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tamarii
ABSTRACT
A strain of Aspergillus tamarii, a filamentous fungus isolated from soil, was able to
produce both -amylase and glucoamylase activities in mineral media supplemented
with 1% (w/v) starch or maltose as the carbon source. Static cultivation led to
significantly higher yields than those obtained using shaking culture. The production
of amylases was tolerant to a wide range of initial culture pH values (from 4 to 10) and
temperature (from 25 to 42oC). Two amylases, one -amylase and one glucoamylase,
were separated by ion exchange chromatography. Both partially purified enzymes had
optimal activities at pH values between 4.5 and 6.0 and were stable under acid conditions
(pH 4.0-7.0). The enzymes exhibited optimal activities at temperatures between 50o
and 60o C and were stable for more than ten hours at 55oC.
Key words: -amylase, Aspergillus tamarii, glucoamylase.
INTRODUCTION
A variety of industries (e.g. food, chemical,
detergent, textile) employ microbial amylolytic
enzymes to convert starch into different sugar
solutions (2,13,15,18). Several types of enzymes are
involved in the degradation of starch, mainly amylase (1,4 -glucan glucanohydrolase, EC
3.2.1.1), -amylase (1,4 -glucan maltohydrolase,
EC 3.2.1.2), and glucoamylase (1,4 -glucan
glucohydrolase, EC 3.2.1.3) (2,8). These enzymes
are common in fungi, and Aspergillus sp and
Rhizopus sp are often used as sources of industrial
amylases (3). Although genetic manipulation by
classical mutation techniques and recombinant DNA
technology are frequently used to increase the
expression levels of a large number of microbial
* Corresponding author. Mailing address: Departamento de Bioqumica, Universidade Estadual de Maring, CEP 87020-900, Maring,
PR, Brasil. E-mail: rmperalta@pbc.uem.br
157
Amylases by A. tamarii
Table 1. Effect of the carbon source on growth and amylase production by Aspergillus tamarii.
Static conditions
biomass
total amylase
(mg)
(U/mg)
28 5
15 2
26030
24 3
25021
25 3
35530
10 2
21523
68 8
28430
15516
31027
18021
29032
17615
28935
17020
38042
14416
Carbon source
(1%, w/v)
none
sucrose
cellobiose
glucose
raffinose
maltose
glycogen
starch
amylopectin
amylose
Shaking conditions
biomass
total amylase
(mg)
(U/mg)
45 5
8 1
31740
10 2
32028
12 2
44050
7 1
32040
20 2
41037
38 5
48051
47 6
44048
42 4
49053
40 5
40037
32 2
Amylase (U/mg)
120
The cultures were grown in 250 ml Erlenmeyer flasks containing 50 ml minimal medium supplemented with various carbon sources at
1% (w/v) under static or shaking conditions for 6 days at 30oC. The mycelia (biomass) were separated by filtration and dried to constant
weigh at 60oC. One unit of total amylase (glucoamylase plus -amylase) was defined as the amount of enzymes that releases 1 mol of
reducing sugar as D-glucose per min. under the assay conditions. The results are presented as specific activity (U/mg extracellular
protein). The results express the mediaSD of three different experiments.
100
80
60
40
20
0
0
8 10
time (days)
350
300
250
200
150
100
50
0
0
8 10
time (days)
Initial
pH
3
4
5
6
7
8
9
10
11
final
pH
4.35
5.38
6.28
6.90
7.10
7.18
7.47
7.68
9.20
biomass
(mg)
ND
24820
27932
29528
25629
25421
21326
20829
ND
glucoamylase -amylase
(U/mg)
(U/mg)
ND
ND
128
303
129
314
136
353
130
325
131
323
125
293
121
284
ND
ND
temperature
(oC)
25
28
30
32
35
37
39
40
42
45
biomass
(mg)
21018
28024
29528
30031
29230
28931
27630
28025
23021
ND
glucoamylase
(U/mg)
12915
12811
13612
13016
12910
12015
12515
12212
11910
ND
-amylase
(U/mg)
304
313
353
325
324
303
282
254
203
ND
-1
II
5
4
3
2
1
0
0
10 20 30 40 50 60 70
fraction number
Amylases by A. tamarii
100
B 100
100
80
80
80
60
60
60
40
20
0
40
40
3
5
pH
40
50
60
70
temperature (oC)
60 120 180
time (min)
Figure 4. Effect of pH (A) and temperature (B) on the activity and effect of temperature on the stability (C) of amylases from Aspergillus
tamarii. A and B: ( ) glucoamylase activity; ( ) -amylase activity; C: ( ) residual glucoamylase and -amylase activities at 55oC;
( ) residual -amylase activitiy at 65oC; ( ) residual glucoamylase activity at 65oC.
161
REFERENCES
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RESUMO
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