ammonium
Total units (U)
sulfate
Total protein
Specific
Recovery or %
Fold
(mg)
activity (U/mg)
yield
purification
fractionation
(%)
Crude broth
25000
120
208.3
100
1.0
0-20
-
-
-
-
-
0-40
-
110
-
-
-
0-50
18500
100
185
74
0.8
0-60
17300
90
192.2
69.2
0.92
0-70
22479
80
280.9
89.9
13
0-80
16200
78
207.6
64.8
0.99
0-90
-
-
-
-
-
Table S2
TableS2: Step wise purification profile of alpha amylase produced by mutant strain of A. oryzae EMS18
Purification steps
Crude broth
Ammonium sulphate
Enzyme
Total protein
Specific activity
Recovery or
Fold
activity (U)
(mg)
(U/mg)
% yield
25000
120
208.3
100
1.0
22479
80
280.9
89.9
1.3
10113.1
18
561.8
40.4
2.6
5963.1
3
1987.7
23.8
9.5
purification
fractionation (70%)
DEAE Sephadex
chromatography
Sephadex G-100
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Figure S1:
Elution pattern on Sephadex – DEAE
4
2.5
7000
6000
2
1.5
4000
3000
1
2000
0.5
1000
0
0
1
2 3
4 5
6 7
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Fractions
Absorbance
Enzyme activiity (U)
Figure S2: The elution profile on Sephadex G-100
Enzyme activity (U) .
Absorbance ( 280 nm)
5000
5
Molecular weight
Electrophoretic mobility of purified alpha-amylase with reference to mobilities of protein marker (SMO
313) Fractions was analyzed on SDS-polyacrylamide gel. The mobility of the purified alpha-amylase
corresponded to a molecular weight of 48kDa (Figure S3).
Figure S3: SDS-PAGE analysis of pooled fractions of ion exchange chromatography and
ammonium sulfate fractionation
Lane 1; Marker, lane 2; Ammonium sulfate fractionation, lane 3; pooled fractions of ion exhange
chromatography.