Amylase Anti Staling

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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 ofa-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

microbiala-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. Todaya 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].

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

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

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lases at length.a-Amylases are one of the most popular

and important form of industrial amylases and the

present review highlights the various aspects of micro-

bial a-amylases.

2. Distribution of a-amylase among microorganisms

a-Amylases are universally distributed throughout the

animal, plant and microbial kingdoms. Over the past

few decades, considerable research has been undertaken

with the extracellular a-amylase being produced by a

wide variety of microorganisms [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 bacterialsources 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 ofa-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 dextrinisingactivity ofa-amylase in terms of decrease in the iodine

colour reaction.

3.1.1. Determination of dextrinising activity

The dextrinising activity of a-amylases employs

soluble starch as substrate and after terminating the

reaction with dilute HCl, iodine solution is added. The

decrease in absorbance at 620 nm is then measured

against a substrate control. One percent decline in

absorbance is considered as one unit of enzyme [8].

The major limitation of this assay is interference of

media components including Luria broth, tryptone,

peptone, corn steep liquor (CSL), etc. and thiol com-

pounds with starch iodine complex. Copper sulphate

and hydrogen peroxide protect the 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 theoriginal 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 inv

olv

es 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]. Themajor defect in this assay is a slow loss in colour

produced and destruction of glucose by constituents of

the DNSA reagent.

To overcome these limitations, a modified method for

the estimation of reducing sugars was developed [16].

Rochelle salts were excluded and 0.05% sodium sulphate

was added to prevent the oxidation of the reagent. Since

then the modified method has been used extensively to

measure reducing sugars without any further modifica-

tions in the procedure.

Alternate methods, which also rely on the estimation

of the reducing sugars are also, employed [17].

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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] orCibacron Blue F3 G-A [19] as an alternative substrate.

The synthesis of these substrates involves two major

steps. Soluble starch is coloured under alkaline condi-

tions using the dye. This is the result of formation of

covalent bonds between starch and dye molecules. The

coloured starch is subsequently cross-linked by the

addition of 1,4-butanediol diglycide ether. This gives

an insoluble network, which swells in water. The

enzymic hydrolysis of such insoluble starch derivatives

yields soluble starch hydrolysates carrying the coloured

marker. This method is simple and sensitive for a-

amylase determination, but even minute quantities ofglucose 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 fora-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 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

fora-amylase. The assay is simple, reliable and accurate

but is expensive as it involves the use of a synthetic

substrate and specific enzymes. Thus the use of this

method is restricted only to very specific tests and not

for routine analysis. A comparison was made for the use

of end blockedp -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 ofa-amylase activity

and can be used as per the requirement. Both these

methods are commercially available as commercial kits,

however, it is found that a-amylases exhibit lower

affinity for low molecular weight substrates [18].

3.4. Decrease in viscosity of the starch suspension

These methods are generally used in the bakery

industry to assess the quality of the flour and not for

estimating a-amylase activity which are based on the

determination of the rheological properties of the

dough. Methods, which fall into this category, are the

falling number test and the Amylograph or Farinograph

test.

3.4.1. Falling number (FN) method

The falling number (FN) method, internationally

standardised [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 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 ofa-amylase by submerged fermenta-

tion (SmF) and solid state fermentation (SSF) has been

thoroughly investigated and is affected by a variety of

physicochemical factors. Most notable among these are

the composition of the growth medium, pH of the

medium, phosphate concentration, inoculum age, tem-

perature, aeration, carbon source and nitrogen source

[5,26]. Most reports among fungi have been limited to a

few species of mesophilic fungi where attempts have

been made to specify the cultural conditions and to

select superior strains of the fungus to produce on a

commercial scale[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.

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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-foldincrease 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 ofA. oryzae DSM 63303 has been

reported[29]. Apart from maltose, in some strains, other

carbon sources as lactose, trehalose, a-methyl-D-glyco-

side also served as inducers ofa-amylase[28]. Not only

the carbon source, but also the mycelial condition/age

affect the synthesis ofa-amylase byA. 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 tocatabolite 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 ofa-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 ofa-amylase while the agro-proces-

sing byproduct, wheat bran has been used for the

economic production ofa-amylase by SSF[5]. The use

of wheat bran in liquid surface fermentation (LSF) for

the production ofa-amylase from Aspergillus fumigatus

and from Clavatia gigantea , respectively, has also been

reported [40,41]. High a-amylase activities from A.

fumigatus have also been reported using a-methyl-D-

glycoside (a synthetic analogue of maltose) as substrate

[42].Use of low molecular weight dextran in combination

with either Tween 80 or Triton X-100 for a-amylase

production in the thermophilic fungus Thermomyces

lanuginosus (ATCC 200065) has been reported [43].

Triton X-100 had no effect, whereas Tween 80 increases

the a-amylase activity 27-fold.

