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Aplicicaciones de Las Amilasas
Aplicicaciones de Las Amilasas
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Abstract
Amylases are one of the most important and oldest industrial enzymes. These comprise hydrolases, which hydrolyse starch
molecules to fine diverse products as dextrins, and progressively smaller polymers composed of glucose units. Large arrays of
amylases are involved in the complete breakdown of starch. However, a-amylases which are the most in demand hydrolyse a-1,4
glycosidic bond in the interior of the molecule. a-Amylase holds the maximum market share of enzyme sales with its major
application in the starch industry as well as its well-known usage in bakery. With the advent of new frontiers in biotechnology, the
spectrum of a-amylase application has also expanded to medicinal and analytical chemistry as well as in automatic dishwashing
detergents, textile desizing and the pulp and paper industry. Amylases are of ubiquitous occurrence, produced by plants, animals
and microorganisms. However, microbial sources are the most preferred one for large scale production. Today a large number of
microbial a-amylases are marketed with applications in different industrial sectors. This review focuses on the microbial amylases
and their application with a biotechnological perspective.
# 2003 Elsevier Science Ltd. All rights reserved.
Keywords: a-Amylase; Baking; Antistaling; Dextrinising activity; Starch liquefaction
1. Introduction
Amylases are enzymes which hydrolyse starch molecules to give diverse products including dextrins and
progressively smaller polymers composed of glucose
units [1]. These enzymes are of great significance in
present day biotechnology with applications ranging
from food, fermentation, textile to paper industries [2].
Although amylases can be derived from several sources,
including plants, animals and microorganisms, microbial enzymes generally meet industrial demands. Today
a large number of microbial amylases are available
commercially and they have almost completely replaced
chemical hydrolysis of starch in starch processing
industry [2].
The history of amylases began in 1811 when the first
starch degrading enzyme was discovered by Kirchhoff.
0032-9592/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0032-9592(03)00053-0
1600
peptone, corn steep liquor (CSL), etc. and thiol compounds with starch iodine complex. Copper sulphate
and hydrogen peroxide protect the starch/iodine colour
in the case of interference by these media components
[9]. Further, zinc sulphate was found to be best for
counteracting the interference of various metal ions.
Various workers [10,11] have successfully used the
original assay procedure in combination with flow
injection analysis (FIA). The flow system comprised of
an injection valve, a peristaltic pump, a photometer with
a flow cell and 570 nm filter and a pen recorder. Samples
are allowed to react with starch in a coil before iodine
was added. Absorbance is then read at 570 nm. This
method has many advantages including high sampling
rates, fast response, flexibility and simple apparatus.
3.1.2. Sandstedt Kneen and Blish (SKB) method
The SKB method [12], is one of the most widely
adopted methods for determination of amylases used in
the baking industry. The potency of most commercial
amylases is described in terms of SKB [12] units. This
method is used generally to express the diastatic strength
of the malt and not for expressing a-amylase activity
alone [13].
3.1.3. Indian pharmacopoeia method
As described in the Indian pharmacopoeia, this
method is used to calculate a-amylase activity in terms
of grams of starch digested by a given volume of enzyme
[14]. This procedure involves incubation of the enzyme
preparation in a range of dilutions in buffered starch
substrate at 40 8C for 1 h. The solutions are then treated
with iodine solution. The tube, which does not show any
blue colour, is then used to calculate activity in terms of
grams of starch digested. This method is usually
employed for estimating a-amylase activity in cereals.
3.2. Increase in reducing sugars or dinitrosalicyclic acid
(DNSA) method
This method determines the increase in reducing
sugars as a result of amylase action on starch [15]. The
major defect in this assay is a slow loss in colour
produced and destruction of glucose by constituents of
the DNSA reagent.
To overcome these limitations, a modified method for
the estimation of reducing sugars was developed [16].
Rochelle salts were excluded and 0.05% sodium sulphate
was added to prevent the oxidation of the reagent. Since
then the modified method has been used extensively to
measure reducing sugars without any further modifications in the procedure.
Alternate methods, which also rely on the estimation
of the reducing sugars are also, employed [17].
