Microbial a-amylases: a biotechnological perspective

Rani Gupta *
,1
, Paresh Gigras, Harapriya Mohapatra, Vineet Kumar Goswami,
Bhavna Chauhan
Department of Microbiology, University of Delhi South Campus, Benito Juarez Marg, New Delhi 110 021, India
Received 3 July 2002; accepted 30 January 2003
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 mole-
cules 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, micro-
bial 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.
This was followed by several reports of digestive
amylases and malt amylases. It was much later in
1930, that Ohlsson suggested the classification of starch
digestive enzymes in malt as a- and b-amylases accord-
ing to the anomeric type of sugars produced by the
enzyme reaction. a-Amylase (1,4-a-D-glucan-glucanhy-
drolase, EC. 3.2.1.1) is a widely distributed secretary
enzyme. a-Amylases of different origin have been
extensively studied.
Amylases can be divided into two categories, endoa-
mylases and exoamylases. Endoamylases catalyse hy-
drolysis in a random manner in the interior of the starch
molecule. This action causes the formation of linear and
branched oligosaccharides of various chain lengths.
Exoamylases hydrolyse from the non-reducing end,
successively resulting in short end products. Today a
large number of enzymes are known which hydrolyse
starch molecule into different products and a combined
action of various enzymes is required to hydrolyse
starch completely.
A number of reviews exist on amylases and their
applications, however, none specifically covers a-amy-
* Corresponding author. Tel.: /91-11-2611-1933; fax: /91-11-
2688-5270.
E-mail address: ranigupta15@rediffmail.com (R. Gupta).
1
E-mail: microzyme@123india.com.
Process Biochemistry 00 (2003) 1Á/18
www.elsevier.com/locate/procbio
0032-9592/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0032-9592(03)00053-0
ARTICLE IN PRESS
lases at length. a-Amylases are one of the most popular
and important form of industrial amylases and the
present review highlights the various aspects of micro-
bial a-amylases.
2. Distribution of a-amylase among microorganisms
a-Amylases are universally distributed throughout the
animal, plant and microbial kingdoms. Over the past
few decades, considerable research has been undertaken
with the extracellular a-amylase being produced by a
wide variety of microorganisms [1Á/5]. The major
advantage of using microorganisms for the production
of amylases is the economical bulk production capacity
and microbes are easy to manipulate to obtain enzymes
of desired characteristics [5]. a-Amylase has been
derived from several fungi, yeasts, bacteria and actino-
mycetes, however, enzymes from fungal and bacterial
sources have dominated applications in industrial sec-
tors [2].
3. Determination of a-amylase activity
a-Amylases are generally assayed using soluble starch
or modified starch as the substrate. a-Amylase catalyses
the hydrolysis of a-1,4 glycosidic linkages in starch to
produce glucose, dextrins and limit dextrins. The reac-
tion is monitored by an increase in the reducing sugar
levels or decrease in the iodine colour of the treated
substrate. Various methods are available for the deter-
mination of a-amylase activity [6]. These are based on
decrease in starchÁ/iodine colour intensity, increase in
reducing sugars, degradation of colour-complexed sub-
strate and decrease in viscosity of the starch suspension.
3.1. Decrease in starchÁ/iodine colour intensity
Starch forms a deep blue complex with iodine [7] and
with progressive hydrolysis of the starch, it changes to
red brown. Several procedures have been described for
the quantitative determination of amylase based on this
property. This method determines the dextrinising
activity of a-amylase in terms of decrease in the iodine
colour reaction.
3.1.1. Determination of dextrinising activity
The dextrinising activity of a-amylases employs
soluble starch as substrate and after terminating the
reaction with dilute HCl, iodine solution is added. The
decrease in absorbance at 620 nm is then measured
against a substrate control. One percent decline in
absorbance is considered as one unit of enzyme [8].
The major limitation of this assay is interference of
media components including Luria broth, tryptone,
peptone, corn steep liquor (CSL), etc. and thiol com-
pounds with starch iodine complex. Copper sulphate
and hydrogen peroxide protect the starchÁ/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 modifica-
tions in the procedure.
Alternate methods, which also rely on the estimation
of the reducing sugars are also, employed [17].
R. Gupta et al. / Process Biochemistry 00 (2003) 1Á/18 2
ARTICLE IN PRESS
3.3. Degradation of colour-complexed substrate
For some years, groups have been working on the
development of a specific a-amylase determination
method based on the use of new types of substrates.
These methods employ starch covalently complexed
with blue dye such as Remazol brilliant Blue R [18] or
Cibacron Blue F3 G-A [19] as an alternative substrate.
The synthesis of these substrates involves two major
steps. Soluble starch is coloured under alkaline condi-
tions using the dye. This is the result of formation of
covalent bonds between starch and dye molecules. The
coloured starch is subsequently cross-linked by the
addition of 1,4-butanediol diglycide ether. This gives
an insoluble network, which swells in water. The
enzymic hydrolysis of such insoluble starch derivatives
yields soluble starch hydrolysates carrying the coloured
marker. This method is simple and sensitive for a-
amylase determination, but even minute quantities of
glucose might lead to erroneous results due to starch
contamination by dextrin substrate [19]. Recently, a
rapid and sensitive microassay based on dye cross linked
starch for a-amylase detection has been reported. It can
successfully detect as low as 0Á/50 ng of enzyme [20].
Other novel substrates such as nitrophenyl derivatives
of maltosaccharides have also been employed. The assay
measures the release of free p-nitrophenyl groups. The
use of nitrophenyl-maltosaccharides in conjunction with
a specific yeast a-glucosidase can be used but these
substrates are rapidly cleaved by glucoamylases com-
monly present in the culture broths. The use of non-
reducing end blocked p-nitrophenyl maltoheptoside
(BPNPG
7
) has also been described [21]. The blocking
group (4,6-O-benzylidene) prevents the hydrolysis of the
substrate by the exo-acting enzymes and is thus specific
for a-amylase. The assay is simple, reliable and accurate
but is expensive as it involves the use of a synthetic
substrate and specific enzymes. Thus the use of this
method is restricted only to very specific tests and not
for routine analysis. A comparison was made for the use
of end blocked p-nitrophenyl maltoheptoside (BPNPG
7
)
with a number of accepted procedures that employ
starch as the substrate. The reaction was monitored
using the starchÁ/iodine colour [21]. There was an
excellent correlation between each of the assay proce-
dures employed. This indicates that all the methods give
an accurate and reliable measure of a-amylase activity
and can be used as per the requirement. Both these
methods are commercially available as commercial kits,
however, it is found that a-amylases exhibit lower
affinity for low molecular weight substrates [18].
3.4. Decrease in viscosity of the starch suspension
These methods are generally used in the bakery
industry to assess the quality of the flour and not for
estimating a-amylase activity which are based on the
determination of the rheological properties of the
dough. Methods, which fall into this category, are the
falling number test and the Amylograph or Farinograph
test.
3.4.1. Falling number (FN) method
The falling number (FN) method, internationally
standardised [22Á/24] is accepted for assessing cereal a-
amylase activity in flourÁ/enzyme preparations at
100 8C. Both cereal and fungal a-amylases are used to
improve the fermentation of flour deficient in amylase
activities. Because fungal a-amylases have low thermo-
stability, they cannot be detected by the standard FN
method at 100 8C [25]. This method has been modified
and standardised [25] for measuring both cereal and
fungal a-amylase activity at 300 8C, by replacing a part
of the flour with pre-gelatinised starch. A falling number
of about 400 indicates a normally malted flour.
3.4.2. Amylograph/Farinograph test
The milling and baking industries generally assess the
diastatic activity of flours by means of an amylograph.
This method is also based on the relationship of peak
viscosity of starch slurry and the enzyme activity level
[23]. The higher the enzyme activity, the thinner is the
hot paste viscosity. When the amylograph is used, values
of 400Á/600 Brabender units of the Farinograph are
considered optimal for bread baking flours (higher
values indicate a lack and lower values indicate an
excess of activity).
4. Physiology of a-amylase production
The production of a-amylase by submerged fermenta-
tion (SmF) and solid state fermentation (SSF) has been
thoroughly investigated and is affected by a variety of
physicochemical factors. Most notable among these are
the composition of the growth medium, pH of the
medium, phosphate concentration, inoculum age, tem-
perature, aeration, carbon source and nitrogen source
[5,26]. Most reports among fungi have been limited to a
few species of mesophilic fungi where attempts have
been made to specify the cultural conditions and to
select superior strains of the fungus to produce on a
commercial scale [2Á/4].
4.1. Physiochemical parameters
The role of various physico-chemical parameters,
including carbon and nitrogen source, surface acting
agents, phosphate, metal ions, temperature, pH and
agitation have been studied.
R. Gupta et al. / Process Biochemistry 00 (2003) 1Á/18 3
ARTICLE IN PRESS
4.1.1. Substrate source: induction of a-amylase
a-Amylase is an inducible enzyme and is generally
induced in the presence of starch or its hydrolytic
product, maltose [27Á/30]. Most reports available on
the induction of a-amylase in different strains of
Aspergillus oryzae suggest that the general inducer
molecule is maltose. There is a report of a 20-fold
increase in enzyme activity when maltose and starch
were used as inducers in A. oryzae (NRC 401013) [31].
Similarly strong a-amylase induction by starch and
maltose in the case of A. oryzae DSM 63303 has been
reported [29]. Apart from maltose, in some strains, other
carbon sources as lactose, trehalose, a-methyl-D-glyco-
side also served as inducers of a-amylase [28]. Not only
the carbon source, but also the mycelial condition/age
affect the synthesis of a-amylase by A. oryzae M-13 [28].
There are reports that 5 days starved non-growing
mycelia were the most appropriate for optimal induction
by maltose. a-Amylase production is also subjected to
catabolite repression by glucose and other sugars, like
most other inducible enzymes [30,32]. However, the role
of glucose in the production of a-amylase in certain
cases is controversial. a-Amylase production by A.
oryzae DSM 63303 was not repressed by glucose rather;
a minimal level of the enzyme was induced in its
presence [29]. However, xylose or fructose have been
classified as strongly repressive although they supported
good growth in Aspergillus nidulans [33].
The carbon sources as glucose and maltose have been
utilised for the production of a-amylase. However, the
use of starch remains promising and ubiquitous. A
number of other non-conventional substrates as lactose
[34], casitone [35,36], fructose [37], oilseed cakes [38] and
starch processing waste water [39] have also been used
for the production of a-amylase while the agro-proces-
sing byproduct, wheat bran has been used for the
economic production of a-amylase by SSF [5]. The use
of wheat bran in liquid surface fermentation (LSF) for
the production of a-amylase from Aspergillus fumigatus
and from Clavatia gigantea, respectively, has also been
reported [40,41]. High a-amylase activities from A.
fumigatus have also been reported using a-methyl-D-
glycoside (a synthetic analogue of maltose) as substrate
[42].
Use of low molecular weight dextran in combination
with either Tween 80 or Triton X-100 for a-amylase
production in the thermophilic fungus Thermomyces
lanuginosus (ATCC 200065) has been reported [43].
Triton X-100 had no effect, whereas Tween 80 increases
the a-amylase activity 27-fold.
4.1.2. Nitrogen sources
Organic nitrogen sources have been preferred for the
production of a-amylase. Yeast extract has been used in
the production of a-amylase from Streptomyces sp. [44],
Bacillus sp. IMD 435 [45] and Halomonas meridiana
[46]. Yeast extract has also been used in conjunction
with other nitrogen sources such as bactopeptone in the
case of Bacillus sp. IMD 434 [47], ammonium sulphate
in the case of Bacillus subtilis [48], ammonium sulphate
and casein for C. gigantea [40] and soybean flour and
meat extract for A. oryzae [49]. Yeast extract increased
the productivity of a-amylase by 110Á/156% in A. oryzae
when used as an additional nitrogen source than when
ammonia was used as a sole source [50]. Various other
organic nitrogen sources have also been reported to
support maximum a-amylase production by various
bacteria and fungi. However, organic nitrogen sources
viz. beef extract, peptone and com steep liquor sup-
ported maximum a-amylase production by bacterial
strains [35,38,51Á/54] soybean meal and casamino acids
by A. oryzae [55]. CSL has also been used for the
economical and efficient production of a-amylase from
a mutant of B. subtilis [56]. Apart from this, various
inorganic salts such as ammonium sulphate for A.
oryzae [30] and A. nidulans [29], ammonium nitrate
for A. oryzae [57] and Vogel salts for A. fumigatus [42]
have been reported to support better a-amylase produc-
tion in fungi.
Amino acids in conjunction with vitamins have also
been reported to affect a-amylase production. However,
no conclusion can be drawn about the role of amino
acids and vitamins in enhancing the a-amylase produc-
tion in different microorganisms as the reports are
highly variable. a-Amylase production by Bacillus
amyloliquefaciens ATCC 23350 increased by a factor
of 300 in the presence of glycine [58]. The effect of
glycine was not only as a nitrogen source rather it
affected a-amylase production by controlling pH and
subsequently amylase production increased. b-Alanine,
DL-nor valine and D-methionine were effective for the
production of alkaline amylase by Bacillus sp. A-40-2.
However, the role of amino compounds was considered
to be neither as nitrogen nor as a carbon source, but as
stimulators of amylase synthesis and excretion [59]. It
has been reported that only asparagine gave good
enzyme yields [57] while the importance of arginine for
a-amylase production from B. subtilis has also been well
documented [60].
4.1.3. Role of phosphate
Phosphate plays an important regulatory role in the
synthesis of primary and secondary metabolites in
microorganisms [61,62] and likewise it affects the growth
of the organism and production of a-amylase. A
significant increase in enzyme production and conidia-
tion in A. oryzae above 0.2 M phosphate levels has been
reported [55]. Similar findings were corroborated in B.
amyloliquefaciens where low levels of phosphate resulted
in severely low cell density and no a-amylase production
[63]. In contrast, high phosphate concentrations were
R. Gupta et al. / Process Biochemistry 00 (2003) 1Á/18 4
ARTICLE IN PRESS
inhibitory to enzyme production by B. amyloliquefaciens
[58].
4.1.4. Role of other ions
K