4.1.2. Nitrogen sources

Organic nitrogen sources have been preferred for the

production ofa-amylase. Yeast extract has been used in

the production ofa-amylase fromStreptomyces 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 ofBacillus sp. IMD 434 [47], ammonium sulphate

in the case ofBacillus 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 ofa-amylase by 110/156% inA. oryzaewhen used as an additional nitrogen source than when

ammonia was used as a sole source [50]. Various other

organic nitrogen sources have also been reported to

support maximum a-amylase production by various

bacteria and fungi. However, organic nitrogen sources

viz. beef extract, peptone and com steep liquor sup-

ported maximum a-amylase production by bacterial

strains [35,38,51/54]soybean meal and casamino acids

by A. oryzae [55]. CSL has also been used for the

economical and efficient production ofa-amylase from

a mutant of B. subtilis [56]. Apart from this, various

inorganic salts such as ammonium sulphate for A.oryzae [30] and A. nidulans [29], ammonium nitrate

for A. oryzae [57]and Vogel salts for A. fumigatus [42]

have been reported to support better a-amylase produc-

tion in fungi.

Amino acids in conjunction with vitamins have also

been reported to affecta-amylase production. However,

no conclusion can be drawn about the role of amino

acids and vitamins in enhancing the a-amylase produc-

tion in different microorganisms as the reports are

highly variable. a-Amylase production by Bacillus

amyloliquefaciens ATCC 23350 increased by a factor

of 300 in the presence of glycine [58]. The effect of

glycine was not only as a nitrogen source rather itaffected 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 fromB. 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 inA. oryzae above 0.2 M phosphate levels has been

reported [55]. Similar findings were corroborated in B.

amyloliquefacienswhere low levels of phosphate resulted

in severely low cell density and noa-amylase production

[63]. In contrast, high phosphate concentrations were


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inhibitory to enzyme production byB. 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 importantrole and production was reduced to 50% when Mg2

was omitted from the medium. Na and Mg2 show

coordinated stimulation of enzyme production by Ba-

cillussp. 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 betweena-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 growth

medium plays an important role by inducing morpho-

logical change in the organism and in enzyme secretion.

The pH change observed during the growth of the

organism also affects product stability in the medium.

Most of the Bacillus strains used commercially for the

production of bacterial a-amylases by SmF have an

optimum pH between 6.0 and 7.0 for growth and

enzyme production. This is also true of strains used in

the production of the enzyme by SSF. In most cases the

pH used is not specified excepting pH 3.2/4.2 in the case

ofA. oryzae DAE 1679[39], 7.0/8.0 inA. 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. TemperatureThe 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 ofa-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 thusinfluences 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 regula-

tion of extracellular enzymes in batch cultures is well

documented[77]. A lot of work on the morphology and

physiology ofa-amylase production byA. oryzae during

batch cultivation has been done. Accordingly, morphol-

ogy of A. oryzae was critically affected by the growth

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

observed that at pH 3.0/3.5, freely dispersed hyphal

elements were formed. In the pH range 4/5, both pellets

and freely dispersed hyphal fragments were observedwhereas 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 ofa-amylase increased rapidly[79]. One report

states that inoculum quantity did not affect morpholo-

gical changes inA. oryzae in air-lift bioreactors and that

pellet size decreased considerably as the air velocity

increased [39]. In the case ofa-amylase production by

Bacillus flavothermus in batch cultivation in a 20 lfermentor, a-amylase production and biomass peaked

twice and highest activity was obtained after 24 h[34]. It

was observed that the kinetics of enzyme synthesis was

more of the growth associated than non-growth asso-

ciated type [35]. Similar findings were cited in another

report with B. amyloliquefaciens [63].

Continuous and fed-batch cultures have been recog-

nised as most effective for the production of the enzyme

[60]and several groups have studied the effectiveness of

these cultures. The production of a-amylase from

B. subtilis TN106 (pAT5) was enhanced substantially

by extending batch cultivation with fed-batch operation


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[60]. The bulk enzyme activity was nearly 54% greater in

a two-stage fed-batch operation at a feed rate of 31.65

ml h1 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 g h1 for a-amylase and

glucoamylase production fromA. oryzae RIB 642 in a

rotary draft tube fermentor (RTF) have been studied[49]. At a feed rate of 1 g h1 the yields ofa-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 h1, the biomass and a-

amylase yields was higher than those obtained in a

laboratory scale jar fermentor.

