Amylases Summary

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: [email protected] (R. Gupta).1 E-mail: [email protected].

Process Biochemistry 38 (2003) 1599�/1616

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

mailto:[email protected]

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 awide 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 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 starch�/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 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 iodinecolour 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 inabsorbance 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 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 iodinewas 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 enzymepreparation 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 colourproduced 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 38 (2003) 1599�/16161600

3.3. Degradation of colour-complexed substrate

For some years, groups have been working on the

development of a specific a-amylase determinationmethod 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. Thecoloured 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 starchcontamination 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 witha 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

(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 accuratebut 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 (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 anexcellent 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 Farinographtest.

3.4.1. Falling number (FN) method

The falling number (FN) method, internationallystandardised [22�/24] is accepted for assessing cereal a-

amylase 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 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 andfungal 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 areconsidered 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 arethe 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 [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.

R. Gupta et al. / Process Biochemistry 38 (2003) 1599�/1616 1601

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 onthe 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, othercarbon 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, likemost 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 theeconomic 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 theproduction 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 sulphateand 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 sourcesviz. beef extract, peptone and com steep liquor sup-

ported 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 nitratefor 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 arehighly 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


inhibitory to enzyme production by B. amyloliquefaciens

[58].

4.1.4. Role of other ions

K�, Na�, Fe2�, Mn2�, Mo2�, Cl�, SO42� had no

effect while Ca2� was inhibitory to amylase production

by A. oryzae EI 212 [57]. Mg2� played an important

role and production was reduced to 50% when Mg2�

was omitted from the medium. Na� and Mg2� 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 Co2� [66], the presence of Co2� 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 growthmedium 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 inthe 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 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 productionis 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 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

[69,72�/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 affectenzyme 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, authorsobserved 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 wasexhausted 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 peakedtwice 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 ofthese cultures. The production of a-amylase from

B. subtilis TN106 (pAT5) was enhanced substantially

by extending batch cultivation with fed-batch operation


[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 ofmaltose at a feed rate of 1�/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 alaboratory 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 givendilution 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 continuouscultivation 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 SSFtechnique 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 relativelycrude 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 prop-

erties.

The purification of a-amylases from microbial sourcesin 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 areavailable 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 [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 tomicroorganism. 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 optimain 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 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

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].


Thermostabilities have not been estimated defactor in

many studies. Thermostabilities as high as 4 h at 100 8Chave 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 CUMC305 [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]. Mo-lecular weights of microbial a-amylases are usually 50�/

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 leastone Ca2� ion [111]. The affinity of Ca2� 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% (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

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/�/ [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

12980

Adsorption on soluble starch (1%) in 10% (NH4)2SO4, washing with Aces (pH

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

�/ [158]

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]


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 �/ �/ 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 (1 h) Ag2�, Hg2� Ca2� Km (0.5 g l�1); Vmax (108.67

mM reducing sugar mg�1

protein min�1

[92]

A. foetidus ATCC

10254

�/ 41.5 5.0/�/ 45 8C/35 8C (60

min)

�/ �/ Km�/2.19 mg ml�1 [163]

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

min)

Ag�, Cu2�, Fe3�, Hg2�, halides Substrate [164]

A. awamori ATCC

22342

4.2 54.0 4.8�/5.0/3.5�/6.5

(24 h)

50 8C/40 8C (60

min)

Hg2�, Pb2�, maltose Km�/1 mg ml�1 [32]

A. chevalieri NSPRI

105

�/ 68.0 5.5/�/ 40 8C/60 8C (15

min)

EDTA, DNP Ca2�, Mg2� Km�/0.19 mg ml�1 [165]

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

min)

Ag�, Cu2�, Hg2�, halides Substrate [164]

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

min)

�/ �/ �/ [41]

A. hennebergi Bloch-

weitz

�/ 50.0 5.5/�/ 50 8C/40 8C (15

min)

�/ �/ �/ [166]

A. niger 3.44 58.0 4.0�/5.0/2.2�/7.0 �//60 8C (15 min) �/ Ca2� Acid stable [167�/

171]

