Microbial a-amylases: a biotechnological perspective

Abstract 

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 

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. 

* 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. 

ARTICLE IN PRESS 

Process Biochemistry 00 (2003) 1 /18 

0032-9592/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. 

doi:10.1016/S0032-9592(03)00053-0 

www.elsevier.com/locate/procbio 

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 according 

to the anomeric type of sugars produced by the 

enzyme reaction. a-Amylase (1,4-a-D-glucan-glucanhydrolase, 

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, endoamylases 

and exoamylases. Endoamylases catalyse hydrolysis 

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-

2 

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 microbial 

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 [1 /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 actinomycetes, 

however, enzymes from fungal and bacterial 

sources have dominated applications in industrial sectors 

[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 reaction 

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 determination 

of a-amylase activity [6]. These are based on 

decrease in starch /iodine colour intensity, increase in 

reducing sugars, degradation of colour-complexed substrate 

and decrease in viscosity of the starch suspension. 

3.1. Decrease in starch /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, 


R. Gupta et al. / Process Biochemistry 00 (2003) 1 /18 

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].

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 conditions 

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 aamylase 

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 0 /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 commonly 

present in the culture broths. The use of nonreducing 

end blocked p-nitrophenyl maltoheptoside 

(BPNPG7) 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 asitinvolves 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 (BPNPG7) 

with a number of accepted procedures that employ 

starch as the substrate. The reaction was monitored 

using the starch /iodine colour [21]. There was an 

excellent correlation between each of the assay procedures 

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 


R. Gupta et al. / Process Biochemistry 00 (2003) 1 /18 3 

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 [22 /24] is accepted for assessing cereal aamylase 

activity in flour /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 thermostability, 

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 400 /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 fermentation 

(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, temperature, 

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 [2 /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.

4 

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 [27 /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-glycoside 

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-processing 

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-Dglycoside 

(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 110 /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 supported 

maximum a-amylase production by bacterial 

strains [35,38,51 /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 production 

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 production 

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 conidiation 

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

inhibitory to enzyme production by B. amyloliquefaciens 

[58]. 

4.1.4. Role of other ions 

K ,Na ,Fe 2 

,Mn 2 

,Mo 2 

,Cl ,SO4 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 Bacillus 

sp. CRP strain [64]. Addition of zeolites to control 

ammonium ions in B. amyloliquefaciens resulted in 

increased yield of a-amylase [65]. Aninverse relationship 

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 morphological 

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 3.2 /4.2 in the case 

of A. oryzae DAE 1679 [39], 7.0 /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 aamylase 

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 

25 /37 8C. Optimum yields of a-amylase were achieved 

at 30 /37 8C for A. oryzae [55,57]. a-Amylase production 

has also been reported at 55 8C by the thermophilic 

fungus Thermomonospora fusca [70] and at 50 8C byT. 

lanuginosus [17]. 

a-Amylase has been produced at a much wider range 

of temperature among the bacteria. Continuous production 

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 

[69,72 /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 regulation 

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, morphology 

of A. oryzae was critically affected by the growth 

pH [78]. In a series of batch experiments, authors 

observed that at pH 3.0 /3.5, freely dispersed hyphal 

elements were formed. In the pH range 4 /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 morphological 

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 associated 

type [35]. Similar findings were cited in another 

report with B. amyloliquefaciens [63]. 

Continuous and fed-batch cultures have been recognised 

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

6 

[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 1 /4 gh 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 aamylase 

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]. Furthermore, 

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 production 

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 

structure /function relationships and biochemical properties. 

The purification of a-amylases from microbial sources 

in most cases has involved classical purification methods. 

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 aamylases 

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 aamylases 

from several microorganisms have been extensively 

studied and described [2 /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, amylopectin, 

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 9 /10.5 reported from an alkalophilic 

Bacillus sp. [85 /88]. Extremely alkalophilic a-amylase 

with pH optima of 11 /12 has been reported from 

Bacillus sp. GM8901 [89]. In some cases, the pH 

optimum was observed to be dependent upon temperature 

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 aamylase 

is related to the growth of the microorganism 

[4]. The lowest temperature optimum is reported to be 

25 /30 8C for F. oxysporum amylase [94] and the highest 

of 100 and 130 8C from archaebacteria, Pyrococcus 

furiosus and Pyrococcus woesei, respectively [95 /97]. 

Temperature optima of enzymes from Micrococcus 

varians are calcium dependent [98] and that from H. 

meridiana is sodium chloride dependent [46].