, Na

, Fe
2
, Mn
2
, Mo
2
, Cl

, SO
4
2
had no
effect while Ca
2
was inhibitory to amylase production
by A. oryzae EI 212 [57]. Mg
2
played an important
role and production was reduced to 50% when Mg
2
was omitted from the medium. Na

and Mg
2
show
coordinated stimulation of enzyme production by Ba-
cillus sp. CRP strain [64]. Addition of zeolites to control
ammonium ions in B. amyloliquefaciens resulted in
increased yield of a-amylase [65]. An inverse relation-
ship between a-amylase production and growth rate was
observed for Streptomyces sp. in the presence and
absence of Co
2
[66], the presence of Co
2
enhancing
the final biomass levels by 13-fold, albeit with a
reduction in enzyme yield.
4.1.5. pH
Among the physical parameters, the pH of the growth
medium plays an important role by inducing morpho-
logical change in the organism and in enzyme secretion.
The pH change observed during the growth of the
organism also affects product stability in the medium.
Most of the Bacillus strains used commercially for the
production of bacterial a-amylases by SmF have an
optimum pH between 6.0 and 7.0 for growth and
enzyme production. This is also true of strains used in
the production of the enzyme by SSF. In most cases the
pH used is not specified excepting pH 3.2Á/4.2 in the case
of A. oryzae DAE 1679 [39], 7.0Á/8.0 in A. oryzae EI 212
[57] and 6.8 for B. amyloliquefaciens MIR-41 [67]. In
fungal processes, the buffering capacity of some media
constituents sometimes eliminates the need for pH
control [68]. The pH values also serves as a valuable
indicator of the initiation and end of enzyme synthesis
[69]. It is reported that A. oryzae 557 accumulated a-
amylase in the mycelia when grown in phosphate or
sulphate deficient medium and was released when the
mycelia were replaced in a medium with alkaline pH
(above 7.2) [28].
4.1.6. Temperature
The influence of temperature on amylase production
is related to the growth of the organism. Among the
fungi, most amylase production studies have been done
with mesophilic fungi within the temperature range of
25Á/37 8C. Optimum yields of a-amylase were achieved
at 30Á/37 8C for A. oryzae [55,57]. a-Amylase produc-
tion has also been reported at 55 8C by the thermophilic
fungus Thermomonospora fusca [70] and at 50 8C by T.
lanuginosus [17].
a-Amylase has been produced at a much wider range
of temperature among the bacteria. Continuous produc-
tion of amylase from B. amyloliquefaciens at 36 8C has
been reported [67]. However, temperatures as high as
80 8C have been used for amylase production from the
hyperthermophile Thermococcus profundus [71].
4.1.7. Agitation
Agitation intensity influences the mixing and oxygen
transfer rates in many fungal fermentations and thus
influences mycelial morphology and product formation
[69,72Á/76]. It has been reported that a higher agitation
speed is sometimes detrimental to mycelial growth and
thus may decrease enzyme production. However, it is
reported that the variations in mycelial morphology as a
consequence of changes in agitation rate do not affect
enzyme production at a constant specific growth rate
[76].
Agitation intensities of up to 300 rpm have normally
been employed for the production of amylase from
various microorganisms as reported in the literature.
5. Fermentation studies on a-amylase production
The effect of environmental conditions on the regula-
tion of extracellular enzymes in batch cultures is well
documented [77]. A lot of work on the morphology and
physiology of a-amylase production by A. oryzae during
batch cultivation has been done. Accordingly, morphol-
ogy of A. oryzae was critically affected by the growth
pH [78]. In a series of batch experiments, authors
observed that at pH 3.0Á/3.5, freely dispersed hyphal
elements were formed. In the pH range 4Á/5, both pellets
and freely dispersed hyphal fragments were observed
whereas at pH higher than 6 pellets were the only
growth forms recorded. Other groups [39,79] have
recorded similar observations for other strains of
A. oryzae. The optimum growth temperature was found
to be 35 8C. It is demonstrated that when glucose was
exhausted the biomass production stopped whereas the
secretion of a-amylase increased rapidly [79]. One report
states that inoculum quantity did not affect morpholo-
gical changes in A. oryzae in air-lift bioreactors and that
pellet size decreased considerably as the air velocity
increased [39]. In the case of a-amylase production by
Bacillus flavothermus in batch cultivation in a 20 l
fermentor, a-amylase production and biomass peaked
twice and highest activity was obtained after 24 h [34]. It
was observed that the kinetics of enzyme synthesis was
more of the growth associated than non-growth asso-
ciated type [35]. Similar findings were cited in another
report with B. amyloliquefaciens [63].
Continuous and fed-batch cultures have been recog-
nised as most effective for the production of the enzyme
[60]and several groups have studied the effectiveness of
these cultures. The production of a-amylase from
B. subtilis TN106 (pAT5) was enhanced substantially
by extending batch cultivation with fed-batch operation
R. Gupta et al. / Process Biochemistry 00 (2003) 1Á/18 5
ARTICLE IN PRESS
[60]. The bulk enzyme activity was nearly 54% greater in
a two-stage fed-batch operation at a feed rate of 31.65
ml h
1
of medium, than that attained in the single stage
batch culture. The effects of controlled feeding of
maltose at a feed rate of 1Á/4 g h
1
for a-amylase and
glucoamylase production from A. oryzae RIB 642 in a
rotary draft tube fermentor (RTF) have been studied
[49]. At a feed rate of 1 g h
1
the yields of a-amylase
were twice than those obtained in batch cultures. When
fed-batch cultivations were performed on a pilot scale
RTF at a feed rate of 24 g h
1
, the biomass and a-
amylase yields was higher than those obtained in a
laboratory scale jar fermentor.
A model to simulate the steady-state values for
biomass yield, residual sugar concentration and specific
rate of a-amylase production has been proposed which
simulated experimental data very well [80]. Further-
more, it was found in chemostat experiments that the
specific rate of a-amylase production decreased by up to
70% with increasing biomass concentration at a given
dilution rate. Shifts in the dilution rate in continuous
culture could be used to obtain different proportions of
the enzymes, by the same strain [66]. It was further
demonstrated that maximum production of a-amylase
occurred in continuous culture at a dilution rate of 0.15
h
1
and amylase activity in the culture was low at
dilution rates above 1.2 h
1
. In contrast, in Bacillus sp.
the switching of growth from batch to continuous
cultivation resulted in the selection of a non a-amylase
producing variant [63]. A decline in enzyme production
was also accompanied by morphological and metabolic
variations during continuous cultivation [81,82].
The industrial exploitation of SSF for enzyme pro-
duction has been confined to processes involving fungi
and it is generally believed that these techniques are not
suitable for bacterial cultivation [5]. The use of SSF
technique in a-amylase production and its specific
advantages over other methods has been discussed
extensively [5].
6. Purification of microbial a-amylases
Industrial enzymes produced in bulk generally require
little downstream processing and hence are relatively
crude preparations. The commercial use of a-amylase
generally does not require purification of the enzyme,
but enzyme applications in pharmaceutical and clinical
sectors require high purity amylases. The enzyme in
purified form is also a prerequisite in studies of
structureÁ/function relationships and biochemical prop-
erties.
The purification of a-amylases from microbial sources
in most cases has involved classical purification meth-
ods. These methods involve separation of the culture
from the fermentation broth, selective concentration by
precipitation using ammonium sulphate or organic
solvents such as chilled acetone. The crude enzyme is
then subjected to chromatography, usually affinity, ion
exchange and/or gel filtration. A number of reviews are
available on purification and characterisation of a-
amylases from a range of microorganisms [1,2,4,26,83].
Table 1 summarises various purification strategies
adopted for microbial a-amylases.
7. Biochemical properties of a-amylases
The enzymic and physicochemical properties of a-
amylases from several microorganisms have been ex-
tensively studied and described [2Á/4,83]. A summary is
presented in Table 2.
7.1. Substrate specificity
As holds true for the other enzymes, the substrate
specificity of a-amylase varies from microorganism to
microorganism. In general, a-amylases display highest
specificity towards starch followed by amylose, amylo-
pectin, cyclodextrin, glycogen and maltotriose.
7.2. pH optima and stability
The pH optima of a-amylases vary from 2 to 12 [4]. a-
Amylases from most bacteria and fungi have pH optima
in the acidic to neutral range [2]. a-Amylase from
Alicyclobacillus acidocaldarius showed an acidic pH
optima of 3 [84], in contrast to the alkaline amylase
with optima of pH 9Á/10.5 reported from an alkalophilic
Bacillus sp. [85Á/88]. Extremely alkalophilic a-amylase
with pH optima of 11Á/12 has been reported from
Bacillus sp. GM8901 [89]. In some cases, the pH
optimum was observed to be dependent upon tempera-
ture as in the case of Bacillus stearothermophilus DONK
BS-1 [90] and on calcium as in the case of B.
stearothermophilus [91].
a-Amylases are generally stable over a wide range of
pH from 4 to 11 [3,4,45,47,85,92], however, a-amylases
with stability in a narrow range have also been reported
[46,86,93].
7.3. Temperature optima and stability
The temperature optimum for the activity of a-
amylase is related to the growth of the microorganism
[4]. The lowest temperature optimum is reported to be
25Á/30 8C for F. oxysporum amylase [94] and the highest
of 100 and 130 8C from archaebacteria, Pyrococcus
furiosus and Pyrococcus woesei , respectively [95Á/97].
Temperature optima of enzymes from Micrococcus
varians are calcium dependent [98] and that from H.
meridiana is sodium chloride dependent [46].
R. Gupta et al. / Process Biochemistry 00 (2003) 1Á/18 6
ARTICLE IN PRESS
Thermostabilities have not been estimated defactor in
many studies. Thermostabilities as high as 4 h at 100 8C
have been reported for Bacillus licheniformis CUMC
305 [86]. Many factors affect thermostability. These
include the presence of calcium, substrate and other
stabilisers [4]. The stabilising effect of starch was
observed in a-amylases from B. licheniformis CUMC
305 [85], Lipomyces kononenkoae [98] and Bacillus sp.
WN 11 [100]. Thermal stabilisation of the enzyme in the
presence of calcium has also been reported from time to
time [100Á/102].
7.4. Molecular weight
Molecular weights of a-amylases vary from about 10
to 210 kDa. The lowest value, 10 kDa for Bacillus
caldolyticus [103] and the highest of 210 kDa for
Chloroflexus aurantiacus has been reported [104]. Mo-
lecular weights of microbial a-amylases are usually 50Á/
60 kDa as shown directly by analysis of cloned a-
amylase genes and deduced amino acid sequences [4].
Carbohydrate moieties raise the molecular weight of
some a-amylases. Glycoproteins have been detected in
A. oryzae [105,106], L. kononenkoae [98], B. stearother-
mophilus [107] and B. subtilis strains [108,109]. Glyco-
sylation of bacterial proteins is rare. A carbohydrate
content as high as 56% has been reported in S. castelii
[110] whereas this is about 10% for other a-amylases [4].
7.5. Inhibitors
Many metal cations, especially heavy metal ions,
sulphydryl group reagents, N-bromosuccinimide, p-
hydroxyl mercuribenzoic acid, iodoacetate, BSA,
EDTA and EGTA inhibit a-amylases.
7.6. Calcium and stability of a-amylase
a-Amylase is a metalloenzyme, which contains at least
one Ca
2
ion [111]. The affinity of Ca
2
to a-amylase is
much stronger than that of other ions. The amount of
bound calcium varies from one to ten. Crystalline Taka-
Table 1
Purification strategies employed for a-amylase
Microorganism Purification strategy Fold purification/
yield (%)
Reference
Fungi and yeast
A. oryzae NRC 401013 DE52-Cellulose (pH 7.0), 70% (NH
4
)
2
SO
4
, Sephacryl S300, 70% (NH
4
)
2
SO
4
,
DE52-Cellulose (pH 7.0)
[31]
A. flavus LINK 50Á/90% (NH
4
)
2
SO
4
, DEAE-Sephadex A50 (pH 6.5) 13.8/70 [92]
Cryptococcus sp. S-2 Ultrafiltration, a-Cyclodextrin coupled with Sepharose 6B (pH 7.0) 140/78 [152]
L. kononenkoae CBS5608 60% (NH
4
)
2
SO
4
, crosslinked starch (pH 8.5), DEAE Bio-Gel A (pH 5.5) 6000/52 [99]
Saccharomyces cerevisiae
YPB-G
Ultrafiltration, b-Cyclodextrin linked Sepharose 6B (Epoxy activated, pH 4.5),
Sephadex G-100 (pH 4.5)
5/2 [153]
Schwanniomyces alluvius
UCD-54-83
Ultrafiltration, DEAE-sephacel (pH 5.6), Sephadex G-150 (pH 5.6) 10.8/17.1 [154]
Thermomonospora curvata Ultrafiltration, 75% ethanol precipitation, Sephadex G-150 (pH 8.0), DEAE
Cellulose, ultrafiltration
66/9 [155]
T. lanuginosus Ultrafiltration, DEAE-Trisacryl (pH 7.0), Phenyl-Sepharose (pH 7.0) [156]
T. lanuginosus IISc91 Ultrafiltration, DEAE-Sephadex A50 (pH 5.0), ultrogel AcA54, DEAE-Sephadex
A50 (pH 8.0), Bio-Gel P-30
112/41 [17]
Bacteria
Bacillus sp. IMD435 a-Cyclodextrin coupled Sepharose 6B (pH 6.0) 744/65 [45]
Bacillus sp. IMD 434 Acetone precipitation, Resource Q (pH 7.0), Phenyl Sepharose CL-4B (pH 7.8) 266/ Á/ [47]
Bacillus sp. WN 11 60% (NH
4
)
2
SO
4
, DEAE Sepharose (pH 5.3), Sephadex G-75 Amy I 65/13, Amy II
40.7/9.5
[100]
B. licheniformis CUMC 305 65% (NH
4
)
2
S0
4
, CM-Cellulose (pH 6.4) 212/42 [86]
B. licheniformis NCIB 6346 DEAE-Cellulose DE52 (pH 5.3) 33/66 [157]
B. stearothermophilus ATCC
12980
Adsorption on soluble starch (1%) in 10% (NH4)
2
SO
4
, washing with Aces (pH
7.5) and 10% (NH
4
)
2
SO
4
, DEAE chromatography (Zetaprep disk), ultrafiltration
Á/ [158]
B. subtilis 60% (NH
4
)
2
SO
4
, Sephacryl-S200 HR (pH 8.0), 60% (NH
4
)
2
SO
4
, S-Sepharose 9/17 [159]
B. subtilis Ultrafiltration 2.5/ Á/ [83]
B. subtilis 65 Sephacryl S-300, CM Sephadex C-50 30.85/24.8 [51]
Lactobacillus plantarum A6 Ultrafiltration, 50Á/80% (NH
4
)
2
SO
4
, ultrafiltration, DEAE-Cellulose 20/35 [160]
Pseudomonas stutzeri Concentrated by drum humidifier, 25% (NH
4
)
2
SO
4
, 70% acetone 1.036/ Á/ [93]
Streptococcus bovis JB1 70% (NH
4
)
2
SO
4
, Sephadex G-25 (pH 7.5), Mono Q 6.9/50 [161]
Thermomonospora curvata
NCIMB 10081
85% (NH4)
2
S0
4
, ultrafiltration, gel filtration (pH 6.0), DEAE-Sephacel (pH 8.O) 300/ Á/ [162]
T. profundus DT5432 80% (NH
4
)
2
SO
4
, DEAE-Toyopearl 650 M (pH 7.5), Superdex 200 HR (pH 7.5) 816/26 [71]
R. Gupta et al. / Process Biochemistry 00 (2003) 1Á/18 7
ARTICLE IN PRESS
Table 2
Properties of some microbial amylases
Source pI Molecular
weight
(kDa)
pH optima/stabi-
lity
Temperature op-
tima/stability
Inhibitors Stabilisers Additional properties Reference
Fungi and yeast
A. oryzae Á/ Á/ 65.4/5.0Á/9.0 50 8C/50 8C (30
min)
Á/ Á/ K
m
(0.13%) [28]
A. flavus LINK 3.5 52.5 6.0/6.0Á/10.0 55 8C/50 8C (1 h) Ag
2
, Hg
2
Ca
2
K
m
(0.5 g l
1
); V
max
(108.67
mM reducing sugar mg
1
protein min
1
[92]
A. foetidus ATCC
10254
Á/ 41.5 5.0/ Á/ 45 8C/35 8C (60
min)
Á/ Á/ K
m
/2.19 mg ml
1
[163]
A. awamori Á/ Á/ 5.0/6.0Á/7.0 40 8C/55 8C (10
min)
Ag