A model to simulate the steady-state values for

biomass yield, residual sugar concentration and specific

rate ofa-amylase production has been proposed which

simulated experimental data very well [80]. Further-

more, it was found in chemostat experiments that the

specific rate ofa-amylase production decreased by up to70% 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

h1 and amylase activity in the culture was low at

dilution rates above 1.2 h1. In contrast, in Bacillus sp.

the switching of growth from batch to continuous

cultivation resulted in the selection of a non a-amylase

producing variant[63].A decline in enzyme production

was also accompanied by morphological and metabolic

variations during continuous cultivation[81,82].The industrial exploitation of SSF for enzyme pro-

duction has been confined to processes involving fungi

and it is generally believed that these techniques are not

suitable for bacterial cultivation [5]. The use of SSF

technique in a-amylase production and its specific

advantages over other methods has been discussed

extensively[5].

6. Purification of microbial a-amylases

Industrial enzymes produced in bulk generally requirelittle 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 prop-

erties.

The purification ofa-amylases from microbial sources

in most cases has involved classical purification meth-

ods. These methods involve separation of the culture

from the fermentation broth, selective concentration by

precipitation using ammonium sulphate or organic

solvents such as chilled acetone. The crude enzyme is

then subjected to chromatography, usually affinity, ion

exchange and/or gel filtration. A number of reviews are

available on purification and characterisation of a-

amylases from a range of microorganisms[1,2,4,26,83].

Table 1 summarises various purification strategiesadopted 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 inTable 2.

7.1. Substrate specificity

As holds true for the other enzymes, the substrate

specificity of a-amylase varies from microorganism to

microorganism. In general, a-amylases display highest

specificity towards starch followed by amylose, amylo-

pectin, cyclodextrin, glycogen and maltotriose.

7.2. pH optima and stability

The pH optima ofa-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 amylasewith 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 ofBacillus 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 forF. 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].


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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 ofa-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 least

one Ca2 ion[111].The affinity of Ca2 toa-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 fora-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]


ARTICLE IN PRESS


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Table 2

Properties of some microbial amylases

Source pI Molecular

weight

(kDa)

pH optima/stabi-

lity

Temperature op-

tima/stability

Inhibitors Stabilisers

Fungi and yeast

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

min)

/ /

A. flavus LINK 3.5 52.5 6.0/6.0/10.0 55 8C/50 8C (1 h) Ag2

, Hg2

Ca2

A. foetidus ATCC

10254

/ 41.5 5.0// 45 8C/35 8C (60

min)

/ /

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

min)

Ag, Cu2, Fe3, Hg2, halides Substrate

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

A. chevalieri NSPRI

105

/ 68.0 5.5// 40 8C/60 8C (15

min)

EDTA, DNP Ca2, Mg2

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

min)

Ag, Cu2, Hg2, halides Substrate

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

min)

/ /

A. hennebergi Bloch-weitz

/ 50.0 5.5// 50 8C/40 8C (15min)

/ /

A. niger 3.44 58.0 4.0/5.0/2.2/7.0 //60 8C (15 min) / Ca2

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 / /

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

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

min)

Ag, Cu2, Fe3, Hg2, halides Substrate

A. oryzae / / 4.8/6.6// 35/37 8C// / /

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

A. oryzae 245 (ATCC

9376)

/ / 5.0/6.0// 30/40 8C// / /

A. usamii / 54.0 3.0/5.5// 60/70 8C// / /

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

(min)

/ /


9/18

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 /

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 /

L. kononenkoae CBS

5608

3.5 76.0 4.5/5.0/5.0/7.0

(1 h)

70 8C// DTT, Cu2, Ag2 Starch

Paecilomyces sp.

ATCC 46889

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

min, /Ca)

/ Ca2

Saccharomyces cer-

evisiae

/ 54.1 5.0// 50 8C// / /

Schwanniomyces al-

luvius UCD 5483

/ 61.9 6.3/4.5/7.5 40 8C/5/40 8C / /

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

h)

/ Ca2

Trichoderma viride / / 5.0/5.5/4.0/7.0 //60 8C (10 min) / /

Bacteria

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

B. licheniformis / 22.5 9.0/6.0/11.0 76 8C/B/60 8C / /

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

B. licheniformis NCIB

6346

/ 62/65 7.0/7.0/10.0 70/90 8C/85 8C

(1 h)

/ /

B. stearothermophilus 4.82 / 4.6/5.1// 55/70 8C// EDTA Ca2

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.

B. stearothermophilus

MFF4

/ / 5.5/6.0// 70/75 8C/half life

5.1 h at 80 8C

/ /

B. subtilis / 48.0 6.5/5/7.0 50 8C/5/50 8C Hg2, Fe3, Al3 Mn2, Co2

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


10/18

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

Bacillus sp. IMD 435 5.6 63.0 6.0 and 6.5 / / /

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

Bacillus sp. US 100 / / 5.6/4.5/8.0 82 8C/90/95 8C / Starch, Ca2

Bacillus sp. WN 11 / / 5.0/8.0// 75/80 8C// / /

Bacillus sp. WN 11 / 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 /

Bacillus sp. XAL 601 / / 9.0// 70 8C// / /

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

H. meridiana DSM

5425

/ / 7.0/5.0/7.0 37 8C// / Ca2

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

acetic acid, Hg2, dimethyl amino-

benzaldehyde

/

Micromonospora mel-

anosporea

7.6 45.0 7.0// 55 8C/40 8C (pH

11/12, 40 min)