Table 1 

Purification strategies employed for a-amylase 

Microorganism Purification strategy Fold purification/ 

yield (%) 

Fungi and yeast 

A. oryzae NRC 401013 DE52-Cellulose (pH 7.0), 70% (NH4)2SO4, Sephacryl S300, 70% (NH4)2SO4, 

DE52-Cellulose (pH 7.0) 

[31] 

A. flavus LINK 50 /90% (NH4)2SO4, 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% (NH4)2SO4, crosslinked starch (pH 8.5), DEAE Bio-Gel A (pH 5.5) 6000/52 [99] 

Saccharomyces cerevisiae Ultrafiltration, b-Cyclodextrin linked Sepharose 6B (Epoxy activated, pH 4.5), 5/2 [153] 

YPB-G 

Sephadex G-100 (pH 4.5) 

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/ / [47] 

Bacillus sp. WN 11 60% (NH4) 2SO4, DEAE Sepharose (pH 5.3), Sephadex G-75 Amy I 65/13, Amy II 

40.7/9.5 

[100] 

B. licheniformis CUMC 305 65% (NH4)2S04, CM-Cellulose (pH 6.4) 212/42 [86] 

B. licheniformis NCIB 6346 DEAE-Cellulose DE52 (pH 5.3) 33/66 [157] 

B. stearothermophilus ATCC Adsorption on soluble starch (1%) in 10% (NH4) 2SO4, washing with Aces (pH / [158] 

12980 

7.5) and 10% (NH4)2SO4, DEAE chromatography (Zetaprep disk), ultrafiltration 

B. subtilis 60% (NH4)2SO4, Sephacryl-S200 HR (pH 8.0), 60% (NH4)2SO4, S-Sepharose 9/17 [159] 

B. subtilis Ultrafiltration 2.5/ / [83] 

B. subtilis 65 Sephacryl S-300, CM Sephadex C-50 30.85/24.8 [51] 

Lactobacillus plantarum A6 Ultrafiltration, 50 /80% (NH4)2SO4, ultrafiltration, DEAE-Cellulose 20/35 [160] 

Pseudomonas stutzeri Concentrated by drum humidifier, 25% (NH4)2SO4, 70% acetone 1.036/ / [93] 

Streptococcus bovis JB1 70% (NH4) 2SO4, Sephadex G-25 (pH 7.5), Mono Q 6.9/50 [161] 

Thermomonospora curvata 

NCIMB 10081 

85% (NH4)2S04, ultrafiltration, gel filtration (pH 6.0), DEAE-Sephacel (pH 8.O) 300/ / [162] 

T. profundus DT5432 80% (NH4)2SO4, DEAE-Toyopearl 650 M (pH 7.5), Superdex 200 HR (pH 7.5) 816/26 [71] 

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 [100 /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]. Molecular 

weights of microbial a-amylases are usually 50 / 

60 kDa as shown directly by analysis of cloned aamylase 

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. stearothermophilus 

[107] and B. subtilis strains [108,109]. Glycosylation 

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, phydroxyl 

mercuribenzoic acid, iodoacetate, BSA, 

EDTA and EGTA inhibit a-amylases. 

7.6. Calcium and stability of a-amylase 

Reference 

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 2 

Properties of some microbial amylases 

Source pI Molecular 

weight 

(kDa) 

pH optima/stability 

Temperature optima/stability 

Inhibitors Stabilisers Additional properties Reference 

Fungi and yeast 

A. oryzae / / 65.4/5.0 /9.0 50 8C/50 8C (30 

min) 

/ / Km (0.13%) [28] 

A. flavus LINK 3.5 52.5 6.0/6.0 /10.0 55 8C/50 8C (1h) Ag 2 

,Hg 2 

Ca 2 

1 

Km (0.5 g l ); Vmax (108.67 

mM reducing sugar mg 1 

protein min 1 

[92] 

A. foetidus ATCC 

/ 41.5 5.0/ / 45 8C/35 8C (60 / / Km /2.19 mg ml 

1 

[163] 

10254 

min) 

A. awamori / / 5.0/6.0 /7.0 40 8C/55 8C (10 

min) 

Ag ,Cu 2 

,Fe 3 

,Hg 2 

, halides Substrate [164] 

A. awamori ATCC 4.2 54.0 4.8 /5.0/3.5 /6.5 50 8C/40 8C (60 Hg 

22342 

(24 h) 

min) 

2 

,Pb 2 

, maltose Km /1mgml 

1 

[32] 

A. chevalieri NSPRI / 68.0 5.5/ / 40 8C/60 8C (15 EDTA, DNP Ca 

105 

min) 