3.75 61.0 5.0�/6.0/5.0�/8.5 �//40 8C (15 min) �/ Ca2�

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

(�/Ca)

60 8C/65 8C (10

min)

Ag�, Al3�, Cu2�, Hg2�, Pb2�,

Zn2�, EDTA

Ca2� NaF and MgSO4 stimula-

tion

[173�/

175]

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

min)

Ag�, Cu2�, Fe3�, Hg2�, halides Substrate �/ [164]

A. oryzae �/ �/ 4.8�/6.6/�/ 35�/37 8C/�/ �/ �/ Km�/7.13, 4.35, 3.12 mM [176]

A. oryzae �/ 53.0 5.0�/5.9/5.8�/7.2

(over a year,

10 8C); 5.0�/8.2

(37 8C, 30 min)

�//60 8C (90 min,

�/Ca) 50 8C (30

min, �/Ca)

PCMB Ca2� ‘‘Taka Diastase’’, ‘‘Taka-

Amylase A’’, Km�/ 29,

2.4%, 4.7, 10.2, 2.4 mM

[177�/

180]

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 Taka-

amylase

[183]

A. oryzae M13 4.0 52.0 5.4/5.0�/9.0 50 8C/5/50 8C(min)

�/ �/ Km (0.13%) [28]

R.

Gu

pta

eta

l./

Pro

cessB

ioch

emistry

38

(2

00

3)

15

99�

/16

16

16

06

Table 2 (Continued )

Source pI Molecular

weight

(kDa)

pH optima/stabi-

lity

Temperature op-

tima/stability


Cryptococcus S-2 4.2 66.0 6.0/�/ 50�/60 8C/90 8C(CaCl2)

Hg2�, Ag2�, Cu2�, Zn2� �/ Raw starch digesting en-

zyme; end products, G1, G2,

G3, G4

[152]

Fusarium vasinfectum

Atk

�/ �/ 4.4�/5.0:5.8:7.8�/

8.0/3.8�/10.0

45�/50 8C/50 8C(30 min)

Cu2�, Mn2�, Zn2� �/ �/ [184]

L. kononenkoae CBS

5608

3.5 76.0 4.5�/5.0/5.0�/7.0

(1 h)

70 8C/�/ DTT, Cu2�, Ag2� Starch Km (0.8 g l�1); Kcat (622

s�1); insensitive to Ca2�;

end products, G3, G4, G5,

G6

[99]

Paecilomyces sp.

ATCC 46889

�/ 69.0 4.0/4.0�/9.0 45 8C/45 8C (10

min, �/Ca)

�/ Ca2� �/ [185]

Saccharomyces cer-

evisiae

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

Schwanniomyces al-

luvius UCD 5483

�/ 61.9 6.3/4.5�/7.5 40 8C/5/40 8C �/ �/ Km (0.364 mg ml�1); end

product, G1

[154]

T. lanuginosus IISc 91 �/ 42.0 5.6/�/ 65 8C/50 8C (�/7

h)

�/ Ca2� A.E. (44 kJ mol�1; Km (2.5

mg ml�1); end product, G2

[17]

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

Bacteria

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

B. licheniformis �/ 22.5 9.0/6.0�/11.0 76 8C/B/60 8C �/ �/ End product, G5 [85]

B. licheniformis

CUMC 305 lichenifor-

mis CUMC 305

�/ 28.0 9.0/7.0�/9.0 90 8C/60 8C (3 h),

100 8C (4 h) in

presence of solu-

ble starch

Hg2�, Cu2�, Ni2�, Zn2�, Ag2�,

Fe2�, Co2�, Cd2�, Al3�, Mn2�,

p- chloromercuribenzoic acid, so-

dium iodoacetate, EDTA

Na2�, Ca2� Mg2�, azide, F�,

SO32�, SO4

2�, S2O32�, MoO4

2�,

WO42�, cysteine, glutathione,

thiourea, b-mercaptoethanol,

sod. glycerophosphate

E.A. (5.1�/105 J mol�1);

Km (1.274 mg ml�1); Vmax

(0.738 mg glucose ml�1

min�1

[86]

