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Effect of pH, temperature and metal ions on amylase activity from Bacillus
subtilis KCX 006
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K. JAYA PRIYA
Department of Biotechnology, Prince Shri Venkateshwara Arts and Science College,
Gowrivakkam, Chennai 601302, Tamil Nadu, India
*Corresponding author
ABSTRACT
Under different growth conditions such as pH, temperature and metal ions, Bacillus
subtilis KCX006 was tested for amylase production and kinetic properties of amylase was
determined. The enzyme activity was more in the stationary phase and the value was
21.78 U/mL (at 30 h) and protein content was 0.303 mg/mL. The maximum amylase activity
(38 U/mL) was also found at stationary phase when the culture was grown at 37°C. The
enzyme was purified to homogeneity with an overall recovery of 24.2% and specific activity
of 4133 U/mg. Either acidic or alkaline condition significantly affected the growth and
enzyme production. Enzyme activity was more in reaction mixture containing NiCl2 and its
residual activity was found to be 117.5%. These results suggest possible application in food
and detergent industries where moderate pH and temperature are employed.
INTRODUCTION
Table 1
Activity of Amylase from Bacillus subtilis KCX006
Table 2
Enzyme Activity at Stationary Phase
Figure 1
Amylase Activity at Different pH
14
12
10
Amylase U/ml
8
6
4
2
0
-2 0 10 20 30 40
Time (h)
pH 4 pH 5 pH 6 pH 7
pH 8 pH 9 pH 10 pH 11
No amylase production was seen at pH 4, 10 and 11. These results indicate that the strain is
not able to tolerate acidic and alkaline pH (Table 3).
Table 3
Amylase Activity at Different pH
1 0 0 0 0 0 0 0 0 0
2 6 0.167 0.153 3.112 5.662 1.077 0.803 0.274 0.180
iii. Optimal temperature for amylase activity the activity was assayed. The result
Thermal stability was checked by incubating obtained was plotted with residual activity
the enzyme at different temperature of 40– versus time shown in Fig. 2 and Table 4.
70°C in a water bath upto 60 min. Samples
were withdrawn at regular 10 min interval and
Figure 2
Temperature Stability of Pure Amylase
80
(%)
60
40
20
0
0 10 20 30 40 50 60 70
Time (min)
40C 50C 60C 70C
Figure 3
Michalis–Menten Kinetic of Pure Enzyme
0.35
y = 0.0123x + 0.0422
0.3 R2 = 0.9487
0.25
0.2
0.15
0.1
0.05
0
0 5 10 15 20 25
The Km was found to be 0.291 mg/mL and Vmax was calculated as 23.69 U/mL
Figure 4
Effect of Metal Ions on Pure Amylase
15 0
Ni
C ont r ol Ba Ca Mn Hg Mg
R e si d u a l 10 0 Zn
Fe
Act ivit y
50 Cu
( %)
Ag
0
Cont r ol Ag Ba Ca Cu Fe
Mn Ni Hg Zn Mg