, Cu
2
, Fe
3
, Hg
2
, halides Substrate [164]
A. awamori ATCC
22342
4.2 54.0 4.8Á/5.0/3.5Á/6.5
(24 h)
50 8C/40 8C (60
min)
Hg
2
, Pb
2
, maltose K
m
/1 mg ml
1
[32]
A. chevalieri NSPRI
105
Á/ 68.0 5.5/ Á/ 40 8C/60 8C (15
min)
EDTA, DNP Ca
2
, Mg
2
K
m
/0.19 mg ml
1
[165]
A. flavus Á/ Á/ 5.25/5.0Á/8.0 50 8C/55 8C (10
min)
Ag

, Cu
2
, Hg
2
, halides Substrate [164]
A. fumigatus Á/ Á/ 6.0/ Á/ 50 8C/60 8C (40
min)
Á/ Á/ Á/ [41]
A. hennebergi Bloch-
weitz
Á/ 50.0 5.5/ Á/ 50 8C/40 8C (15
min)
Á/ Á/ Á/ [166]
A. niger 3.44 58.0 4.0Á/5.0/2.2Á/7.0 Á//60 8C (15 min) Á/ Ca
2
Acid stable [167Á/
171]
3.75 61.0 5.0Á/6.0/5.0Á/8.5 Á//40 8C (15 min) Á/ Ca
2
A. niger ATCC 13469 Á/ Á/ 5.0/4.0Á/6.0 50 8C/ B/60 8C Á/ Á/ Á/ [172]
A. niger van Tieghem
CFTRI 1105
Á/ 56.23 5.0; 6.0/5.2Á/6.0
(/Ca); 5.8Á/7.0
(/Ca)
60 8C/65 8C (10
min)
Ag

, Al
3
, Cu
2
, Hg
2
, Pb
2
,
Zn
2
, EDTA
Ca
2
NaF and MgSO
4
stimula-
tion
[173Á/
175]
A. oryzae Á/ Á/ 5.0/6.0Á/8.0 40 8C/55 8C (10
min)
Ag

, Cu
2
, Fe
3
, Hg
2
, halides Substrate Á/ [164]
A. oryzae Á/ Á/ 4.8Á/6.6/ Á/ 35Á/37 8C/ Á/ Á/ Á/ K
m
/7.13, 4.35, 3.12 mM [176]
A. oryzae Á/ 53.0 5.0Á/5.9/5.8Á/7.2
(over a year,
10 8C); 5.0Á/8.2
(37 8C, 30 min)
Á//60 8C (90 min,
/Ca) 50 8C (30
min, /Ca)
PCMB Ca
2
‘‘Taka Diastase’’, ‘‘Taka-
Amylase A’’, K
m
/ 29,
2.4%, 4.7, 10.2, 2.4 mM
[177Á/
180]
A. oryzae 245 (ATCC
9376)
Á/ Á/ 5.0Á/6.0/ Á/ 30Á/40 8C/ Á/ Á/ Á/ K
m
/4.16 mg ml
1
[181,182]
A. usamii Á/ 54.0 3.0Á/5.5/ Á/ 60Á/70 8C/ Á/ Á/ Á/ Higher thermal stability
than commercial Taka-
amylase
[183]
A. oryzae M13 4.0 52.0 5.4/5.0Á/9.0 50 8C/ 5/50 8C
(min)
Á/ Á/ K
m
(0.13%) [28]
R
.
G
u
p
t
a
e
t
a
l
.
/
P
r
o
c
e
s
s
B
i
o
c
h
e
m
i
s
t
r
y
0
0
(
2
0
0
3
)
1
Á
/
1
8
8
A
R
T
I
C
L
E
I
N
P
R
E
S
S
Table 2 (Continued)
Source pI Molecular
weight
(kDa)
pH optima/stabi-
lity
Temperature op-
tima/stability
Inhibitors Stabilisers Additional properties Reference
Cryptococcus S-2 4.2 66.0 6.0/ Á/ 50Á/60 8C/90 8C
(CaCl
2
)
Hg
2
, Ag
2
, Cu
2
, Zn
2
Á/ Raw starch digesting en-
zyme; end products, G
1
, G
2
,
G
3
, G
4
[152]
Fusarium vasinfectum
Atk
Á/ Á/ 4.4Á/5.0:5.8:7.8Á/
8.0/3.8Á/10.0
45Á/50 8C/50 8C
(30 min)
Cu
2
, Mn
2
, Zn
2
Á/ Á/ [184]
L. kononenkoae CBS
5608
3.5 76.0 4.5Á/5.0/5.0Á/7.0
(1 h)
70 8C/ Á/ DTT, Cu
2
, Ag
2
Starch K
m
(0.8 g l
1
); K
cat
(622
s
1
); insensitive to Ca
2
;
end products, G
3
, G
4
, G
5
,
G
6
[99]
Paecilomyces sp.
ATCC 46889
Á/ 69.0 4.0/4.0Á/9.0 45 8C/45 8C (10
min, /Ca)
Á/ Ca
2
Á/ [185]
Saccharomyces cer-
evisiae
Á/ 54.1 5.0/ Á/ 50 8C/ Á/ Á/ Á/ End products, G
1
, G
2
, G
3
[153]
Schwanniomyces al-
luvius UCD 5483
Á/ 61.9 6.3/4.5Á/7.5 40 8C/ 5/40 8C Á/ Á/ K
m
(0.364 mg ml
1
); end
product, G
1
[154]
T. lanuginosus IISc 91 Á/ 42.0 5.6/ Á/ 65 8C/50 8C ( /7
h)
Á/ Ca
2
A.E. (44 kJ mol
1
; K
m
(2.5
mg ml
1
); end product, G
2
[17]
Trichoderma viride Á/ Á/ 5.0Á/5.5/4.0Á/7.0 Á//60 8C (10 min) Á/ Á/ Á/ [186]
Bacteria
B. brevis HPD 31 Á/ Á/ 6.0/4.5Á/9.0 45Á/55 8C/ Á/ Á/ Á/ Á/ [187]
B. licheniformis Á/ 22.5 9.0/6.0Á/11.0 76 8C/ B/60 8C Á/ Á/ End product, G
5
[85]
B. licheniformis
CUMC 305 lichenifor-
mis CUMC 305
Á/ 28.0 9.0/7.0Á/9.0 90 8C/60 8C (3 h),
100 8C (4 h) in
presence of solu-
ble starch
Hg
2
, Cu
2
, Ni
2
, Zn
2
, Ag
2
,
Fe
2
, Co
2
, Cd
2
, Al
3
, Mn
2
,
p- chloromercuribenzoic acid, so-
dium iodoacetate, EDTA
Na
2
, Ca
2
Mg
2
, azide, F

