1. Characterisation of a metal ion dependent, detergent activated α-amylase from halotolerant Bacillus acquimaris
Anupama A and Jayaraman G
School of Bio-Sciences and Technology, VIT University, Vellore – 632 014.
Introduction
On the advent of the current trends in various industries the use of en-
zymes at extreme conditions like high and low temperatures as well as
pHs is of utmost use. In view of the present scenario, α-amylases occupy
25 % of the world’s enzyme market. The organisms that survive in ex-
treme conditions are called Extremophiles and the enzymes from them are
known as Extremozymes. Recently several halotolerant strains have been
isolated from Kumta coastal area, Karnataka, India. In this study one of
the bacterial strain named Bacillus aquimaris VITP4 (GenBank ID:
FJ687490) (was explored for the production of the enzyme α-amylase.
Materials and Methods
Production of α-amylase
B. aquimaris strain VITP4 was cultured on Zobell Marine Agar media + 1
% soluble starch
Capability of α-amylase production - using KI reagent
Clear zone of hydrolysis was seen along the culture margin - positive for
α-amylase production
Production of α-amylase using Zobell marine broth + 1 % soluble starch
Conditions - 37 o
C, 150 rpm and 48 hr of incubation
Assay of α-amylase
Optimization of the parameters of production medium
Media pH 7.0 to 9.0 samples harvesting every 6 hr interval
Assay of α–amylase
Temperature 30 to 60 o
C sample harvesting every 6 hr interval
Assay of α–amylase
Molecular weight determination 12 % SDS - PAGE performed with
Molecular weight markers.
Silver staining was carried out to visualize the resolved bands.
Effect of Salt concentration, metal ions and other additives of enzyme
activity
1.Assay mixture to determine the effect of salt contains:0M - 5M NaCl in
20 mM Sodium phosphate buffer (pH 6.9).
2.Assay mixture to determine the effect of metal ions : various conc. of
CaCl2 is added to the assay mixture.
3. Assay mixture to determine the effect of additives : various additives
added.
Incubation time 15 min at optimum temp. and pH - residual activity de-
termined.
The activity of the enzyme without any additive taken as 100 %.
α-amylase assay procedure
Using Dinitrosalicylic acid
method - to measure the
amount of reducing sugars
produced (540 nm)
By estimating the reduc-
tion in the starch-iodine
color intensity, reagent
used KI solution (550 nm)
Results
Initial confirmation
B. aquimaris strain VITP4
streaked to confirm the
presence of amylase using
KI reagent.
Enzyme activity study
Study of enzyme activity
Revealed at 24 hr of
Incubation production
Of –amylase is high.
Optimum pH and
Temperature for α–
amylase production
pH - 8.0 (49.7%)
Temperature - 40 o
C
(54.3 %)
Optimum pH and tem-
perature for α-amylase
activity
pH- 8.0 (83.5 %)
Temperature - 40 o
C
(86.5 %)
Thermostability of α-
amylase Highest half-life Effect of Salt concentration Tolerable
of the enzyme was observed at its optimum temp. Salt conc. is 3 M with 75 % relative activ-
i.e.,40 o
C (156±5). activity.
Molecular weight determina-
tion
SDS-PAGE (12%) made us unde-
rstand the approximate molecu-
lar weight of α-amylase as
54 kDa.
Myosin (205 kDa), Phosphorylase (97.4 kDa), bovine
serum albumin (66 kDa), ovalbumin (43 kDa). Lane 2:
Crude α-amylase sample.
2 mM 5 mM 10 mM
Tween20 42 ± 1 6 ± 0.3 -
CTAB 4 ± 0.2 28 ± 0.5 160 ± 1.5
SDS 2 ± 0.1 - -
EDTA 20 ± 0.4 - -
β-
mercap-
toethanol
48 ± 1.2 36 ± 0.7 18 ± 0.4
Temperature (o
C) Relative activity (%)
after 1 h incubation
t1/2 (min)
40 94 ± 1 156 ± 5
50 93 ± 120 ± 4
60 89 ± 1.5 108 ± 3
70 83 ± 1.5 103 ± 1
80 84 ± 1.7 73.2 ± 0.7
90 80 ± 1.9 43.5 ± 0.7
Time (h)
0 10 20 30 40
log10CFU/ml
5
10
15
20
25
30
OD600nm
0.0
0.5
1.0
1.5
2.0
2.5
3.0
pH
7.0 7.5 8.0 8.5 9.0
Enzymeactivity(%)
0
10
20
30
40
50
60
Temperature (oC)
30 35 40 45 50 55 60
Enzymeactivity(%)
0
10
20
30
40
50
60
Temperature (oC)
20 30 40 50 60 70 80 90
Enzymeactivity(%)
0
20
40
60
80
100
pH
4 5 6 7 8 9 10 11
Enzymeactivity(%)
10
20
30
40
50
60
70
80
90
Time (h)
0 10 20 30 40 50 60
Enzymeactivity(%)
0
10
20
30
40
50
60
[Salt] (M)
0 1 2 3 4 5 6
Relativeactivity(%)
0
20
40
60
80
100
120
[metal ion]
c 0.01 0.1 1 10
Relativeactivity(%)
0
20
40
60
80
100
120
Growth Kinetics Study of growth kinet-
ics revealed logarithmic growth of the bac-
teria with 6 hr of exponential phase.
Effect of calcium ion and other additives:
Lower concentration of calcium ion en-
hanced the enzyme activity to 105 % rela-
tive activity when compared with no
metal ion. This says that B. aquimaris
strain VITP4 α-amylase is calcium de-
pendent.
Other additives like CTAB at higher con-
centrations enhances the activity to 160 %
SDS, EDTA, Tween 20 in
hibits the enzyme activity
as their concentration in-
creases.
Discussion Conclusion
B. aquimaris strain VITP4 can produce α-amy- α-amylase produced is metal ion dependent
lase at optimum pH 8.0 and temperature 40 o
C. thermostable and detergent activated. It can
The characterization of the enzyme shows opt. be used in various industrial applications.
activity at pH 8.0 and temperature 40 o
C again. Purification of the enzyme is in progress
The thermostability studies revealed maximum which could make the enzyme apt for
half-life at opt. temp. and tolerable salt conc. wide array of uses.
is 3 M. Requirement of Ca ion to enhance the
activity was observed. Molecular weight was Acknowledgements The authors thank the
approximated as 54 kDa by SDS-PAGE. VIT University for providing the facility
and support.