2 

,Mg 2 

Km /0.19 mg ml 

1 

[165] 

A. flavus / / 5.25/5.0 /8.0 50 8C/55 8C (10 

min) 

Ag ,Cu 2 

,Hg 2 

, halides Substrate [164] 

A. fumigatus / / 6.0/ / 50 8C/60 8C (40 

min) 

/ / / [41] 

A. hennebergi Bloch- / 50.0 5.5/ / 50 8C/40 8C (15 / / / [166] 

weitz 

min) 

A. niger 3.44 58.0 4.0 /5.0/2.2 /7.0 //60 8C (15 min) / Ca 2 

Acid stable [167 / 

171] 

3.75 61.0 5.0 /6.0/5.0 /8.5 //40 8C (15 min) / Ca 2 

A. niger ATCC 13469 / / 5.0/4.0 /6.0 50 8C/B/60 8C / / / [172] 

A. niger van Tieghem 

CFTRI 1105 

/ 56.23 5.0; 6.0/5.2 /6.0 

( /Ca); 5.8 /7.0 

60 8C/65 8C (10 

min) 

Ag ,Al 

( /Ca) 

3 

,Cu 2 

,Hg 2 

,Pb 2 

Zn 

, 

2 

, EDTA 

Ca 2 

NaF and MgSO4 stimulation 

[173 / 

175] 

A. oryzae / / 5.0/6.0 /8.0 40 8C/55 8C (10 

min) 

Ag ,Cu 2 

,Fe 3 

,Hg 2 

, halides Substrate / [164] 

A. oryzae 

A. oryzae 

/ 

/ 

/ 

53.0 

4.8 /6.6/ / 

5.0 /5.9/5.8 /7.2 

35 /37 8C/ / 

//60 8C (90 min, 

/ 

PCMB 

/ 

Ca 

Km /7.13, 4.35, 3.12 mM [176] 

(over a year, 

/Ca) 50 8C (30 

10 8C); 5.0 /8.2 

(37 8C, 30 min) 

min, /Ca) 

2 

‘‘Taka Diastase’’, ‘‘Taka- [177 / 

Amylase A’’, Km / 29, 180] 

2.4%, 4.7, 10.2, 2.4 mM 

A. oryzae 245 (ATCC 

9376) 

/ / 5.0 /6.0/ / 30 /40 8C/ / / / Km /4.16 mg ml 

1 

[181,182] 

A. usamii / 54.0 3.0 /5.5/ / 60 /70 8C/ / / / Higher thermal stability 

than commercial Takaamylase 

[183] 

A. oryzae M13 4.0 52.0 5.4/5.0 /9.0 50 8C/5/50 8C 

(min) 

/ / Km (0.13%) [28] 

8 


ARTICLE IN PRESS

Table 2 (Continued) 


weight 

(kDa) 



Cryptococcus S-2 4.2 66.0 6.0/ / 50 /60 8C/90 8C 

(CaCl2) 

Fusarium vasinfectum 

Atk 

L. kononenkoae CBS 

5608 

/ / 4.4 /5.0:5.8:7.8 / 

8.0/3.8 /10.0 

3.5 76.0 4.5 /5.0/5.0 /7.0 

(1 h) 


Hg 2 

Cu 2 

,Ag 2 

,Mn 2 

45 /50 8C/50 8C 

(30 min) 

70 8C/ / DTT, Cu 2 

,Cu 2 

,Zn 2 

,Ag 2 

,Zn 2 

/ Raw starch digesting enzyme; 

end products, G1, G2, 

G3,G4 [152] 

/ / [184] 

1 

Starch Km (0.8 g l ); Kcat (622 

1 2 

s ); insensitive toCa ; 

end products, G3, G4, G5, 

Paecilomyces sp. 

/ 69.0 4.0/4.0 /9.0 45 8C/45 8C (10 

/ [185] 

ATCC 46889 

min, /Ca) 

Saccharomyces cerevisiae 

/ 54.1 5.0/ / 50 8C/ / / / End products, G1, G2, G3 [153] 

Schwanniomyces al- / 61.9 6.3/4.5 /7.5 40 8C/5/40 8C / / Km (0.364 mg ml 

1 

); end [154] 

luvius UCD 5483 

T. lanuginosus IISc 91 / 42.0 5.6/ / 65 8C/50 8C ( 

h) 

/7 / Ca 

product, G1 2 

1 

A.E. (44 kJ mol ;Km (2.5 

1 

mg ml ); end product, G2 

[17] 