,
SO
3
2
, SO
4
2
, S
2
O
3
2
, MoO
4
2
,
WO
4
2
, cysteine, glutathione,
thiourea, b-mercaptoethanol,
sod. glycerophosphate
E.A. (5.1/10
5
J mol
1
);
K
m
(1.274 mg ml
1
); V
max
(0.738 mg glucose ml
1
min
1
[86]
B. licheniformis NCIB
6346
Á/ 62Á/65 7.0/7.0Á/10.0 70Á/90 8C/85 8C
(1 h)
Á/ Á/ End products, G
1
, G
2
, G
3
,
G
5
[157]
B. stearothermophilus 4.82 Á/ 4.6Á/5.1/ Á/ 55Á/70 8C/ Á/ EDTA Ca
2
Higher affinity for branched
chain substrate; E.A. (14
kcal); extremely resistant to
heat inactivation; effect of
EDTA reversed by Ca
2
[101]
B. stearothermophilus
ATCC 12980
8.8 59.0 5.0Á/6.0/6.0Á/7.5
(1 h, 80 8C)
70Á/80 8C/(5 days)
70 8C or (45 min)
90 8C
Cd
2
, Cu
2
, Hg
2
, Pb
2
, Zn
2
,
denaturation by 6 M urea
Ca
2
, Na
2
, B.S.A. K
m
/14 mg ml
1
; enzyme
active after acetone and
ethanol treatment
[4]
B. stearothermophilus
MFF4
Á/ Á/ 5.5Á/6.0/ Á/ 70Á/75 8C/half life
5.1 h at 80 8C
Á/ Á/ Ca
2
enhances thermo-
stability
[102]
B. subtilis Á/ 48.0 6.5/ 5/7.0 50 8C/ 5/50 8C Hg
2
, Fe
3
, Al
3
Mn
2
, Co
2
K
m
(3.845 mg ml
1
); V
max
(585.1 mg); end product, G
2
[159]
B. subtilis 65 Á/ 68.0 6.0/6.0Á/9.0 60 8C/60 8C (5
min)
Cu
2
, Fe
3
, Mn
2
, Hg
2
, Zn
2
,
Pb
2
, Al
3
, Cd
2
, Ag
2
, EDTA
Ca
2
End products, G
1
, G
2
[51]
R
.
G
u
p
t
a
e
t
a
l
.
/
P
r
o
c
e
s
s
B
i
o
c
h
e
m
i
s
t
r
y
0
0
(
2
0
0
3
)
1
Á
/
1
8
9
A
R
T
I
C
L
E
I
N
P
R
E
S
S
Table 2 (Continued)
Source pI Molecular
weight
(kDa)
pH optima/stabi-
lity
Temperature op-
tima/stability
Inhibitors Stabilisers Additional properties Reference
B. licheniformis M27 Á/ 56.0 6.5Á/7.0 and 8.5Á/
9.0/ 5/7.0 and ]/
7.5
85Á/90 8C/ /
90 8C
Á/ Ca
2
E.A. (25 kJ mol
1
); ther-
mostability dependent upon
pH stability
[188]
Bacillus sp. IMD 435 5.6 63.0 6.0 and 6.5 Á/ Á/ Á/ End products, G
1
, G
2
, G
3
,
G
4
[45]
Bacillus sp. IMD 434 5.9 69.2 6.0/4.0Á/9.0 65 8C/40 8C (1 h) N-Bromosuccinimide, p-hydroxy-
mercuribenzoic acid
Cysteine, DTT End products, G
1
, G
2
; spe-
cificity for raw starch; K
m
(
1.9 mm)
[47]
Bacillus sp. US 100 Á/ Á/ 5.6/4.5Á/8.0 82 8C/90Á/95 8C Á/ Starch, Ca
2
Half-life increases to 110 8C
in presence of 20% (w/v)
substrate
[189]
Bacillus sp. WN 11 Á/ Á/ 5.0Á/8.0/ Á/ 75Á/80 8C/ Á/ Á/ Á/ No requirement for Ca
2
;
starch increases temperature
stability
[100]
Bacillus sp. WN 11 Á/ Amy 1-
76.0, Amy
2-53.0
5.5/5.5Á/9.0 (1 h) 75Á/80 8C/80 8C
(4 h)
Fe
3
, Hg
2
, Cu
2
Á/ End products, G
1
, G
2
, G
3
,
G
4
[190]
Bacillus sp. XAL 601 Á/ Á/ 9.0/ Á/ 70 8C/ Á/ Á/ Á/ Adsorbs to raw starch or
cellulose hydrolysis pro-
ducts, G
2
and G
4
[87]
Escherichia coli 48.0 6.5/ 5/7.0 50 8C/ B/70 8C Hg
2
, Fe
3
, Al
3
Mn
2
, Co
2
Á/ [159]
H. meridiana DSM
5425
Á/ Á/ 7.0/5.0Á/7.0 37 8C/ Á/ Á/ Ca
2
End products, G
2
, G
3
;
showed activity in 30% salts
[46]
L. plantarum A6 Á/ 50.0 5.5/ /3.0Á/ B/8.0 65 8C/ Á/ N-bromosuccinimide, iodine,
acetic acid, Hg
2
, dimethyl amino-
benzaldehyde
Á/ K
m
(2.38 g l
1
); A.E. (30.9
kJ mol
1
)
[160]
Micromonospora mel-
anosporea
7.6 45.0 7.0/ Á/ 55 8C/40 8C (pH
11Á/12, 40 min)
Á/ Á/ Á/ [191]
M. melanosporea 7.6 45.0 7.0/6.0Á/12.0 55 8C/ Á/ Á/ Á/ End product, G
1
[191]
Pseudomonas stutzeri Á/ 12.5 8.0/7.0Á/9.5 47 8C/40 8C (1 h) Á/ Ca
2
E.A. (13 400 and 5200 cal
mol
1
; end product, G
4
[93]
Streptococcus bovis
JB1
4.5 77.0 5.0Á/6.0/5.5Á/8.5 Á//50 8C (1 h) Hg
2
, p-chloromercuribenzoic
acid (both reversible by DTT)
Á/ K
m
(0.88 mg ml
1
); K
cat
(2510 mmol reducing sugar
mg
1
protein); end pro-
ducts, G
2
, G
3
, G
4
[161]
Streptomyces sp. IMD
2679
8.9(1),
8.7(2),
7.2(3)
47.8 5.5/ Á/ 60 8C/ Á/, 60Á/
65 8C/ Á/, 65 8C/ Á/
Á/ Á/ End products, G
1
, G
3
; K
m
(8.0Á/8.2 mM)
[44]
T. profundus DT5432 Á/ 42.0 5.5Á/6.0/5.9Á/9.8 80 8C/80 8C (3 h),
90 8C (15 min)
Iodoacetic acid, N-bromosuccinic
acid, SDS, guanidine hydrochlor-
ide
Ca
2
End products, G
2
, G
3
; K
m
(0.23%)
[71]
Thermomonospora
curvata
6.2 60.9 6.0/ Á/ 65 8C/ Á/ Á/ Á/ End product, G
2
; low affi-
nity for G
3
[162]
R
.
G
u
p
t
a
e
t
a
l
.
/
P
r
o
c
e
s
s
B
i
o
c
h
e
m
i
s
t
r
y
0
0
(
2
0
0
3
)
1
Á
/
1
8
1
0
A
R
T
I
C
L
E
I
N
P
R
E
S
S
amylase A (TAA) contains ten Ca
2
ions but only one
is tightly bound [112]. In other systems usually one
Ca
2
ion is sufficient to stabilise the enzyme. Ca
2
can
be removed from amylases by dialysis against EDTA or
by electrodialysis. Calcium free enzymes can be reacti-
vated by adding Ca
2
ions. Some studies have been
carried out on the ability of other ions to replace Ca
2
as Sr
2
in B. caldolyticus amylase [113]. Ca
2
in TAA
has been substituted by Sr
2
and Mg
2
in successive
crystallisation in the absence of Ca
2
and in excess of
Sr
2
and Mg
2
[114]. EDTA inactivated TAA can be
reactivated by Sr
2
, Mg
2
and Ba
2
[114]. In the
presence of Ca
2
, a-amylases are much more thermo-
stable than without it [4,115]. a-Amylase from A. oryzae
EI 212 is inactivated in the presence of Ca
2
, but retains
activity after EDTA treatment [116]. There are also
reports where Ca
2
did not have any effect on the
enzyme [117].
8. Industrial applications of a-amylase
Amylases are among the most important hydrolytic
enzymes for all starch based industries, and the com-
mercialisation of amylases is oldest with first use in
1984, as a pharmaceutical aid for the treatment of
digestive disorders. In the present day scenario, amy-
lases find application in all the industrial processes such
as in food, detergents, textiles and in paper industry, for
the hydrolysis of starch. In this light, microbial amylases
have completely replaced chemical hydrolysis in the
starch processing industry. They can also be of potential
use in the pharmaceutical and fine chemical industries.
Today, amylases have the major world market share of
enzymes [118]. Several different amylase preparations
are available with various enzyme manufacturers for
specific use in varied industries. A comprehensive
account on commercial applications of a-amylases is
quoted by Godfrey and West [119]. Various applications
of a-amylase are dealt here in brief.
8.1. Bread and baking industry and as an antistaling
agent
The baking industry has made use of these enzymes
for hundreds of years to manufacture a wide variety of
high quality products. For decades, enzymes such as
malt and microbial a-amylases have been widely used in
the baking industry [120,121]. These enzymes were used
in bread and rolls to give these products a higher
volume, better colour and a softer crumb. It is the
malt preparation that has led the way and opened the
opportunities for many enzymes to be used commer-
cially in baking. Today, many enzyme preparations such
as proteases, lipases, xylanases, pullulanases, pentosa-
nases, cellullases, glucose oxidases, lipoxygenases etc. T
a
b
l
e
2
(
C
o
n
t
i
n
u
e
d
)
S
o
u
r
c
e
p
I
M
o
l
e
c
u
l
a
r
w
e
i
g
h
t
(
k
D
a
)
p
H
o
p
t
i
m
a
/
s
t
a
b
i
-
l
i
t
y
T
e
m
p
e
r
a
t
u
r
e
o
p
-
t
i
m
a
/
s
t
a
b
i
l
i
t
y
I
n
h
i
b
i
t
o
r
s
S
t
a
b
i
l
i
s
e
r
s
A
d
d
i
t
i
o
n
a
l
p
r
o
p
e
r
t
i
e
s
R
e
f
e
r
e
n
c
e
T
.
c
u
r
v
a
t
a
Á
/
6
2
.
0
5
.
5
Á
/
6
.
0
/
a
c
t
i
v
a
t
e
d
a
t
p
H
7
.
0
Á
/
8
.
0
6
5
8
C
/
Á
/
B
.
S
.
A
.
Á
/
E
n
d
p
r
o
d
u
c
t
s
,
G
4
,
G
5
;
K
m
(
0
.
3
m
g
m
l