B. licheniformis NCIB

6346

�/ 62�/65 7.0/7.0�/10.0 70�/90 8C/85 8C(1 h)

�/ �/ End products, G1, G2, G3,

G5

[157]

B. stearothermophilus 4.82 �/ 4.6�/5.1/�/ 55�/70 8C/�/ EDTA Ca2� Higher affinity for branched

chain substrate; E.A. (14

kcal); extremely resistant to

heat inactivation; effect of

EDTA reversed by Ca2�

[101]

B. stearothermophilus

ATCC 12980

8.8 59.0 5.0�/6.0/6.0�/7.5

(1 h, 80 8C)

70�/80 8C/(5 days)

70 8C or (45 min)

90 8C

Cd2�, Cu2�, Hg2�, Pb2�, Zn2�,

denaturation by 6 M urea

Ca2�, Na2�, B.S.A. Km�/14 mg ml�1; enzyme

active after acetone and

ethanol treatment

[4]

B. stearothermophilus

MFF4

�/ �/ 5.5�/6.0/�/ 70�/75 8C/half life

5.1 h at 80 8C�/ �/ Ca2� enhances thermo-

stability

[102]

B. subtilis �/ 48.0 6.5/5/7.0 50 8C/5/50 8C Hg2�, Fe3�, Al3� Mn2�, Co2� Km (3.845 mg ml�1); Vmax

(585.1 mg); end product, G2

[159]

B. subtilis 65 �/ 68.0 6.0/6.0�/9.0 60 8C/60 8C (5

min)

Cu2�, Fe3�, Mn2�, Hg2�, Zn2�,

Pb2�, Al3�, Cd2�, Ag2�, EDTA

Ca2� End products, G1, G2 [51]

R.

Gu

pta

eta

l./

Pro

cessB

ioch

emistry

38

(2

00

3)

15

99�

/16

16

16

07

Table 2 (Continued )

Source pI Molecular

weight

(kDa)

pH optima/stabi-

lity

Temperature op-

tima/stability


B. licheniformis M27 �/ 56.0 6.5�/7.0 and 8.5�/

9.0/5/7.0 and ]/

7.5

85�/90 8C/�/

90 8C�/ Ca2� 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 �/ �/ �/ End products, G1, G2, G3,

G4

[45]

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

mercuribenzoic acid

Cysteine, DTT End products, G1, G2; spe-

cificity for raw starch; Km (

1.9 mm)

[47]

Bacillus sp. US 100 �/ �/ 5.6/4.5�/8.0 82 8C/90�/95 8C �/ Starch, Ca2� Half-life increases to 110 8Cin presence of 20% (w/v)

substrate

[189]

Bacillus sp. WN 11 �/ �/ 5.0�/8.0/�/ 75�/80 8C/�/ �/ �/ No requirement for Ca2�;

starch increases temperature

stability

[100]

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)

Fe3�, Hg2�, Cu2� �/ End products, G1, G2, G3,

G4

[190]

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

cellulose hydrolysis pro-

ducts, G2 and G4

[87]

Escherichia coli 48.0 6.5/5/7.0 50 8C/B/70 8C Hg2�, Fe3�, Al3� Mn2�, Co2� �/ [159]

H. meridiana DSM

5425

�/ �/ 7.0/5.0�/7.0 37 8C/�/ �/ Ca2� End products, G2, G3;

showed activity in 30% salts

[46]

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

acetic acid, Hg2�, dimethyl amino-

benzaldehyde

�/ Km (2.38 g l�1); A.E. (30.9

kJ mol�1)

[160]

Micromonospora mel-

anosporea

7.6 45.0 7.0/�/ 55 8C/40 8C (pH

11�/12, 40 min)

�/ �/ �/ [191]

M. melanosporea 7.6 45.0 7.0/6.0�/12.0 55 8C/�/ �/ �/ End product, G1 [191]

Pseudomonas stutzeri �/ 12.5 8.0/7.0�/9.5 47 8C/40 8C (1 h) �/ Ca2� E.A. (13 400 and 5200 cal

mol�1; end product, G4

[93]