1601
1602
1603
1604
1605
Table 1
Purification strategies employed for a-amylase
Microorganism
Purification strategy
Fold purification/
yield (%)
Reference
[31]
13.8/70
140/78
6000/52
5/2
[92]
[152]
[99]
[153]
10.8/17.1
[154]
[155]
744/65
266/ /
Amy I 65/13, Amy II
40.7/9.5
212/42
33/66
/
[45]
[47]
[100]
9/17
2.5/ /
30.85/24.8
20/35
1.036/ /
6.9/50
300/ /
[159]
[83]
[51]
[160]
[93]
[161]
[162]
80% (NH4)2SO4, DEAE-Toyopearl 650 M (pH 7.5), Superdex 200 HR (pH 7.5) 816/26
[156]
[17]
[86]
[157]
[158]
[71]
1606
Table 2
Properties of some microbial amylases
pI
Molecular
weight
(kDa)
Inhibitors
Stabilisers
Additional properties
Reference
/
/
65.4/5.0 /9.0
/
/
Km (0.13%)
[28]
A. flavus LINK
3.5
52.5
6.0/6.0 /10.0
Ag2 , Hg2
Ca2
A. foetidus ATCC
10254
A. awamori
/
41.5
5.0/ /
(60
/
/
/
/
5.0/6.0 /7.0
(10
A. awamori ATCC
22342
A. chevalieri NSPRI
105
A. flavus
4.2
54.0
(60
/
68.0
(15
EDTA, DNP
Ca2 , Mg2
/
/
5.25/5.0 /8.0
(10
Substrate
A. fumigatus
/
/
6.0/ /
(40
/
/
/
[41]
A. hennebergi Blochweitz
A. niger
/
50.0
5.5/ /
(15
/
/
/
[166]
3.44
58.0
min)
/
Ca2
Acid stable
[167 /
171]
3.75
A. niger ATCC 13469 /
A. niger van Tieghem /
CFTRI 1105
61.0
/
56.23
/
/
Ag , Al3 , Cu2 , Hg2 , Pb2 ,
Zn2 , EDTA
Ca2
/
Ca2
/
NaF and MgSO4 stimulation
[172]
[173 /
175]
A. oryzae
/
/
/
[164]
A. oryzae
A. oryzae
/
/
/
53.0
40 8C/55 8C (10
min)
35 /37 8C/ /
//60 8C (90 min,
/Ca) 50 8C (30
min, /Ca)
/
PCMB
/
Ca2
[176]
[177 /
180]
30 /40 8C/ /
/
/
Km /4.16 mg ml 1
[181,182]
[183]
50 8C/50 8C (30
min)
55 8C/50 8C (1 h)
45 8C/35 8C
min)
40 8C/55 8C
min)
50 8C/40 8C
min)
40 8C/60 8C
min)
50 8C/55 8C
min)
50 8C/60 8C
min)
50 8C/40 8C
min)
//60 8C (15
/
4.8 /6.6/ /
5.0 /5.9/5.8 /7.2
(over a year,
10 8C); 5.0 /8.2
(37 8C, 30 min)
5.0 /6.0/ /
54.0
3.0 /5.5/ /
60 /70 8C/ /
/
/
A. oryzae M13
52.0
5.4/5.0 /9.0
50 8C/ 5/50 8C
(min)
/
/
4.0
[164]
Km /1 mg ml 1
[32]
Km /0.19 mg ml 1
[165]
[164]
[28]
Source
Table 2 (Continued )
pI
Molecular
weight
(kDa)
Inhibitors
Stabilisers
Additional properties
Cryptococcus S-2
4.2
66.0
6.0/ /
Fusarium vasinfectum
Atk
L. kononenkoae CBS
5608
/
/
3.5
76.0
/
50 /60 8C/90 8C
(CaCl2)
/
4.4 /5.0:5.8:7.8 /
8.0/3.8 /10.0
4.5 /5.0/5.0 /7.0
(1 h)
45 /50 8C/50 8C
(30 min)
70 8C/ /
/
Starch
69.0
4.0/4.0 /9.0
/
/
54.1
5.0/ /
45 8C/45 8C (10
min, /Ca)
50 8C/ /
[99]
Ca2
/
/
[153]
Paecilomyces sp.