1
)
[
1
5
5
]
T
.
f
u
s
c
a
Y
X
Á
/
Á
/
6
.
0
/
Á
/
6
0
8
C
/
B
/
6
5
8
C
Á
/
S
t
a
r
c
h
,
C
a
2

E
n
d
p
r
o
d
u
c
t
s
,
G
3
,
G
4
,
G
6
;
K
m
(
3
.
3
m
g
m
l

1
)
;
E
.
A
.
(
5
9
k
J
m
o
l

1
)
[
7
0
]
G
1
,
g
l
u
c
o
s
e
;
G
2
,
m
a
l
t
o
s
e
;
G
3
,
m
a
l
t
o
t
r
i
o
s
e
;
G
4
,
m
a
l
t
o
t
e
t
r
a
o
s
e
;
G
5
,
m
a
l
t
o
p
e
n
t
a
o
s
e
;
E
.
A
.
,
e
n
z
y
m
e
a
c
t
i
v
a
t
i
o
n
e
n
e
r
g
y
;
k
c
a
l
,
k
i
l
o
c
a
l
o
r
i
e
s
;
k
J
,
k
i
l
o
j
o
u
l
e
s
.
R. Gupta et al. / Process Biochemistry 00 (2003) 1Á/18 11
ARTICLE IN PRESS
are being used in the bread industry for varied purposes
[13,99,121Á/123], but none had been able to replace a-
amylases.
Till date, the a-amylases used in baking have been
cereal enzymes from barley malt and microbial enzymes
from fungi and bacteria [124,125]. Fungal a-amylases
have been permitted as bread additives since 1955 in the
US and in 1963 in UK after confirmation of their GRAS
status [126]. Presently they are used all over the world to
different extents. Supplementation of flour with exo-
genous fungal a-amylase having higher activities is
common in the present day modern and continuous
baking process [126]. a-Amylase supplementation in
flour not only enhances the rate of fermentation and
reduces the viscosity of dough (resulting in improve-
ments in the volume and texture of the product, but also
generates additional sugar in the dough, which improves
the taste, crust colour and toasting qualities of the bread
[127]. One of the new applications of a-amylase in the
industry has been in retarding the staling of baked
products, which reduces the shelf life of these products.
Upon storage the crumb becomes dry and firm, the
crust loses its crispness and the flavour of the bread
deteriorates. All these undesirable changes in the bread
are together known as staling. The importance of
retrogradation of starch fraction in bread staling has
been emphasised [128]. A loss of more than US $1
billion is incurred in USA alone every year due to the
staling of bread.
Conventionally various additives are used to prevent
staling and improve the texture and flavour of baked
products. Additives include chemicals, small sugars,
enzymes/their combinations, milk powder; emulsifiers,
monoglycerides/diglycerides, sugar esters, lecithin, etc;
granulated fat, anti-oxidant (ascorbic acid or potassium
borate), sugars/salts [129]. Recently emphasis has
been given to the use of enzymes in dough improve-
ment/as anti-staling agents, e.g. a-amylase [130,131],
branching enzymes [132] and debranching enzymes
[133], maltogenic amylases [134], b-amylases [135]
amyloglucosidases [136]. Pullulanases and a-amylase
combination are used for efficient antistaling property
[133]. However, a slight excess of a-amylases was also
used which is undesirable as it causes stickiness in bread
[134]. Therefore, a recent trend is to use intermediate
temperature stable (ITS) a-amylases [13,124,125,137].
They are active after starch gelatinisation and become
inactive much before the completion of the baking
process. Further, the dextrin with 4Á/9 degree of poly-
merisation produced by these shows the anti-staling
properties. Although a wide variety of microbial a-
amylases is known, a-amylase with ‘ITS’ property has
been reported from only a few microorganisms
[99,123,138,139].
8.2. Starch liquefaction and saccharification
The major market for a-amylases lies in the produc-
tion of starch hydrolysates such as glucose and fructose.
Starch is converted into high fructose corn syrups
(HFCS). Because of their high sweetening property,
these are used in huge quantities in the beverage
industry as sweeteners for soft drinks. The process
requires the use of a highly thermostable a-amylase for
starch liquefaction. The use of enzyme in starch
liquefaction is well established and has been extensively
reviewed [2,140].
8.3. Textile desizing
Modern production processes for textiles introduce a
considerable strain on the warp during weaving. The
yarn must, therefore, be prevented from breaking. For
this purpose a removable protective layer is applied to
the threads. The materials that are used for this size
layer are quite different. Starch is a very attractive size,
because it is cheap, easily available in most regions of
the world, and it can be removed quite easily. Good
desizing of starch sized textiles is achieved by the
application of a-amylases, which selectively remove the
size and do not attack the fibres. It also randomly
cleaves the starch into dextrins that are water soluble
and can be removed by washing. The use of a-amylases
in warp sizing of textile fibres for manufacturing fibres
with great strength has been reported [141].
8.4. Paper industry
The use of a-amylase for the production of low
viscosity, high molecular weight starch for coating of
paper is reported [142]. The use of amylases in the pulp
and paper industry is in the modification of starches for
coated paper. As for textiles, sizing of paper is
performed to protect the paper against mechanical
damage during processing. It also improves the quality
of the finished paper. The size enhances the stiffness and
strength in paper. It also improves the erasibilty and is a
good coating for the paper. Starch is also a good sizing
agent for the finishing of paper. Starch is added to the
paper in the size press and paper picks up the starch by
passing through two rollers that transfer the starch
slurry. The temperature of this process lies in the range
of 45Á/60 8C. A constant viscosity of the starch is
required for reproducible results at this stage. The mill
also has the flexibility of varying the starch viscosity for
different paper grades. The viscosity of the natural
starch is too high for paper sizing and is adjusted by
partially degrading the polymer with a-amylases in a
batch or continuous processes. The conditions depend
upon the source of starch and the a-amylase used [143].
A number of amylases exist for use in the paper
R. Gupta et al. / Process Biochemistry 00 (2003) 1Á/18 12
ARTICLE IN PRESS
industry, which include Amizyme

(PMP Fermentation
Products, Peoria, USA), Termamyl

, Fungamyl,
BAN

(Novozymes, Denmark) and a-amylase
G9995

(Enzyme Biosystems, USA).
8.5. Detergent applications
Enzymes now comprise as one of the ingredients of
modern compact detergents. The main advantage of
enzyme application in detergents is due to much milder
conditions than with enzyme free detergents. The early
automatic dishwashing detergents were very harsh,
caused injury when ingested and were not compatible
with delicate china and wooden dishware. This forced
the detergent industries to search for milder and more
efficient solutions [144]. Enzymes also allow lowering of
washing temperatures. a-Amylases have been used in
powder laundry detergents since 1975. Nowadays, 90%
of all liquid detergents contain a-amylase [145] and the
demand for a-amylases for automatic dishwashing
detergents is growing. One of the limitations of a-
amylases in detergents is that the enzyme shows
sensitivity to calcium and stability is severely compro-
mised in a low calcium environment. In addition, most
wild-type a-amylases are sensitive to oxidants which are
generally a component of detergent formulations. Sta-
bility against oxidants in household detergents was
achieved by utilising successful strategies followed with
other enzymes such as protease. Recently scientists from
the two major detergent enzyme suppliers Novozymes
and Genencore International have used protein engi-
neering to improve the bleach stability of the amylases
[146Á/148]. They independently replaced oxidation sen-
sitive amino acids with other amino acids. The replace-
ment of met at position 197 by leu in B. licheniformis
amylase resulted in an amylase with improved resistance
against oxidative compounds. This improved oxidation
stability resulted in better storage stability and perfor-
mance of the mutant enzyme in the bleach containing
detergent formulations. Genencore International and
Novozyme have introduced these new products in the
market under the trade names Purafect OxAm