Trichoderma viride 

Bacteria 

/ / 5.0 /5.5/4.0 /7.0 //60 8C (10 min) / / / [186] 

B. brevis HPD 31 / / 6.0/4.5 /9.0 45 /55 8C/ / / / / [187] 

B. licheniformis 

B. licheniformis 

/ 

/ 

22.5 

28.0 

9.0/6.0 /11.0 

9.0/7.0 /9.0 

76 8C/B/60 8C 

90 8C/60 8C (3 h), 

/ 

Hg 

/ End product, G5 [85] 

CUMC 305 licheniformis 

CUMC 305 

100 8C (4h)in 

presence of soluble 

starch 

2 

,Cu 2 

,Ni 2 

,Zn 2 

,Ag 2 

, 

Fe 2 

,Co 2 

,Cd 2 

,Al 3 

,Mn 2 

Na 

, 

p- chloromercuribenzoic acid, sodium 

iodoacetate, EDTA 

2 

,Ca 2 Mg 2 

, azide, F , 

2 2 2 2 

SO3 ,SO4 ,S2O3 ,MoO4 WO4 

, 

2 

E.A. (5.1 /10 

, cysteine, glutathione, 

thiourea, b-mercaptoethanol, 

sod. glycerophosphate 

5 Jmol 

1 

); 

1 

Km (1.274 mg ml ); Vmax 

1 

(0.738 mg glucose ml 

min 1 

[86] 

B. licheniformis NCIB / 62 /65 7.0/7.0 /10.0 70 /90 8C/85 8C / / End products, G1,G2,G3, [157] 

6346 

B. stearothermophilus 4.82 / 4.6 /5.1/ / 

(1 h) 

55 /70 8C/ / EDTA Ca 

G5 

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 8.8 59.0 5.0 /6.0/6.0 /7.5 70 /80 8C/(5 days) Cd 

ATCC 12980 

(1 h, 80 8C) 70 8C or (45 min) 

90 8C 

2 

,Cu 2 

,Hg 2 

,Pb 2 

,Zn 2 

, Ca 

denaturation by 6 M urea 

2 

,Na 2 

, B.S.A. Km /14 mg ml 

1 

; enzyme [4] 

active after acetone and 

ethanol treatment 

B. stearothermophilus / / 5.5 /6.0/ / 70 /75 8C/half life / / Ca 

MFF4 

5.1 h at 80 8C 

2 

enhances thermo- [102] 

stability 

B. subtilis / 48.0 6.5/5/7.0 50 8C/5/50 8C Hg 2 

,Fe 3 

,Al 3 

Mn 2 

,Co 2 

Km (3.845 mg ml 

1 

); Vmax [159] 

(585.1 mg); end product, G2 

B. subtilis 65 / 68.0 6.0/6.0 /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, G1,G2 [51] 

/ Ca 2 

G6 

[99] 


R. Gupta et al. / Process Biochemistry 00 (2003) 1 /18 9



weight 

(kDa) 




B. licheniformis M27 / 56.0 6.5 /7.0 and 8.5 / 85 /90 8C/ / 

/ Ca 

9.0/5/7.0 and ]/ 90 8C 

7.5 

2 

E.A. (25 kJ mol 

1 

); thermostability 

dependent upon 

pH stability 

Bacillus sp. IMD 435 5.6 63.0 6.0 and 6.5 / / / End products, G1, G2, G3, 

Bacillus sp. IMD 434 5.9 69.2 6.0/4.0 /9.0 65 8C/40 8C (1h) N -Bromosuccinimide, p-hydroxymercuribenzoic 

acid 

Bacillus sp. US 100 / / 5.6/4.5 /8.0 82 8C/90 /95 8C / Starch, Ca 2 

Cysteine, DTT End products, G1, G2; specificity 

for raw starch; Km ( 

1.9 mm) 

Half-life increases to 110 8C 

in presence of 20% (w/v) 

substrate 

; 

starch increases temperature 

stability 

/ End products, G1,G2,G3, Bacillus sp. WN 11 / / 5.0 /8.0/ / 75 /80 8C/ / / / No requirement for Ca 2 

Bacillus sp. WN 11 / Amy 1- 

76.0, Amy 

2-53.0 

5.5/5.5 /9.0 (1 h) 75 /80 8C/80 8C 

(4 h) 

Fe 3 

,Hg 2 

Bacillus sp. XAL 601 / / 9.0/ / 70 8C/ / / / Adsorbs to raw starch or 

cellulose hydrolysis products, 

G2 and G4 

[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 

/ [159] 