Streptococcus bovis

JB1

4.5 77.0 5.0�/6.0/5.5�/8.5 �//50 8C (1 h) Hg2�, p -chloromercuribenzoic

acid (both reversible by DTT)

�/ Km (0.88 mg ml�1); Kcat

(2510 mmol reducing sugar

mg�1 protein); end pro-

ducts, G2, G3, G4

[161]

Streptomyces sp. IMD

2679

8.9(1),

8.7(2),

7.2(3)

47.8 5.5/�/ 60 8C/�/, 60�/

65 8C/�/, 65 8C/�/

�/ �/ End products, G1, G3; Km

(8.0�/8.2 mM)

[44]

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

90 8C (15 min)

Iodoacetic acid, N -bromosuccinic

acid, SDS, guanidine hydrochlor-

ide

Ca2� End products, G2, G3; Km

(0.23%)

[71]

Thermomonospora

curvata

6.2 60.9 6.0/�/ 65 8C/�/ �/ �/ End product, G2; low affi-

nity for G3

[162]

R.

Gu

pta

eta

l./

Pro

cessB

ioch

emistry

38

(2

00

3)

15

99�

/16

16

16

08

amylase A (TAA) contains ten Ca2� ions but only one

is tightly bound [112]. In other systems usually one

Ca2� ion is sufficient to stabilise the enzyme. Ca2� can

be removed from amylases by dialysis against EDTA orby electrodialysis. Calcium free enzymes can be reacti-

vated by adding Ca2� ions. Some studies have been

carried out on the ability of other ions to replace Ca2�

as Sr2� in B. caldolyticus amylase [113]. Ca2� in TAA

has been substituted by Sr2� and Mg2� in successive

crystallisation in the absence of Ca2� and in excess of

Sr2� and Mg2� [114]. EDTA inactivated TAA can be

reactivated by Sr2�, Mg2� and Ba2� [114]. In thepresence of Ca2�, 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 Ca2�, but retains

activity after EDTA treatment [116]. There are also

reports where Ca2� 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, forthe 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 comprehensiveaccount 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 enzymesfor 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.Tab

le2

(Co

nti

nued

)

So

urc

ep

IM

ole

cula

r

wei

gh

t

(kD

a)

pH

op

tim

a/s

tab

i-

lity

Tem

per

atu

reo

p-

tim

a/s

tab

ilit

y

Inh

ibit

ors

Sta

bil

iser

sA

dd

itio

nal

pro

per

ties

Ref

eren

ce

T.

curv

ata

�/6

2.0

5.5�/6

.0/a

ctiv

ate

d

at

pH

7.0� /

8.0

658C

/�/

B.S

.A.

�/E

nd

pro

du

cts,

G4,

G5;

Km

(0.3

mg

ml�

1)

[15

5]

T.

fusc

aY

X�/

�/6

.0/�

/6

08C

/B/6

58C

�/S

tarc

h,

Ca

2�

En

dp

rod

uct

s,G

3,

G4,

G6;

Km

(3.3

mg

ml�

1);

E.A

.(5

9

kJ

mo

l�1)

[70

]

G1,

glu

cose

;G

2,

ma

lto

se;

G3,

ma

lto

trio

se;

G4,

ma

lto

tetr

ao

se;

G5,

ma

lto

pen

tao

se;

E.A

.,en

zym

ea

ctiv

ati

on

ener

gy

;k

cal,

kil

oca

lori

es;

kJ,

kil

ojo

ule

s.


are being used in the bread industry for varied purposes

[13,99,121�/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 4�/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 fibreswith 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 forcoated 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 bypassing 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 abatch 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 compatiblewith 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 fromthe two major detergent enzyme suppliers Novozymes

and Genencore International have used protein engi-

neering to improve the bleach stability of the amylases

[146�/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 oxidationstability 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 Expressclinical 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 areamong 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 forthese 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 orthrough 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, 1965:273�/304.

[2] Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R.

Advances in microbial amylases. Biotechnol Appl Biochem

2000;31:135�/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, 1979:45�/

108.