/ /

M. melanosporea 7.6 45.0 7.0/6.0/12.0 55 8C// / /

Pseudomonas stutzeri / 12.5 8.0/7.0/9.5 47 8C/40 8C (1 h) / Ca2

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)

/

Streptomyces sp. IMD

2679

8.9(1),

8.7(2),

7.2(3)

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

65 8C//, 65 8C//

/ /

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

Thermomonospora

curvata

6.2 60.9 6.0// 65 8C// / /


11/18

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 or

by 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 the

presence of Ca2, a-amylases are much more thermo-

stable than without it[4,115].a-Amylase fromA. 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, 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 potentialuse 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

ofa-amylase are dealt here in brief.

8.1. Bread and baking industry and as an antistaling

agent

The baking industry has made use of these enzymes

for hundreds of years to manufacture a wide variety of

high quality products. For decades, enzymes such as

malt and microbial a-amylases have been widely used in

the baking industry[120,121]. These enzymes were used

in bread and rolls to give these products a higher

volume, better colour and a softer crumb. It is the

malt preparation that has led the way and opened the

opportunities for many enzymes to be used commer-

cially in baking. Today, many enzyme preparations such

as proteases, lipases, xylanases, pullulanases, pentosa-

nases, cellullases, glucose oxidases, lipoxygenases etc.Table2(Continued)

Source

pI

Molecular

weight

(kDa)

pHoptima/stabi-

lity

Temperatureop-

tima/stability

I

nhibitors

Stabilisers

Additionalproperties

Reference

T.curvata

/

62.0

5.5/6.0/activated

atpH7.0/8.0

658C//

B

.S.A.

/

Endproducts,G4,G5;Km

(0.3mgml1)

[155]

T.

fuscaYX

/

/

6.0//

608C/B/658C

/

Starch,

Ca2

Endproducts,G3,G4,G6;

Km

(3.3mgml1

);E.A.(59

kJmol1)

[70]

G1,glucose;G2,maltose;G3,maltotriose;G4,maltotetraose;G5,maltopentaose;E.A.,enzymeactivationenergy;kcal,kilocalories;kJ,kilojoules.


ARTICLE IN PRESS


12/18

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 ofa-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 beverageindustry 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 tothe 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 ofa-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 ofa-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 thepaper 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


ARTICLE IN PRESS


13/18

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 thedemand for a-amylases for automatic dishwashing

detergents is growing. One of the limitations of a-

amylases in detergents is that the enzyme shows

sensitivity to calcium and stability is severely compro-

mised in a low calcium environment. In addition, most

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

generally a component of detergent formulations. Sta-

bility against oxidants in household detergents was

achieved by utilising successful strategies followed with

other enzymes such as protease. Recently scientists from

the two major detergent enzyme suppliers Novozymes

and Genencore International have used protein engi-

neering to improve the bleach stability of the amylases[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 oxidation

stability resulted in better storage stability and perfor-

mance of the mutant enzyme in the bleach containing

detergent formulations. Genencore International and

Novozyme have introduced these new products in the

market under the trade names Purafect OxAm and

Duramyl, respectively.

8.6. Analysis in medicinal and clinical chemistry

With the advent of new frontiers in biotechnology, the

spectrum of amylase applications has expanded into

many other fields, such as clinical, medicinal and

analytical chemistry. There are several processes in the

medicinal and clinical areas that involve the application

of amylases. The application of a liquid stable reagent,

based on a-amylase for the Ciba Corning Express

clinical chemistry system has been described [149]. A

process for the detection of higher oligosaccharides,

which involved the application of amylase was also

developed [96]. This method was claimed to be more

efficient than the silver nitrate test. Biosensors with an

electrolyte isolator semiconductor capacitor (EIS-CAP)

transducer for process monitoring were also developed

[150].

9. Conclusions

As evident from the foregoing review, amylases are

among the most important enzymes used in industrial

processes. Although, the use of amylases, a-amylases in

particular, in starch liquefaction and other starch based

industries has been prevalent for many decades and a

number of microbial sources exist for the efficient

production of this enzyme, the commercial production

of this enzyme has been limited to only a few selected

strains of fungi and bacteria. Moreov

er, 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 efficienta-amylases

in various sectors, which can be achieved either by

chemical modification of the existing enzymes or

through protein engineering. In the light of modern

biotechnology, a-amylases are now gaining importance

in biopharmaceutical applications. Still, their applica-

tion in food and starch based industries is the major

market and thus the demand of a-amylases wouldalways 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

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