((2011)) B
((
))
http://www.basra-science-journal.org:
ISSN 1817 2695
**
-
*
-
-
*
*
alfekaiki@yahoo.com
2011-9-25
2011-3-30
-
.
0.025
7.4
%70-30
.
G-100
40000
.20.72
–
20.71
. SDS
.
20
(7-5 )
5
º 80
0.001
.
/
º 90
30
%30
%0.55
255.75
–
Km
–
:
-1
3)
(
Hydrolysis
)Amylolytic Enzymes
-
-
.(1)
. (13)
92
(10
...
-
:
.(18)
.
-
.1
0.5
Hordeumvulagare
-
.
/
(
2
/ )
-2001)
99
( 2002
-
Disc (Disc PAGE)
Poly
acrylamide
gel
.
-
electrophoresis
(7)
(10)
3-2
-
1-2
Activity of Amylases
Determination
(20)
99
:
(7 )
0.1
4-2
(5)
°40
2
2
.
.(
0.01
/
)4
. (9)
(30 20 10)
.(19)
2-2
(90-60)
100
20
0.025
) (5/1)
. ( 3)
5-2
pH
(7.4)
.
30
(9-4)
pH
/
. % (70 – 20 )
(14)
40
(
(×802.5)
6-2
93
Sephadex G-100
((2011)) B
((
7-2
(Km)
(14)
))
(14)
40
Vmax
30
(9-4)
(14)
.
-2
(1)
10.8
:
G-100
(1)
7.4
0.025
(
15
280
( /
.( /
/
70
–
.
) 950
) (5:1)
( /
4042
) 9700
(
) 9160
/
)
-
–
- 30 )
99% (70
.
(III , II, I)
20
II,
III
/
-
(17)
.
II , I
(
/
) 13909
(
3.44
30
.(1)
6700
(
)15300
/
) 83750
( /
31.54
– 30)
)
(4)
-
.
%20.27
(
20.71
94
/
(%60
) 8.6
...
-
:
99-
(%)
-
/
)
(
(
/
)
(
(1 )
/
( )
)
100
1
970000
4042
2.4
9700
100
31.54
3.44
306000
13909
1.1
15300
20
20.72
20.71
201000
83750
0.08
6700
30
280
" ( /
% 70-30
G100
)
10000
0.18
9000
0.16
8000
0.14
7000
0.12
6000
0.1
5000
0.08
4000
0.06
3000
0.04
2000
0.02
1000
(280)
0
0
1
5
9
13
17
21
Sephadex G7.4
25
29
33
37
41
45
)
49
53
99
/
2
57
61
65
69
73
-
77
81
85
89
: ( 1)
(2.5×80)
100
0.025
(
/ ) 0.5
-
1-3
(2)
%(70 –30)
(A-2)
(C-2)
/
)
0.2
(6)
.
.
(B-2)
95
((2011)) B
((
))
.
-
- amylase
+
B
C
-
.
C
A
B
A
:(2)
99-
=A
% 70 – 30
= B
=C
G-100
-
-
.
2-3
SDS- Gel
Electrophorsis
-
.(12)
(3)
48000
.
55200
(Relative mobility )
(4)
-
SDS
.
SDS-PAGE
.
..
45000
-
96
40000
...
-
:
4.9
y = -0.8816x + 4.8872
Bovin Serum Albumin (67KD)
4.8
4.7
Ovah albumin (43 KD)
-amylase( 40 KD)
4.6
Pepsin ( 34 KD)
Log (MW )
4.5
4.4
Trypsin inhibitior (20 KD)
4.3
4.2
Lysozyme (14 KD)
4.1
0
0.1
0.2
0.3
0.4
(Rm )
0.5
99-
(
0.6
)
0.7
0.8
0.9
-
1
(3)
SDS
3-3
99º(70–20)
(4)
(14)
( /
)39000
50
( /
) 20000
70
(9)
.