[4] Vihinen M, Mantsala P. Microbial amylolytic enzymes. Crit Rev

Biochem Mol Biol 1989;24:329�/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, 1990:1�/56.

[6] Priest FG. Extracellular enzyme synthesis in the genus Bacillus .

Bacteriol Rev 1977;41:711�/53.

[7] Hollo J, Szeitli J. The reaction of starch with iodine. In: Rodley

JA, editor. Starch and its derivatives, 4th ed. Chapman & Hall,

1968:203�/46.


[8] Fuwa H. A new method for microdetermination of amylase

activity by the use of amylose as substrate. J Biochem

1954;41:583�/603.

[9] Manonmani HK, Kunhi AAM. Interference of thiol compounds

with dextrinizing activity assay of a-amylase by starch�/iodine

color reaction: modification of the method to eliminate this

interference. World J Microbiol Biotechnol 1999;15:485�/7.

[10] Hansen PW. Determination of fungal a-amylase by flow

injection analysis. Anal Chim Acta 1984;158:375�/7.

[11] Carlsen M, Marcher J, Nielsen J. An improved FIA-method for

measuring a-amylase in cultivation media. Biotechnol Tech

1994;8:479�/82.

[12] Sandstedt RM, Kneen E, Blish MJ. A standardised Wohlgemuth

procedure for a-amylase activity. Cereal Chem 1939;16:712�/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, 1993:152�/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;1:149�/

58.

[16] Miller GL. Use of dinitrosalicylic acid reagent for determination

of reducing sugar. Anal Chem 1959;31:426�/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;21:653�/72.

[18] Ceska M, Hultman E, Ingelman BGA. A new method for

determination of a-amylase. Experentia 1969;25:555�/6.

[19] Dhawale MR, Wilson JJ, Khachatourians GG, Ingledew WM.

Improved method for detection of starch hydrolysis. Appl

Environ Microbiol 1982;44:747�/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;48:4540�/3.

[21] Sheehan H, McCleary BV. A new procedure for the measure-

ment of fungal and bacterial a-amylase. Biotechnol Tech

1988;2:289�/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;61:108�/11.

[26] Fogarty WM, Kelly CT. Starch degrading enzymes of microbial

origin. Prog Ind Microbiol 1979;15:87�/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;25:1�/6.

[28] Yabuki M, Ono N, Hoshino K, Fukui S. Rapid induction of a-

amylase by non-growing mycelia of Aspergillus oryzae . Appl


[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;26:47�/

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;141:2449�/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;62:678�/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,

1977:131�/46.

[34] Kelly CT, Bolton DJ, Fogarty WM. Biphasic production of a-

amylase of Bacillus flavothermus in batch fermentation. Bio-

technol Lett 1997;19:75�/7.

[35] Emanuilova EI, Toda K. a-Amylase production in batch and

continuous cultures by Bacillus caldolyticus . Appl Microbiol

Biotechnol 1984;19:301�/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;30:125�/9.

[37] Welker NE, Campbell LL. Effect of carbon sources on forma-

tion of a-amylase by Bacillus stearothermophilus . J Bacteriol

1963;86:681�/6.

[38] Krishnan T, Chandra AK. Effect of oilseed cakes on a-amylase

production by Bacillus licheniformis CUMC-305. Appl Environ

Microbiol 1982;44:270�/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;34:335�/40.

[40] Kekos D, Galiotou-Panayotou M, Macris BJ. Some nutritional

factors affecting a-amylase production by Calvatia gigantea .

Appl Microbiol Biotechnol 1987;26:527�/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;39:681�/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;167:139�/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;23:249�/52.

[44] McMahon HEM, Kelly CT, Fogarty WM. High maltose

producing amylolytic system of a Streptomyces sp. Biotechnol

Lett 1999;21:23�/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;35:27�/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;183:67�/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;21:111�/5.

[48] Dercova K, Augustin J, Krajcova D. Cell growth and a-amylase

production characteristics of Bacillus subtilis . Folia Microbiol

1992;37:17�/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;76:459�/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;53:278�/81.


[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;54:1516�/22.