and
Duramyl

, respectively.
8.6. Analysis in medicinal and clinical chemistry
With the advent of new frontiers in biotechnology, the
spectrum of amylase applications has expanded into
many other fields, such as clinical, medicinal and
analytical chemistry. There are several processes in the
medicinal and clinical areas that involve the application
of amylases. The application of a liquid stable reagent,
based on a-amylase for the Ciba Corning Express
clinical chemistry system has been described [149]. A
process for the detection of higher oligosaccharides,
which involved the application of amylase was also
developed [96]. This method was claimed to be more
efficient than the silver nitrate test. Biosensors with an
electrolyte isolator semiconductor capacitor (EIS-CAP)
transducer for process monitoring were also developed
[150].
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 applica-
tions 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 applica-
tion in food and starch based industries is the major
market and thus the demand of a-amylases would
always be high in these sectors.
References
[1] Windish WW, Mhatre NS. Microbial amylases. In: Wayne WU,
editor. Advances in applied microbiology, vol. 7. New York:
Academic Press, 1965:273Á/304.
[2] Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R.
Advances in microbial amylases. Biotechnol Appl Biochem
2000;31:135Á/52.
[3] Fogarty WM, Kelly CT. Developments in microbial extracel-
lular enzymes. In: Wiseman A, editor. Topics in enzyme and
fermentation biotechnology, vol. 3. New York: Wiley, 1979:45Á/
108.
[4] Vihinen M, Mantsala P. Microbial amylolytic enzymes. Crit Rev
Biochem Mol Biol 1989;24:329Á/418.
[5] Lonsane BK, Ramesh MV. Production of bacterial thermostable
a-amylase by solid state fermentation: a potential tool for
achieving economy in enzyme production and starch hydrolysis.
In: Advances in applied microbiology, vol. 35. San Diego:
California Academic Press, 1990:1Á/56.
[6] Priest FG. Extracellular enzyme synthesis in the genus Bacillus .
Bacteriol Rev 1977;41:711Á/53.
[7] Hollo J, Szeitli J. The reaction of starch with iodine. In: Rodley
JA, editor. Starch and its derivatives, 4th ed. Chapman & Hall,
1968:203Á/46.
R. Gupta et al. / Process Biochemistry 00 (2003) 1Á/18 13
ARTICLE IN PRESS
[8] Fuwa H. A new method for microdetermination of amylase
activity by the use of amylose as substrate. J Biochem
1954;41:583Á/603.
[9] Manonmani HK, Kunhi AAM. Interference of thiol compounds
with dextrinizing activity assay of a-amylase by starchÁ/iodine
color reaction: modification of the method to eliminate this
interference. World J Microbiol Biotechnol 1999;15:485Á/7.
[10] Hansen PW. Determination of fungal a-amylase by flow
injection analysis. Anal Chim Acta 1984;158:375Á/7.
[11] Carlsen M, Marcher J, Nielsen J. An improved FIA-method for
measuring a-amylase in cultivation media. Biotechnol Tech
1994;8:479Á/82.
[12] Sandstedt RM, Kneen E, Blish MJ. A standardised Wohlgemuth
procedure for a-amylase activity. Cereal Chem 1939;16:712Á/23.
[13] Kulp K. Enzymes as dough improvers. In: Kamel BS, Stauffer
CE, editors. Advances in baking technology. New York: Blackie
Academic and Professional, VCH Publishers, 1993:152Á/78.
[14] Indian Standard. Colorimetric determination of alpha-amylase
activity in cereals and cereal products. IS 10524. Indian
Standards Institution, 1982. New Delhi.
[15] Bernfeld P. Amylases, a and b. Methods Enzymol 1955;1:149Á/
58.
[16] Miller GL. Use of dinitrosalicylic acid reagent for determination
of reducing sugar. Anal Chem 1959;31:426Á/8.
[17] Mishra RS, Maheshwari R. Amylases of the thermophilic fungus
Thermomyces lanuginosus : their purification, properties, action
on starch and response to heat. J Biosci 1996;21:653Á/72.
[18] Ceska M, Hultman E, Ingelman BGA. A new method for
determination of a-amylase. Experentia 1969;25:555Á/6.
[19] Dhawale MR, Wilson JJ, Khachatourians GG, Ingledew WM.
Improved method for detection of starch hydrolysis. Appl
Environ Microbiol 1982;44:747Á/50.
[20] Wong DWS, Batt SB, Robertson GH. Microassay for rapid
screening of alpha-amylase activity. J Agric Food Chem
Washington, DC: Am Chem Soc 2000;48:4540Á/3.
[21] Sheehan H, McCleary BV. A new procedure for the measure-
ment of fungal and bacterial a-amylase. Biotechnol Tech
1988;2:289Á/92.
[22] International Association for Cereal Chemistry. Standard meth-
ods of the ICC. 1968: Method 104, approved 1960; Method 105,
approved 1980; Method 107, approved 1968; Method 108,
approved 1968. Verlag Moritz Schafer, Detmold, West Ger-
many.
[23] American Association of Cereal Chemists. Approved methods of
the AACC.1972: 7th ed. Method 56-81B, approved November
1972. The Association, St. Paul, MN.
[24] International Organization for Standardization. International
standard ISO 3093, approved 1974. The Secretriat: Magyar
Szabvanyugyi Hivatal, 1450, Budapest 9, Pf 24, Hungary.
[25] Perten H. A modified falling number method suitable for
measuring both cereal and fungal a-amylase activity. Cereal
Chem 1984;61:108Á/11.
[26] Fogarty WM, Kelly CT. Starch degrading enzymes of microbial
origin. Prog Ind Microbiol 1979;15:87Á/150.
[27] Tonomura K, Suzuki H, Nakamura N, Kuraya K, Tanabe O.
On the inducers of a-amylase formation in Aspergillus oryzae.
Agric Biol Chem 1961;25:1Á/6.
[28] Yabuki M, Ono N, Hoshino K, Fukui S. Rapid induction of a-
amylase by non-growing mycelia of Aspergillus oryzae. Appl
Environ Microbiol 1977;34:1Á/6.
[29] Lachmund A, Urmann U, Minol K, Wirsel S, Ruttkowski E.
Regulation of a-amylase formation in Aspergillus oryzae and
Aspergillus nidulans transformants. Curr Microbiol 1993;26:47Á/
51.
[30] Morkeberg R, Carlsen M, Neilsen J. Induction and repression of
a-amylase production in batch and continuous cultures of
Aspergillus oryzae. Microbiology 1995;141:2449Á/54.
[31] Eratt JA, Douglas PE, Moranelli F, Seligy VL. The induction of
a-amylase by starch in Aspergillus oryzae: evidence for con-
trolled mRNA expression. Can J Biochem Cell Biol
1984;62:678Á/90.
[32] Bhella RS, Altosaar I. Purification and some properties of the
extracellular a-amylase from Aspergillus awamori . Can J Micro-
biol 1985;31:149.
[33] Arst HN, Bailey CR. The regulation of carbon metabolism in
Aspergillus nidulans . In: Smith JE, Pateman JA, editors. Genetics
and physiology of Aspergillus . New York: Academic Press,
1977:131Á/46.
[34] Kelly CT, Bolton DJ, Fogarty WM. Biphasic production of a-
amylase of Bacillus flavothermus in batch fermentation. Bio-
technol Lett 1997;19:75Á/7.
[35] Emanuilova EI, Toda K. a-Amylase production in batch and
continuous cultures by Bacillus caldolyticus . Appl Microbiol
Biotechnol 1984;19:301Á/5.
[36] Cheng CY, Yabe I, Toda K. Selective growth of a mutant in
continuous culture of Bacillus caldolyticus for production of a-
amylase. Appl Microbiol Biotechnol 1989;30:125Á/9.
[37] Welker NE, Campbell LL. Effect of carbon sources on forma-
tion of a-amylase by Bacillus stearothermophilus . J Bacteriol
1963;86:681Á/6.
[38] Krishnan T, Chandra AK. Effect of oilseed cakes on a-amylase
production by Bacillus licheniformis CUMC-305. Appl Environ
Microbiol 1982;44:270Á/4.
[39] Jin B, Van Leeuwen JH, Patel B. Mycelial morphology and
fungal protein production from starch processing wastewater in
submerged cultures of Aspergillus oryzae. Process Biochem
1999;34:335Á/40.
[40] Kekos D, Galiotou-Panayotou M, Macris BJ. Some nutritional
factors affecting a-amylase production by Calvatia gigantea.
Appl Microbiol Biotechnol 1987;26:527Á/30.
[41] Domingues CM, Peralta RM. Production of amylase by soil
fungi and partial biochemical characterization of amylase of a
selected strain (Aspergillus fumigatus fresenius ), Can J Microbiol
1993;39:681Á/5.
[42] Goto CE, Barbosa EP, Kistner LCL, Moreira FG, Lenartoviez
V, Peralta RM. Production of amylase by Aspergillus fumigatus
utilizing a-methyl D-glucoside, a synthetic analog of maltose as
substrate. FEMS Microbiol Lett 1998;167:139Á/43.
[43] Arnesen S, Eriksen SH, Olsen J, Jensen B. Increased production
of alpha amylase from Thermomyces lanuginosus by the addition
of Tween-80. Enzyme Microb Technol 1998;23:249Á/52.
[44] McMahon HEM, Kelly CT, Fogarty WM. High maltose
producing amylolytic system of a Streptomyces sp. Biotechnol
Lett 1999;21:23Á/6.
[45] Hamilton LM, Kelly CT, Fogarty WM. Production and proper-
ties of the raw starch-digesting a-amylase of Bacillus sp. IMD
435. Process Biochem 1999;35:27Á/31.
[46] Coronado MJ, Vargas C, Hofemeister J, Ventosa A, Nieto JJ.
Production and biochemical characterization of an a-amylase
from the moderate halophile Halomonas meridiana. FEMS
Microbiol Lett 2000;183:67Á/71.
[47] Hamilton LM, Kelly CT, Fogarty WM. Purification and proper-
ties of the raw starch degrading a-amylase of Bacillus sp.
IMD434. Biotechol Lett 1999;21:111Á/5.
[48] Dercova K, Augustin J, Krajcova D. Cell growth and a-amylase
production characteristics of Bacillus subtilis . Folia Microbiol
1992;37:17Á/23.
[49] Imai Y, Suzuki M, Masamoto M, Nagayasu K. Amylase
production by Aspergillus oryzae in a new kind of fermentor
with a rotary draft tube. J Ferment Bioeng 1993;76:459Á/64.
[50] Pedersen H, Nielsen J. The influence of nitrogen sources on the
a-amylase productivity of Aspergillus oryzae in continuous
cultures. Appl Microbiol Biotechnol 2000;53:278Á/81.
R. Gupta et al. / Process Biochemistry 00 (2003) 1Á/18 14
ARTICLE IN PRESS
[51] Hayashida S, Teramoto Y, Inoue T. Production and character-
istics of raw potato starch digesting a-amylase from Bacillus
subtilis 65. Appl Environ Microbiol 1988;54:1516Á/22.
[52] Rukhaiyar R, Srivastava SK. Effect of various carbon substrate
on a-amylase production from Bacillus sp. J Microb Biotechnol
1995;10:76Á/82.
[53] Yoshigi N, Chikano T, Kamimura M. Production of an
extracellular a-amylase from Bacillus cereus NY-14. J Jpn Soc
Starch Sci 1985;32:217Á/21.
[54] Cheng CY, Yabe I, Toda K. Predominant growth of a-amylase
regulation mutant in continuous culture of Bacillus caldolyticus .
J Ferment Bioeng 1989;67:176Á/81.
[55] Ueno S, Miyama M, Ohashi Y, Izumiya M, Kusaka I. Secretory
enzyme production and conidiation of Aspergillus oryzae in
submerged liquid culture. Appl Microbiol Biotechnol
1987;26:273Á/6.
[56] Shah NK, Upadhyay CM, Nehete PN, Kothari RM, Hegde MV.
An economical, upgraded, stabilized and efficient preparation of
a-amylase. J Biotechnol 1990;16:97Á/108.
[57] Kundu AK, Das S, Gupta TK. Influence of culture and
nutritional conditions on the production of amylase by the
submerged culture of Aspergillus oryzae. J Ferment Technol
1973;51:142Á/50.
[58] Zhang Q, Tsukagoshi N, Miyashiro S, Udaka S. Increased
production of a-amylase by Bacillus amyloliquefaciens in the
presence of glycine. Appl Environ Microbiol 1983;46:293Á/5.
[59] Ikura Y, Horikoshi K. Effect of amino compounds on alkaline
amylase production by alkalophilic Bacillus sp. J Ferment
Technol 1987;65:707Á/9.
[60] Lee J, Parulekar SJ. Enhanced production of a-amylase in fed-
batch cultures of Bacillus subtilis TN 106[pAT5]. Biotechnol
Bioeng 1993;42:1142Á/50.
[61] Dean ACR. Influence of environment on the control of enzyme