50000
40000
20000
(
/
)
30000
10000
0
0
5
10
15
20
25
30
35
40
45
50
55
60
( )
( 99 -
–
97
(4)
65
70
75
((2011)) B
((
))
4-3
º(60-45)
-
º65
(5)
99
30 20
%70
%95
º (75 – 45)
º75
30
(30
20
10 )
%10
10
20
30
120
100
80
(%)
60
40
20
0
1
45
.
2
50
3
55
4
60
5
65
99
6
70
-
7
75
:(5)
5-3
pH
–
99
. (9 -3)
(ES)
(5)
(6)
. (EP)
5
.
(5 –3)
.(
/
)14350
-
(11)
98
(5)
...
-
:
–
.
16000
14000
12000
)
10000
8000
/
6000
4000
2000
0
0
1
2
3
4
99-
5
6
7
8
-
9
10
:(6)
6-3
pH
(7)
.
% 49.9
%100
(4)
(7-5)
120
100
80
(%)
60
40
20
0
0
99
1
2
3
4
5
–
6
7
8
:(7)
99
9
10
((2011)) B
((
))
(9)
. (6 -5)
-
% (12.6)
(4)
(8 -4)
. (6.5 -6)
(5)
(14)
%90
(15 )
%85
-
(7)
. ( /
(12)
7-3
) 0.83
-
%0.33
Km
.
Substrate
.A,B,C,D8)
Vmax
Vmax Km
(
)
/
% 0.55
255.75
(2)
(16)
B.
licheniformis
R5
%0.44
13.22
.(
(13)
.
/
%0.28
)
0.43
% 0.27
-
100
(4)
.
...
-
:
Vmax = 1 / intercept (y)
Vmax = Km/ intercept (- x )
0.016
0.007
0.014
0.006
0.012
0.005
0.01
1 /v
[ S ] /V
0.004
0.008
0.003
- km = intercept (0.002
x)
0.006
0.004
-Km = 1
0.002
0.001
0
0
-6 -5 -4 -3 -2 -1
0
1
2
3
4
5
-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8
6
1 1.2
[s]
1/[S]
(A) 1/V :Lineweaver– Burk Reciprocal Plot
(B)
[S] /V versus :Hanse – Woolf Plot
[S]
versus 1/[S]
300
600
250
500
Intercept (y)=Vmax
Km= Vmax / intercept ( y)
200
V /[ S ]
400
150
V
300
100
200
Vmax = Intercept (x )
Km= Vmax / intercept ( x)
50
100
0
0
0
100
200
300
400
500
600
0
50
V/[s]
(C) V :Woolf – Augustinsson – HofsteePlot
100
150
200
250
V
(D)
V/[S] versus V :Eadie – Scatshard Plot
versus V/ [S]
-
99
101
(8)
300
((2011)) B
((
))
.(2002)
and
some properties of Bgalactosidase
from
the
thermophilic bacterium
10. Meredith, W. O. S.; Anderson, J.
A. and Handson, L.E.(1962).
Evaluation of malting barley. (C. F.
Barley and Malt, ed. by A. H. Cook.
PP 207-269. Academic Press
London and New York).
11. Nielsen, J.E.; Borchert, T.V. and
Uriend, G. (2001). The determinants
of - amylase pH – activity profiles.
Protein Engineering. 14 (7): 505512.
12. Rahman, M. Mahbubar. and
AbSar , Nurul. (2001). Purification
, Characterization and Effect of
Physico–chemical Agents on the
Stability of Amylase from Mango–
pulp. Pakiistan Journal of Biological
Sciences 4 (1) :98- 102 .
13. Rahman,M.;Habibur, Md.; Salim,
Uddin.; Nural, Islam.; Farjana.
Nikkon and M. Fida .Hasan.
(2001). Comparative Analysis on the
Purified Amylases from Healthy and
Diseased Sugarcane Juice . Pakistan
Journal of Biological Sciences 4 (6):
728-732.
14. Segel, I.H. (1976). Biochemical
calculations. 2nd edition, John and
sons. Inc. New York.