[52] Rukhaiyar R, Srivastava SK. Effect of various carbon substrate

on a-amylase production from Bacillus sp. J Microb Biotechnol

1995;10:76�/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;32:217�/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;67:176�/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;26:273�/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;16:97�/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;51:142�/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;46:293�/5.

[59] Ikura Y, Horikoshi K. Effect of amino compounds on alkaline

amylase production by alkalophilic Bacillus sp. J Ferment

Technol 1987;65:707�/9.

[60] Lee J, Parulekar SJ. Enhanced production of a-amylase in fed-

batch cultures of Bacillus subtilis TN 106[pAT5]. Biotechnol

Bioeng 1993;42:1142�/50.

[61] Dean ACR. Influence of environment on the control of enzyme

synthesis. J Appl Chem Biotechnol 1972;22:245�/59.

[62] Mertz FP, Doolin LE. The effect of phosphate on the biosynth-

esis of vanilomycin. Can J Microbiol 1973;19:263�/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;32:51�/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;25:404�/8.

[65] Pazlarova J, Votruba J. Use of zeolite to control ammonium in

Bacillus amyloliquifaciens a-amylase fermentations. Appl Micro-

biol Biotechnol 1996;45:314�/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;48:504�/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;38:84�/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, 1983:421�/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;31:490�/4.

[70] Busch JE, Stutzenberger FJ. Amylolytic activity of Thermomo-

nospora fusca . World J Microbiol Biotechnol 1997;13:637�/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;61:1502�/6.

[72] Bhavaraju SM, Blanch HW. A model for pellet breakup in

fungal fermentations. J Ferment Technol 1976;54:466�/8.

[73] Justen P, Paul GC, Nienow AW, Thomas CR. Dependence of

mycelial morphology on impeller type and agitation intensity.

Biotechnol Bioeng 1996;52:672�/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;65:638�/48.

[75] Cui YQ, Van der Lans RGJM, Luyben KCAM. Effect of

agitation intensities on fungal morphology of submerged fer-

mentation. Biotechnol Bioeng 1997;55:715�/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;62:434�/46.

[77] Priest FG. Products and applications. In: Harwood CR, editor.

Biotechnology handbooks, Bacillus . New York, London: Ple-

num Press, 1989:293�/320.

[78] Carlsen M, Nielsen J, Villadser J. Growth and a-amylase

production by Aspergillus oryzae during continuous cultivations.

J Biotechnol 1996;45:81�/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;19:257�/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;57:321�/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;73:35�/

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;31:288�/92.

[83] Bohdziewicz J. Ultrafiltration of technical amylolytic enzymes.

Process Biochem 1996;31:185�/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;226:981�/91.

[85] Saito NA. Thermophilic extracellular a-amylase from Bacillus

licheniformis . Arch Biochem Biophys 1973;155:290�/8.

[86] Krishnan T, Chandra AK. Purification and characterization of

a-amylase from Bacillus licheniformis CUMC 305. Appl Environ

Microbiol 1983;46:430�/7.

[87] Lee SP, Morikawa M, Takagi M, Imanaka T. Cloning of the

aapT gene and characterization of its product, a-amylase�/

pullulanase (AapT), from thermophilic and alkaliphilic Bacillus

sp. Strain XAL601. Appl Environ Microbiol 1994;60:3764�/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, 1996:332�/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


[90] Ogasahara K, Imanishi A, Isemura T. Studies on thermophilic a-

amylase from Bacillus stearothermophilus . I. Some general and


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;39:392�/8.

[93] Robyt J, Ackerman RJ. Isolation, purification and characteriza-

tion of a maltotetraose producing amylase from Pseudomonas

stutzeri . Arch Biochem Biophys 1971;145:105�/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, 1980:115�/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;59:1339�/40.

[97] Ray RR, Jana DC, Nanda G. Immobilization of b-amylase from

Bacillus megaterium B6 into gelatin film by cross-linking. J Appl

Bacteriol 1995;79:157�/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;73:41�/9.

[100] Mamo G, Gashe BA, Gessesse A. A highly thermostable

amylase from a newly isolated thermophillic Bacillus sp.

WN11. J Appl Microbiol 1999;86:557�/60.