synthesis. J Appl Chem Biotechnol 1972;22:245Á/59.
[62] Mertz FP, Doolin LE. The effect of phosphate on the biosynth-
esis of vanilomycin. Can J Microbiol 1973;19:263Á/70.
[63] Hillier P, Wase DAJ, Emery AN, Solomons GL. Instability of a-
amylase production and morphological variation in continuous
culture of Bacillus amyloliquefaciens is associated with plasmid
loss. Process Biochem 1997;32:51Á/9.
[64] Wu WX, Mabinadji J, Betrand TF, Wu WX. Effect of culture
conditions on the production of an extracellular thermostable
alpha-amylase from an isolate of Bacillus sp. J Zhejiang Univ
Agric Life Sci 1999;25:404Á/8.
[65] Pazlarova J, Votruba J. Use of zeolite to control ammonium in
Bacillus amyloliquifaciens a-amylase fermentations. Appl Micro-
biol Biotechnol 1996;45:314Á/8.
[66] McMahon HEM, Kelly CT, Fogarty WM. Effect of growth rate
on a-amylase production by Streptomyces sp. IMD 2679. Appl
Microbiol Biotechnol 1997;48:504Á/9.
[67] Castro PML, Hayter PM, Ison AP, Bull AT. Application of
statistical design to the optimization of culture medium for
recombinant interferon-gamma production by Chinese hamster
ovary cells. Appl Microbiol Biotechnol 1992;38:84Á/90.
[68] Chahal DS. Growth characteristics of microorganisms in solid
state fermentation for upgrading of protein values of lignocellu-
loses and cellulase production. In: Blanch HW, Papoutsakis ET,
Stephanopoulos G, editors. Foundations of biochemical engi-
neering kinetics and thermodynamics in biological systems.
American Chemical Society, Washington DC. ACS symposium
series, No. 207, 1983:421Á/42.
[69] Friedrich J, Cimerman A, Steiner W. Submerged production of
pectinolytic enzymes by Aspergillus niger : effect of different
aeration/agitation regimes. Appl Microbiol Biotechnol
1989;31:490Á/4.
[70] Busch JE, Stutzenberger FJ. Amylolytic activity of Thermomo-
nospora fusca. World J Microbiol Biotechnol 1997;13:637Á/42.
[71] Chung YC, Kobayashi T, Kanai H, Akiba T, Kudo T.
Purification and properties of extracellular amylase from the
hyperthermophilic archeon Thermococcus profundus DT5432.
Appl Environ Microbiol 1995;61:1502Á/6.
[72] Bhavaraju SM, Blanch HW. A model for pellet breakup in
fungal fermentations. J Ferment Technol 1976;54:466Á/8.
[73] Justen P, Paul GC, Nienow AW, Thomas CR. Dependence of
mycelial morphology on impeller type and agitation intensity.
Biotechnol Bioeng 1996;52:672Á/84.
[74] Bocking SP, Wiebe MG, Robson GD, Hansen K, Christiansen
LH, Trinci APJ. Effect of branch frequency in Aspergillus oryzae
on protein secretion and culture viscosity. Biotechnol Bioeng
1999;65:638Á/48.
[75] Cui YQ, Van der Lans RGJM, Luyben KCAM. Effect of
agitation intensities on fungal morphology of submerged fer-
mentation. Biotechnol Bioeng 1997;55:715Á/26.
[76] Amanullah A, Blair R, Nienow AW, Thomas CR. Effects of
agitation intensity on mycelial morphology and protein produc-
tion in chemostat cultures of recombinant Aspergillus oryzae.
Biotechnol Bioeng 1999;62:434Á/46.
[77] Priest FG. Products and applications. In: Harwood CR, editor.
Biotechnology handbooks, Bacillus . New York, London: Ple-
num Press, 1989:293Á/320.
[78] Carlsen M, Nielsen J, Villadser J. Growth and a-amylase
production by Aspergillus oryzae during continuous cultivations.
J Biotechnol 1996;45:81Á/93.
[79] Spohr A, Carlsen M, Nielsen J, Villadsen J. Morphological
characterization of recombinant strains of Aspergillus oryzae
producing a-amylase during batch cultivations. Biotechnol Lett
1997;19:257Á/61.
[80] Agger T, Spohr AB, Carlsen M, Nielsen J. Growth and product
formation of Aspergillus oryzae during submerged cultivations:
verification of a morphologically structured model using fluor-
escent probes. Biotechnol Bioeng 1998;57:321Á/9.
[81] Heineken FG, O’Conner RJ. Continuous culture studies on the
biosynthesis of alkaline protease, neutral protease and a-amylase
by Bacillus subtilis NRRL-B3411. J Gen Microbiol 1972;73:35Á/
44.
[82] Delgado G, Topete M, Galindo E. Interaction of cultural
conditions and end product distribution in Bacillus subtilis
grown in shake flasks. J Microbiol Biotechnol 1989;31:288Á/92.
[83] Bohdziewicz J. Ultrafiltration of technical amylolytic enzymes.
Process Biochem 1996;31:185Á/91.
[84] Schwermann B, Pfau K, Liliensiek B, Schleyer M, Fischer T,
Bakker EP. Purification, properties and structural aspects of a
thermoacidophilic a-amylase from Alicyclobacillus acidocaldar-
ius ATCC 27009. Insight into acidostability of proteins. Eur J
Biochem 1994;226:981Á/91.
[85] Saito NA. Thermophilic extracellular a-amylase from Bacillus
licheniformis . Arch Biochem Biophys 1973;155:290Á/8.
[86] Krishnan T, Chandra AK. Purification and characterization of
a-amylase from Bacillus licheniformis CUMC 305. Appl Environ
Microbiol 1983;46:430Á/7.
[87] Lee SP, Morikawa M, Takagi M, Imanaka T. Cloning of the
aapT gene and characterization of its product, a-amylaseÁ/
pullulanase (AapT), from thermophilic and alkaliphilic Bacillus
sp. Strain XAL601. Appl Environ Microbiol 1994;60:3764Á/73.
[88] Shinke R, Aoki K, Murokam S, Inoue T, Babat T. Alkaline and
thermophilic amylases of industrial use. In: Dordick JS, Russell
AJ, editors. Enzyme engineering XIII, vol. 799. New York:
Annals of the New York Academy of Sciences, 1996:332Á/40.
[89] Kim TU, Gu BG, Jeong JY, Byun SM, Shin YC. Purification
and characterization of a maltotetraose forming alkaline a-
amylase from an alkalophilic Bacillus sp. GM8901. Appl
Environ Microbiol 1995;61:3105Á/12.
[90] Ogasahara K, Imanishi A, Isemura T. Studies on thermophilic a-
amylase from Bacillus stearothermophilus . I. Some general and
R. Gupta et al. / Process Biochemistry 00 (2003) 1Á/18 15
ARTICLE IN PRESS
physico-chemical properties of thermophilic a-amylase. J Bio-
chem 1970;67:65.
[91] Pfueller SL, Elliott WH. The extracellular a-amylase of Bacillus
stearothermophilus . J Biol Chem 1969;244:48.
[92] Khoo SL, Amirul A-A, Kamaruzaman M, Nazalan N, Azizan
MN. Purification and characterization of a-amylase from
Aspergillus flavus . Folia Microbiol 1994;39:392Á/8.
[93] Robyt J, Ackerman RJ. Isolation, purification and characteriza-
tion of a maltotetraose producing amylase from Pseudomonas
stutzeri . Arch Biochem Biophys 1971;145:105Á/14.
[94] Chary SJ, Reddy SM. Starch degrading enzymes of two species
of Fusarium. Folia Microbiol 1985;30:452.
[95] Fogarty WM, Kelly CT. Economic microbiology. In: Rose AH,
editor. Microbial enzymes and bioconversions, vol. 5. London:
Academic Press, 1980:115Á/70.
[96] Giri NY, Mohan AR, Rao LV, Rao CP. Immobilization of a-
amylase complex in detection of higher oligosaccharides on
paper. Curr Sci 1990;59:1339Á/40.
[97] Ray RR, Jana DC, Nanda G. Immobilization of b-amylase from
Bacillus megaterium B
6
into gelatin film by cross-linking. J Appl
Bacteriol 1995;79:157Á/62.
[98] Kobayashi T, Kamekura M, Kanlayakrit W, Ohnishi H.
Production, purification and characterization of an amylase
from the moderate halophile Micrococcus varians subspecies
halophilus . Microbios 1986;46:165.
[99] Prieto JA, Bort BR, Martinez J, Randez-Gil F, Buesa C, Sanz P.
Purification and characterization of a new a-amylase of inter-
mediate thermal stability from the yeast Lipomyces kononen-
koae. Biochem Cell Biol 1995;73:41Á/9.
[100] Mamo G, Gashe BA, Gessesse A. A highly thermostable
amylase from a newly isolated thermophillic Bacillus sp.
WN11. J Appl Microbiol 1999;86:557Á/60.
[101] Manning GB, Campbell LL. Thermostable a-amylase of Bacillus
stearothermophilus . I. Crystallization and some general proper-
ties. J Biol Chem 1961;236:2952Á/7.
[102] Wind RD, Buitelaar RM, Eggink G, Huizing HJ, Dijkhuizen L.
Characterization of a new Bacillus stearothermophilus isolate: a
highly thermostable a-amylase producing strain. Appl Microbiol
Biotechnol 1994;41:155Á/62.
[103] Grootegoed JA, Lauwers AM, Heinen W. Separation and partial
purification of extracellular amylase and protease from Bacillus
caldolyticus . Arch Microbiol 1973;90:223.
[104] Ratanakhanokchai K, Kaneko J, Kamio Y, Izaki K. Purification
and properties of a maltotetraose and maltotriose producing
amylase from Chloroflexus aurantiacus . Appl Environ Microbiol
1992;58:2490Á/4.
[105] McKelvy J, Lee YC. Microheterogeneity of the carbohydrate
group of Aspergillus oryzae a-amylase. Arch Biochem Biophys
1969;132:99Á/110.
[106] Eriksen SH, Jensen B, Olsen J. Effect of N-linked glycosylation
on secretion, activity and stability of a-amylase from Aspergillus
oryzae. Curr Microbiol 1998;37:117Á/22.
[107] Srivastava RAK. Studies on extracellular and intracellular
purified amylases from a thermophilic Bacillus stearothermophi-
lus . Enzyme Microb Technol 1984;6:422.
[108] Yamane K, Yamaguchi K, Maruo B. Purification and properties
of a cross-reacting material related to a-amylase and biochemical
comparison with parental a-amylase. Biochim Biophys Acta
1973;295:323.
[109] Matsuzaki H, Yamane K, Yamaguchi K, Nagata Y, Maruo B.
Hybrid a-amylase produced by transformants of Bacillus subtilis
I. Immunological and chemical properties of a-amylases pro-
duced by the parental strain and the transformants. Biochim
Biophys Acta 1974;365:235Á/47.
[110] Sills AM, Sauder ME, Stewart GG. Isolation and characteriza-
tion of the amylolytic system of Schwanniomyces castelli . J Inst
Brew 1984;90:311.
[111] Vallee BL, Stein EA, Summerwell WM, Fischer EM. Metal
content of a-amylases of various origins. J Biol Chem
1959;231:2901Á/5.
[112] Oikawa A, Maeda A. The role of calcium in Taka amylase A. J
Biochem 1957;44:745.
[113] Heinen W, Lauwers AM. Amylase activity and stability at high
and low temperature depending on calcium and other divalent
cations. Experientia 1975;26:77.
[114] Oikawa A. The role of calcium in Taka amylase A II. The
exchange reaction. Can J Biochem 1959;46:463.
[115] Robyt J, French D. Action pattern and specificity of an amylase
from Bacillus subtilis . Arch Biochem Biophys 1963;100:451Á/67.
[116] Kundu AK, Das S. Production of amylase in liquid culture by a
strain of Aspergillus oryzae. Appl Microbiol 1970;19:598.
[117] Laderman KA, Davis BR, Krutzsch HC, Lewis MS, Griko YV,
Privalov PL, Anfinsen CB. The purification and characterization
of an extremely thermostable a-amylase from hypothermophilic
archaebacterium Pyrococcus furiosus. J Biol Chem
1993;268:24394Á/401.
[118] Aehle W, Misset O. Enzymes for industrial applications. In:
Rehm HJ, Reed G, editors. Biotechnology, 2nd ed. Germany:
Wiley-VCH, 1999:189Á/216.
[119] Godfrey T, West S. In: Godfrey T, West S, editors. Industrial
enzymology. 2nd ed. New York: Stockton Press, 1996. p.
91,105Á/31,192,339Á/56,361Á/71.
[120] Hamer RJ. Enzymes in the baking industry. In: Tucker GA,
Woods LFJ, editors. Enzymes in food processing. Galsgow:
Blackie Academic and Professional, 1995:190Á/222.
[121] Si JQ. Enzymes, baking, bread making. In: Flickinger MC, Drew
SW, editors. Encyclopedia of bioprocess technology: fermenta-
tion, biocatalysis and bioseparation, vol. 2. Wiley, 1999:947Á/58.
[122] Pintauro ND. Bread and baked goods. In: Pintauro ND, editor.
Food processing enzymes */recent developments. Food technol-
ogy review No. 52. Park Ridge, NJ, USA: Noyes Data
Corporation, 1979.