15. Straathof, A.; Panke, S. and
Schmid, A. (2002.)The productionof
fine
chemicals
by
biotransformations,Curr.
Opin.
Biotechnol, 13,548–556.
16. Thoma ,J.A. (1971) . In Enzymes
,3rd.ed ., Vol 5 Boyer , P.D.,
Academic Press
( New Your NY:
1971 ) pp .115- 189.
17. Vetere,
A.
and
Paoletti,
S.(1998).Separation
and
characterization ofthree
–
galactosidase from
Bacillus
circulans . Biochem. Biophys. Acta.
1380:223-231.
.1
.
. (2004 )
Bacillus licheniformis
.
.2
R5
.
3.
4.
5.
6.
7.
8.
9.
102
.
–
.
Abdul- Hussain , A .S. and
Varriano-Marston , E. (1982) .
Amylolysis of pear Millet starch and
Its fractions by pear Millet alphaamylase ., Cereal Chem .59(5) 351355 th
Bastos, Joao luiz. pinheiro .; josé,
tarquinio.
Prisco.,
and
enéas,Gomes. Filho.
(1994) .
Purification and Characterization of
a Cotyledonary
-amylase from
Cowpea Seedlings . R. Bras. Fisiol.
Veg., 6(1):33-39.
Crabb, W.D. and Shetty, J.K.
(1999). Commodity scale production
of sugars from starches. Current
Option in Microbiology. 2: 252-256.
Fullbrook , P .D .( 1983) . Kinetics (
Practical applied Kinetics ) . Chapter
2 .pp. 67- 70 In: Godforg, T and
Riechelt ,J.( 1983) Industrial
Enzymology . The Nature press .
U.K.
Garfin , D.E. ( 1990) . Purification
Procedures Electrophoretic Methods
. In Methods in Enzymology ( ed,
Murray ,E. D. and Dentscher , P.J.
Vol. 128 : 425 – 411 .
Lowry, O.H.; Rosobrough, N.;
Far, A.L.; and Randall, R.J.
(1951). Protein measurement with
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193: 265-275.
Maciunska, J.; Czyz, B. and
Synowiecki, J. (1998). Isolation
...
-
:
Mercel Dekker. Inc. New York,
USA
20. Wilson, J. J. and Ingledew, W. M.(
1982). Isolation and characterization
of the amylolytic enzymes of
Schwanniomycesalluvius.
Appl.
Environ. Microbiol. 44:301-307.
18. Weselake, J. Randall ., Alexander
,W.MacGregor ., Robert ,D .Hill
(1983) An Endogenous - Amylase
Inhibitor in Barley Kernels . Plant
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Enzymology for the food science.
Purification and Characterization of alpha-amylase produced from a local
Malt Barley
*Ghyath .H.Majeed *Ali .A.Sahi **Dhia .F .Alfekaiki
* Food sciences Dept. – Agric.college - Basra Univ.
alfekaiki@yahoo.com
summary
The study included , alpha amylase was extracted from malt barley local by Imidazol
buffer of pH 7.4 , 0.025 M. Alpha-amylase was purified by number of steps which induced
precipitation by ammonium sulphate saturated concentration was 30- 70 % and dialysis was
performed against the same buffer . The result extract was passed through a column of
sephdex G-100 . Purification was 20.71 fold and enzyme yield was 20.72%. Molecular
weight of alpha – amylase was 40000 Dalton as estimated by SDS –Polyacrylamide
electrophoresis. Optimum pH for activity and stability of enzyme was 5 and (5-7),
respectively . Heat stability of enzyme increased in the presence of Ca++ ione, at a
concentration of 0. 001 M , when the enzyme retained all of its activity when heated to 80 °C
for 20 min while 30 % of the activity remained at 90°C for 30 min . Kinetic constants study
when using starch as a substrate showed that Km was 0.55% and Vmax was 255.75 unit /ml
Key words ;alpha amylase – enzyme purification –malt barley
103