[101] Manning GB, Campbell LL. Thermostable a-amylase of Bacillus

stearothermophilus . I. Crystallization and some general proper-

ties. J Biol Chem 1961;236:2952�/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;41:155�/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;58:2490�/4.

[105] McKelvy J, Lee YC. Microheterogeneity of the carbohydrate

group of Aspergillus oryzae a-amylase. Arch Biochem Biophys

1969;132:99�/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;37:117�/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;365:235�/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;231:2901�/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;100:451�/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;268:24394�/401.

[118] Aehle W, Misset O. Enzymes for industrial applications. In:

Rehm HJ, Reed G, editors. Biotechnology, 2nd ed. Germany:

Wiley-VCH, 1999:189�/216.

[119] Godfrey T, West S. In: Godfrey T, West S, editors. Industrial

enzymology. 2nd ed. New York: Stockton Press, 1996. p.

91,105�/31,192,339�/56,361�/71.

[120] Hamer RJ. Enzymes in the baking industry. In: Tucker GA,

Woods LFJ, editors. Enzymes in food processing. Galsgow:

Blackie Academic and Professional, 1995:190�/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, 1999:947�/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;62:3712�/5.

[124] Hebeda RE, Bowles LK, Teague WM. Developments in enzymes

for retarding staling of baked goods. Cereal Foods World

1990;35:453�/7.

[125] Hebeda RE, Bowles LK, Teague WM. Use of intermediate

temperature stability enzymes for retarding staling in baked

goods. Cereal Foods World 1991;36:618�/24.

[126] Pritchard PA. Studies on the bread improving mechanisms of

fungal a-amylase. J Biol Educ 1992;26:12�/8.

[127] Van Dam HW, Hille JDR. Yeast and enzymes in bread making.

Cereal Foods World 1992;37:245�/52.

[128] Kulp K, Ponte GJ. Staling white pan bread: fundamental causes.

Crit Rev Food Sci Nutr 1981;15:1�/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.


[135] Wursch P, Gumy D. Inhibition of amylopectin retrogradation by

partial beta amylolysis. Carbohydr Res 1994;256:129�/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):203�/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;94:137�/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,

1996:327�/38.

[144] Van Ee JH, van Rijswijk WC, Bollier M. Enzymatic automated

dishwash detergents. Chim Oggi 1992;10:21�/4.

[145] Kottwitz B, Upadek H, Carrer G. Applications and benefits of

enzymes in detergent. Chim Oggi 1994;12:21�/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;33:175�/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;1546:1�/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;318:989�/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;15:561�/4.

[154] Wilson JJ, Ingledew M. Isolation and characterization of

Schwanniomyces alluvius amylolytic enzymes, Appl Environ

Mirobiol 1982;44:301�/7.

[155] Glymph JL, Stutzenberger FJ. Production, purification and

characterization of a-amylase from Thermomonospora curvata .

Appl Environ Microbiol 1977;34:391�/7.

[156] Jenson B, Olsen J, Allermann K. Purification of extracellular

amylolytic enzymes from the thermophilic fungus Thermomyces

lanuginosus . Can J Microbiol 1988;34:218�/23.

[157] Morgan FJ, Priest FG. Characterization of a thermostable a-

amylase from Bacillus licheniformis NCIB 6346. J Appl Bacteriol

1981;50:107�/14.

[158] Vihinen M, Mantsala P. Characterization of a thermostable

Bacillus stearothermophilus a-amylase. Biotechnol Appl Bio-

chem 1990;12:427�/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


[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;75:276�/82.

[161] Freer SN. Purification and characterization of the extracellular

a-amylase from Streptococcus bovis JB 1. Appl Environ Micro-

biol 1993;59:1398�/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;39:31�/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;32:110�/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.


[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;32:104�/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;60:177�/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;11:83�/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;42:233�/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;24:584�/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;25:433�/8.

[191] Kelly TT, Collins BS, Fogarty WM, Doyle EM. Mechanisms of

action of the a-amylase of Micromonospora melanosporea . Appl



Amylases Summary

Documents

Transcript of Amylases Summary