[123] Monfort A, Blasco A, Preito JA, Sanz P. Combined expression
of Aspergillus nidulans endoxylanase X-24 and Aspergillus
oryzae a-amylase in industrial baker’s yeast and their use in
bread making. Appl Environ Microbiol 1996;62:3712Á/5.
[124] Hebeda RE, Bowles LK, Teague WM. Developments in enzymes
for retarding staling of baked goods. Cereal Foods World
1990;35:453Á/7.
[125] Hebeda RE, Bowles LK, Teague WM. Use of intermediate
temperature stability enzymes for retarding staling in baked
goods. Cereal Foods World 1991;36:618Á/24.
[126] Pritchard PA. Studies on the bread improving mechanisms of
fungal a-amylase. J Biol Educ 1992;26:12Á/8.
[127] Van Dam HW, Hille JDR. Yeast and enzymes in bread making.
Cereal Foods World 1992;37:245Á/52.
[128] Kulp K, Ponte GJ. Staling white pan bread: fundamental causes.
Crit Rev Food Sci Nutr 1981;15:1Á/48.
[129] Spendler T, Jorgensen O. Use of a branching enzyme in baking.
1997. Patent Application WO97/41736.
[130] De Stefanis VA, Turner EW. Modified enzyme system to inhibit
bread firming method for preparing same and use of same in
bread and other bakery products. 1981. Patent Application
US4299848.
[131] Cole MS. Antistaling baking composition. 1982. Patent Applica-
tion US4320151.
[132] Okada S, Kitahata S, Yoshikawa S, Sugimoto T, Sugimoto K.
Process for the production of branching enzyme and a method
for improving the qualities of food products therewith. 1984.
Patent Application US4454161.
[133] Carroll JO, Boyce COL, Wong TM, Starace CA. Bread
antistaling method. 1987. Patent Application US4654216.
[134] Olesen T. Antistaling process and agent. 1991. Patent Applica-
tion WO9104669.
R. Gupta et al. / Process Biochemistry 00 (2003) 1Á/18 16
ARTICLE IN PRESS
[135] Wursch P, Gumy D. Inhibition of amylopectin retrogradation by
partial beta amylolysis. Carbohydr Res 1994;256:129Á/37.
[136] Vidal FD, Gerrity AB. Antistaling agent for bakery products.
1979. Patent application US4160848.
[137] Ahuja A, Gupta R, Saxena RK, Gigras P. An antistaling enzyme
from microbes for baked products. In: Crowther JS, Marthi B,
editors. Proceedings of the microbiological safety of processed
foods. New Delhi: Oxford and IBH Publishing Co. Pvt. Ltd,
1998:127.
[138] Kraus JK, Hebeda RE. Method for retarding staling of baked
goods. 1993. US patent 5,209,938.
[139] Gigras P, Sahai V, Gupta R. Statistical media optimization and
production of ITS alpha amylase from A. oryzae in a bioreactor.
Curr Microbiol 2002;45(3):203Á/8.
[140] Van der Maarel MJEC, Van der Veen B, Uitdehaag JCM,
Leemhuis H, Dijkhuizen L. Properties and applications of starch
converting enzymes of alpha amylase family. J Biotechnol
2002;94:137Á/55.
[141] Hendriksen HV, Pedersen S, Bisgard-Frantzen H. A process for
textile warp sizing using enzymatically modified starches. 1999.
Patent Application WO 99/35325.
[142] Bruinenberg PM, Hulst AC, Faber A, Voogd RH. A process for
surface sizing or coating of paper. 1996. European Patent
Application EP 0,690,170 A1.
[143] Tolan JS. Pulp and paper. In: Godfrey T, West S, editors.
Industrial enzymology, 2nd ed. New York: Stockton Press,
1996:327Á/38.
[144] Van Ee JH, van Rijswijk WC, Bollier M. Enzymatic automated
dishwash detergents. Chim Oggi 1992;10:21Á/4.
[145] Kottwitz B, Upadek H, Carrer G. Applications and benefits of
enzymes in detergent. Chim Oggi 1994;12:21Á/4.
[146] Svendsen A, Bisgaard-Frantzen H. PCT patent publication. WO
94/0, 1994.
[147] Tierny L, Danko S, Dauberman J, Vaha-Vahe P, Winetzky D.
Performance advantages of novel a-amylases in automatic
dishwashing. Am Oil Chem Soc 86th San Antonio Annual
meeting, 1995.
[148] Bisgaard-Frantzen H, Borchert T, Svendsen A, Thellersen MH,
Van Der Zee P. PCT Patent Application. WO 95/10603, 1995.
[149] Becks S, Bielawaski C, Henton D, Padala R, Burrows K, Slaby
R. Application of a liquid stable amylase reagent on the Ciba
Corning Express clinical chemistry system. Clin Chem
1995;41:S186.
[150] Menzel C, Lerch T, Schneider K, Weidemann R, Tollnick C,
Kretymer G, Scheper T, Schugert K. Application of biosensors
with an electrolyte isolator semiconductor capacitor (EIS-CAP)
transducer for process monitoring. Process Biochem
1998;33:175Á/80.
[151] Mac Gregor EA, Janecek S, Svensson B. Relationship of
sequence and structure to specificity in the alpha amylase family
of enzymes. Biochim Biophys Acta 2001;1546:1Á/20.
[152] Iefuji P, Chino M, Kato M, Iimura Y. Raw-starch-digesting and
thermostable a-amylase from the yeast Cryptococcus sp. S-2:
purification, characterization, cloning and sequencing. Biochem
J 1996;318:989Á/96.
[153] De Moraes LMP, Astolfi-Filho S, Ulhao CJ. Purification and
some properties of an a-amylase glucoamylase fusion protein
from Saccharomyces cerevisiae. World J Microbiol Biotechnol
1999;15:561Á/4.
[154] Wilson JJ, Ingledew M. Isolation and characterization of
Schwanniomyces alluvius amylolytic enzymes, Appl Environ
Mirobiol 1982;44:301Á/7.
[155] Glymph JL, Stutzenberger FJ. Production, purification and
characterization of a-amylase from Thermomonospora curvata.
Appl Environ Microbiol 1977;34:391Á/7.
[156] Jenson B, Olsen J, Allermann K. Purification of extracellular
amylolytic enzymes from the thermophilic fungus Thermomyces
lanuginosus . Can J Microbiol 1988;34:218Á/23.
[157] Morgan FJ, Priest FG. Characterization of a thermostable a-
amylase from Bacillus licheniformis NCIB 6346. J Appl Bacteriol
1981;50:107Á/14.
[158] Vihinen M, Mantsala P. Characterization of a thermostable
Bacillus stearothermophilus a-amylase. Biotechnol Appl Bio-
chem 1990;12:427Á/35.
[159] Marco JL, Bataus LA, Valencia FF, Ulho CJ, Astolfi-Filho S,
Felix CR. Purification and characterization of a truncated
Bacillus subtilis a-amylase produced by Escherichia coli . Appl
Microbiol Biotechnol 1996;44:746Á/52.
[160] Giraud E, Gosselin L, Marin B, Parada JL, Raimbault M.
Purification and characterization of an extracellular amylase
from Lactobacillus plantarum strain A6. J Appl Bacteriol
1993;75:276Á/82.
[161] Freer SN. Purification and characterization of the extracellular
a-amylase from Streptococcus bovis JB 1. Appl Environ Micro-
biol 1993;59:1398Á/402.
[162] Collins BS, Kelly CT, Fogarty WM, Doyle EM. The high
maltose producing a-amylase of the thermophilic actinomycete,
Thermomonospora curvata. Appl Microbiol Biotechnol
1993;39:31Á/5.
[163] Michelena VV, Castillo FJ. Production of amylase by Aspergillus
foetidus on rice flour medium and characterization of the
enzyme. J Appl Bacteriol 1984;56:395.
[164] Perevozchenko II, Tsyperovich AS. Comparative investigation
of the properties of a-amylases of mold fungi of the genus
Aspergillus . Appl Biochem Microbiol 1972;8:7.
[165] Olutiola PO. a-Amylolytic activity of Aspergillus chevalieri from
mouldy maize seeds. Indian Phytopathol 1982;35:428.
[166] Alazard D, Baldensperger JF. Amylolytic enzymes from Asper-
gellus hennebergi (A. niger group): purification and character-
ization of amylases from solid and liquid cultures. Carbohydr
Res 1982;107:231.
[167] Minoda Y, Yamada K. Acid-stable a-amylase of black Asper-
gilli . I. Detection and purification of acid-stable dextrinizing
amylase. Agric Biol Chem 1963;27:806.
[168] Minoda Y, Arai M, Torigoe Y, Yamada K. Acid-stable a-
amylase of black Aspergilli . III. Separation of acid-stable a-
amylase and acid-unstable a-amylase from the same mold
amylase preparation. Agric Biol Chem 1968;32:110Á/4.
[169] Minoda Y, Arai M, Yamada K. Acid-stable a-amylase of black
Aspergilli . V. Amino acid composition and amino-terminal
amino acid. Agric Biol Chem 1969;33:572.
[170] Arai M, Koyano T, Ozawa H, Minoda Y, Yamada K. Acid-
stable a-amylase of black Aspergilli . IV. Some physicochemical
properties. Agric Biol Chem 1968;32:507.
[171] Arai M, Minoda Y, Yamada K. Acid-stable a-amylase of black
Aspergilli . VI. Carbohydrate and metal content. Agric Biol
Chem 1969;33:922.
[172] Bhumibhamon O. Production of amyloglucosidase by sub-
merged culture. Thailand J Agric Sci 1983;16:173.
[173] Ramachandran N, Skreekantiah KR, Murthy VS. Studies on the
thermophilic amylolytic enzymes of a strain of Aspergillus niger .
Starch 1978;30:272.
[174] Ramachandran N, Skreekantiah KR, Murthy VS. Influence of
media composition on the production of a-amylase and amy-
loglucosidase by a strain of Aspergillus niger . Starch
1979;31:134.
[175] Ramasesh N, Skreekantiah KR, Murthy VS. Purification and
characterization of a thermophilic a-amylase of Aspergillus niger
van Tieghem. Starch 1982;34:274.
[176] Jodal I, Kandra L, Harangi J, Nanasi P, Szejtli J. Hydrolysis of
cyclodextrin by Aspergillus oryzae a-amylase. Starch
1984;36:140.
R. Gupta et al. / Process Biochemistry 00 (2003) 1Á/18 17
ARTICLE IN PRESS
[177] Hanrahan VM, Caldwell ML. A study of action of Taka
amylase. J Am Chem Soc 1953;75:2191.
[178] Hanrahan VM, Caldwell ML. Additional studies of the proper-
ties of Taka amylase. J Am Chem Soc 1953;75:4030.
[179] Minoda Y, Koyano T, Arai M, Yamada K. Acid-stable a-
amylase of black Aspergilli. II. Some general properties. Agric
Biol Chem 1968;32:104Á/9.
[180] Suetsugu N, Koyama S, Takeo KI, Kuge T. Kinetic studies on
the hydrolyses of a, b- and g-cyclodextrins by Taka amylase A. J
Biochem 1974;76:57.
[181] Toda H, Kondo K, Narita K. The complete amino acid sequence
of Taka-amylase A. Proc Jpn Acad 1982;58:208.
[182] Sills AM, Sauder ME, Steward GG. Amylase activity in certain
yeasts and a fungal species. Dev Ind Microbiol 1983;24:295.
[183] Suganuma T, Tahara N, Kitahara K, Nagahama T, Inuzuka K.
N-terminal sequence of amino acids and some properties of an
acid stable a-amylase from citric acid Koji (Aspergillus usamii
var.). Biosci Biotech Biochem 1996;60:177Á/9.
[184] Narayanan AS, Shanmugasundaram ERB. Studies on amylase
of Fusarium vasinfectum. Arch Biochem Biophys 1967;118:317.
[185] Zenin CT, Park YK. Purification and characterization of acid a-
amylase from Paecilomyces sp. J Ferment Technol 1983;61:109.
[186] Schellart JA, Visser FMW, Zandstva T, Middlehover WJ. Starch
degradation by the mold Trichoderma viride I. The mechanism
of degradation. Antonie Van Leeuwenhock J Microbiol Serol
1976;42:229.
[187] Ebisu S, Mori M, Takagi H, Kadowaki K, Yamagata H,
Tsukagoshi N, Udaka S. Production of a fungal protein, Taka
amylase A, by protein-producing Bacillus brevis HPD31. J Ind
Microbiol 1993;11:83Á/8.
[188] Ramesh MV, Lonsane BK. Characteristics and novel features of
thermostable a-amylase produced by Bacillus licheniformis M-27
under solid state fermentation. Starch 1990;42:233Á/8.
[189] Ali MB, Mezghani M, Bejar S. A thermostable alpha amylase
producing maltohexose from a newly isolated Bacillus sp.
US100: study of activity and molecular cloning of the corre-
sponding gene. Enzyme Microb Technol 1999;24:584Á/9.
[190] Mamo G, Gessesse A. Purification and charcterization of two
raw starch degrading thermostable a-amylase from a thermo-
phillic Bacillus . Enzyme Microb Technol 1999;25:433Á/8.
[191] Kelly TT, Collins BS, Fogarty WM, Doyle EM. Mechanisms of
action of the a-amylase of Micromonospora melanosporea. Appl
Microbiol Biotechnol 1993;39:599Á/603.
R. Gupta et al. / Process Biochemistry 00 (2003) 1Á/18 18
ARTICLE IN PRESS