ATCC 46889
Saccharomyces cerevisiae
Schwanniomyces alluvius UCD 5483
T. lanuginosus IISc 91
/
61.9
6.3/4.5 /7.5
40 8C/ 5/40 8C
/
/
/
42.0
5.6/ /
/
Ca2
Trichoderma viride
/
/
65 8C/50 8C ( /7
h)
//60 8C (10 min)
/
/
/
22.5
28.0
6.0/4.5 /9.0
9.0/6.0 /11.0
9.0/7.0 /9.0
45 /55 8C/ /
76 8C/ B/60 8C
90 8C/60 8C (3 h),
100 8C (4 h) in
presence of soluble starch
/
/
Hg2 , Cu2 , Ni2 , Zn2 , Ag2 ,
Fe2 , Co2 , Cd2 , Al3 , Mn2 ,
p- chloromercuribenzoic acid, sodium iodoacetate, EDTA
B. licheniformis NCIB /
6346
B. stearothermophilus 4.82
62 /65
7.0/7.0 /10.0
/
/
4.6 /5.1/ /
70 /90 8C/85 8C
(1 h)
55 /70 8C/ /
/
/
Na2 , Ca2 Mg2 , azide, F ,
2
2
2
SO2
3 , SO4 , S2O3 , MoO4 ,
2
WO4 , cysteine, glutathione,
thiourea, b-mercaptoethanol,
sod. glycerophosphate
/
EDTA
Ca2
B. stearothermophilus
ATCC 12980
8.8
59.0
B. stearothermophilus
MFF4
B. subtilis
/
/
5.5 /6.0/ /
/
48.0
6.5/5/7.0
B. subtilis 65
/
68.0
6.0/6.0 /9.0
Bacteria
B. brevis HPD 31
/
B. licheniformis
/
B. licheniformis
/
CUMC 305 licheniformis CUMC 305
Reference
60 8C/60 8C (5
min)
/
/
Mn2 , Co2
[185]
/
End product, G5
E.A. (5.1/105 J mol 1);
Km (1.274 mg ml 1); Vmax
(0.738 mg glucose ml 1
min1
[187]
[85]
[86]
[157]
[101]
Source
[4]
[102]
[159]
[51]
1607
1608
Table 2 (Continued )
Source
pI
Molecular
weight
(kDa)
Inhibitors
Stabilisers
Additional properties
Reference
B. licheniformis M27
/
56.0
/
Ca2
5.6
63.0
/
/
5.9
69.2
6.0/4.0 /9.0
65 8C/40 8C (1 h)
Cysteine, DTT
/
/
5.6/4.5 /8.0
82 8C/90 /95 8C
/
Starch, Ca2
Bacillus sp. WN 11
/
/
5.0 /8.0/ /
75 /80 8C/ /
/
/
Bacillus sp. WN 11
/
/
/
[188]
9.0/ /
70 8C/ /
/
/
/
48.0
/
6.5/5/7.0
7.0/5.0 /7.0
50 8C/ B/70 8C
37 8C/ /
Mn2 , Co2
Ca2
/
50.0
Micromonospora melanosporea
M. melanosporea
Pseudomonas stutzeri
7.6
45.0
7.0/ /
7.6
/
45.0
12.5
7.0/6.0 /12.0
8.0/7.0 /9.5
55 8C/40 8C (pH
11 /12, 40 min)
55 8C/ /
47 8C/40 8C (1 h)
Streptococcus bovis
JB1
4.5
77.0
47.8
Thermomonospora
curvata
6.2
/
N -bromosuccinimide, iodine,
acetic acid, Hg2 , dimethyl aminobenzaldehyde
/
/
[47]
[189]
[100]
[190]
[87]
[159]
[46]
[160]
/
[191]
End product, G1
E.A. (13 400 and 5200 cal
mol 1; end product, G4
Km (0.88 mg ml 1); Kcat
(2510 mmol reducing sugar
mg1 protein); end products, G2, G3, G4
End products, G1, G3; Km
(8.0 /8.2 mM)
[191]
[93]
Ca2
[71]
/
[162]
/
/
/
Ca2
//50 8C (1 h)
Hg2 , p -chloromercuribenzoic
acid (both reversible by DTT)
/
5.5/ /
60 8C/ /, 60 /
65 8C/ /, 65 8C/ /
/
/
42.0
60.9
6.0/ /
[161]
[44]
Escherichia coli
H. meridiana DSM
5425
L. plantarum A6
[45]
Starch, Ca2
/
/
/
T. fusca YX
G1, glucose; G2, maltose; G3, maltotriose; G4, maltotetraose; G5, maltopentaose; E.A., enzyme activation energy; kcal, kilo calories; kJ, kilo joules.
62.0
Inhibitors
pH optima/stabi- Temperature oplity
tima/stability
Molecular
weight
(kDa)
pI
Source
Table 2 (Continued )
/
Additional properties
Stabilisers
Reference
1609
1610
1611
9. Conclusions
As evident from the foregoing review, amylases are
among the most important enzymes used in industrial
processes. Although, the use of amylases, a-amylases in
particular, in starch liquefaction and other starch based
industries has been prevalent for many decades and a
number of microbial sources exist for the efficient
production of this enzyme, the commercial production
of this enzyme has been limited to only a few selected
strains of fungi and bacteria. Moreover, the demand for
these enzymes is further limited with specific applications as in the food industry, wherein fungal a-amylases
are preferred over other microbial sources due to their
more accepted GRAS status. Structural conformation
plays an important role on amylase activity [151].
Further there arises a need for more efficient a-amylases
in various sectors, which can be achieved either by
chemical modification of the existing enzymes or
through protein engineering. In the light of modern
biotechnology, a-amylases are now gaining importance
in biopharmaceutical applications. Still, their application in food and starch based industries is the major
market and thus the demand of a-amylases would
always be high in these sectors.
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