H. meridiana DSM 

/ / 7.0/5.0 /7.0 37 8C/ / / Ca 

5425 

2 

End products, G2,G3; [46] 

showed activity in 30% salts 

L. plantarum A6 / 50.0 5.5/ /3.0 /B/8.0 65 8C/ / N -bromosuccinimide, iodine, 

acetic acid, Hg 2 

, dimethyl aminobenzaldehyde 

/ Km (2.38 g l 

1 

kJ mol ) 

1 

); A.E. (30.9 [160] 

Micromonospora mel- 7.6 45.0 7.0/ / 55 8C/40 8C (pH / / / [191] 

anosporea 

11 /12, 40 min) 

M. melanosporea 

Pseudomonas stutzeri 

7.6 

/ 

45.0 

12.5 

7.0/6.0 /12.0 

8.0/7.0 /9.5 

55 8C/ / 

47 8C/40 8C (1h) 

/ 

/ 

/ 

Ca 

End product, G1 [191] 

2 

E.A. (13 400 and 5200 cal [93] 

mol 

1 

; end product, G4 

Streptococcus bovis 

JB1 

Streptomyces sp. IMD 

2679 

,Cu 2 

4.5 77.0 5.0 /6.0/5.5 /8.5 //50 8C (1h) Hg 2 

, p-chloromercuribenzoic 

acid (both reversible by DTT) 

8.9(1), 

8.7(2), 

7.2(3) 

47.8 5.5/ / 60 8C/ /, 60 / 

65 8C/ /, 658C/ / 

T. profundus DT5432 / 42.0 5.5 /6.0/5.9 /9.8 80 8C/80 8C (3 h), 

90 8C (15 min) 

Thermomonospora 

curvata 

G4 

G4 

/ Km (0.88 mg ml 

1 ); Kcat 

(2510 mmol reducing sugar 

mg 1 protein); end products, 

G2, G3, G4 

/ / End products, G 1,G 3;K m 

(8.0 /8.2 mM) 

Iodoacetic acid, N-bromosuccinic 

acid, SDS, guanidine hydrochloride 

Ca 2 

End products, G2, G3; Km 

(0.23%) 

6.2 60.9 6.0/ / 65 8C/ / / / End product, G2; low affinity 

for G3 

[188] 

[45] 

[47] 

[189] 

[100] 

[190] 

[161] 

[44] 

[71] 

[162] 

10 


ARTICLE IN PRESS






weight 

(kDa) 

[155] 

[70] 

T. curvata / 62.0 5.5 /6.0/activated 65 8C/ / B.S.A. / End products, G4, G5; Km 

1 

at pH 7.0 /8.0 

(0.3 mg ml ) 

T. fusca YX / / 6.0/ / 60 8C/B/65 8C / Starch, Ca 2 

End products, G3,G4,G6; 1 

Km (3.3 mg ml ); E.A. (59 

1 

kJ mol ) 

G1, glucose; G2, maltose; G3, maltotriose; G4, maltotetraose; G5, maltopentaose; E.A., enzyme activation energy; kcal, kilo calories; kJ, kilo joules. 



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 reactivated 

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 thermostable 

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 

enzyme [117]. 

did not have any effect on the 

8. Industrial applications of a-amylase 

Amylases are among the most important hydrolytic 

enzymes for all starch based industries, and the commercialisation 

of amylases is oldest with first use in 

1984, as a pharmaceutical aid for the treatment of 

digestive disorders. In the present day scenario, amylases 

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 commercially 

in baking. Today, many enzyme preparations such 

as proteases, lipases, xylanases, pullulanases, pentosanases, 

cellullases, glucose oxidases, lipoxygenases etc.

12 

are being used in the bread industry for varied purposes 

[13,99,121 /123], but none had been able to replace aamylases. 

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 exogenous 

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 improvements 

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 improvement/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 4 /9 degree of polymerisation 

produced by these shows the anti-staling 

properties. Although a wide variety of microbial aamylases 

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 production 

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 45 /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

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 aamylases 

in detergents is that the enzyme shows 

sensitivity to calcium and stability is severely compromised 

in a low calcium environment. In addition, most 

wild-type a-amylases are sensitive to oxidants which are 

generally a component of detergent formulations. Stability 

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 engineering 

to improve the bleach stability of the amylases 

[146 /148]. They independently replaced oxidation sensitive 

amino acids with other amino acids. The replacement 

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 performance 

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 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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Microbial a-amylases: a biotechnological perspective

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