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Process Biochemistry 38 (2003) 1599 Á/1616 www.elsevier.

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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 of a-amylase application has also expanded to medicinal and analytical chemistry as well as in automatic dishwashing detergents, textile desizing and the pulp and paper industry. Amylases are of ubiquitous occurrence, produced by plants, animals and microorganisms. However, microbial sources are the most preferred one for large scale production. Today a large number of microbial a-amylases are marketed with applications in different industrial sectors. This review focuses on the microbial amylases and their application with a biotechnological perspective. # 2003 Elsevier Science Ltd. All rights reserved.
Keywords: a-Amylase; Baking; Antistaling; Dextrinising activity; Starch liquefaction

1. Introduction Amylases are enzymes which hydrolyse starch molecules to give diverse products including dextrins and progressively smaller polymers composed of glucose units [1]. These enzymes are of great significance in present day biotechnology with applications ranging from food, fermentation, textile to paper industries [2]. Although amylases can be derived from several sources, including plants, animals and microorganisms, microbial enzymes generally meet industrial demands. Today a large number of microbial amylases are available commercially and they have almost completely replaced chemical hydrolysis of starch in starch processing industry [2]. The history of amylases began in 1811 when the first starch degrading enzyme was discovered by Kirchhoff.

* Corresponding author. Tel.: '/91-11-2611-1933; fax: '/91-112688-5270. E-mail address: ranigupta15@rediffmail.com (R. Gupta). 1 E-mail: microzyme@123india.com.

This was followed by several reports of digestive amylases and malt amylases. It was much later in 1930, that Ohlsson suggested the classification of starch digestive enzymes in malt as a- and b-amylases according to the anomeric type of sugars produced by the enzyme reaction. a-Amylase (1,4-a-D-glucan-glucanhydrolase, EC. 3.2.1.1) is a widely distributed secretary enzyme. a-Amylases of different origin have been extensively studied. Amylases can be divided into two categories, endoamylases and exoamylases. Endoamylases catalyse hydrolysis in a random manner in the interior of the starch molecule. This action causes the formation of linear and branched oligosaccharides of various chain lengths. Exoamylases hydrolyse from the non-reducing end, successively resulting in short end products. Today a large number of enzymes are known which hydrolyse starch molecule into different products and a combined action of various enzymes is required to hydrolyse starch completely. A number of reviews exist on amylases and their applications, however, none specifically covers a-amy-

0032-9592/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0032-9592(03)00053-0

is then used to calculate activity in terms of grams of starch digested. These are based on decrease in starchÁ/iodine colour intensity. enzymes from fungal and bacterial sources have dominated applications in industrial sectors [2]. a-Amylase catalyses the hydrolysis of a-1. Various workers [10. 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]. degradation of colour-complexed substrate and decrease in viscosity of the starch suspension. / Process Biochemistry 38 (2003) 1599 Á/1616 lases at length. The decrease in absorbance at 620 nm is then measured against a substrate control. The major limitation of this assay is interference of media components including Luria broth. Alternate methods.1600 R. To overcome these limitations. employed [17]. it changes to red brown. peptone. a-Amylases are one of the most popular and important form of industrial amylases and the present review highlights the various aspects of microbial a-amylases. 3. bacteria and actinomycetes. The tube. Decrease in starch Á/iodine colour intensity Starch forms a deep blue complex with iodine [7] and with progressive hydrolysis of the starch. . etc. a-Amylase has been derived from several fungi. Absorbance is then read at 570 nm. This method determines the dextrinising activity of a-amylase in terms of decrease in the iodine colour reaction.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]. Further. plant and microbial kingdoms.2. Since then the modified method has been used extensively to measure reducing sugars without any further modifications in the procedure.1. Distribution of a-amylase among microorganisms a-Amylases are universally distributed throughout the animal. yeasts. This method has many advantages including high sampling rates. fast response. Several procedures have been described for the quantitative determination of amylase based on this property. 3. increase in reducing sugars. Various methods are available for the determination of a-amylase activity [6]. and thiol compounds with starch iodine complex. Determination of dextrinising activity The dextrinising activity of a-amylases employs soluble starch as substrate and after terminating the reaction with dilute HCl. Sandstedt Kneen and Blish (SKB) method The SKB method [12]. This method is usually employed for estimating a-amylase activity in cereals. zinc sulphate was found to be best for counteracting the interference of various metal ions. This method is used generally to express the diastatic strength of the malt and not for expressing a-amylase activity alone [13]. 3. Determination of a-amylase activity a-Amylases are generally assayed using soluble starch or modified starch as the substrate.11] have successfully used the original assay procedure in combination with flow injection analysis (FIA). Over the past few decades. The reaction is monitored by an increase in the reducing sugar levels or decrease in the iodine colour of the treated substrate. flexibility and simple apparatus. 3. One percent decline in absorbance is considered as one unit of enzyme [8]. corn steep liquor (CSL).1. tryptone. Gupta et al.1. The major defect in this assay is a slow loss in colour produced and destruction of glucose by constituents of the DNSA reagent. which does not show any blue colour. The potency of most commercial amylases is described in terms of SKB [12] units. iodine solution is added. this method is used to calculate a-amylase activity in terms of grams of starch digested by a given volume of enzyme [14]. This procedure involves incubation of the enzyme preparation in a range of dilutions in buffered starch substrate at 40 8C for 1 h. The solutions are then treated with iodine solution.3. 2. dextrins and limit dextrins. Samples are allowed to react with starch in a coil before iodine was added.4 glycosidic linkages in starch to produce glucose.1. 3.05% sodium sulphate was added to prevent the oxidation of the reagent. a peristaltic pump.1. a photometer with a flow cell and 570 nm filter and a pen recorder. considerable research has been undertaken with the extracellular a-amylase being produced by a wide variety of microorganisms [1 Á/5]. 3. a modified method for the estimation of reducing sugars was developed [16]. The flow system comprised of an injection valve. Rochelle salts were excluded and 0. Indian pharmacopoeia method As described in the Indian pharmacopoeia. is one of the most widely adopted methods for determination of amylases used in the baking industry. however. Copper sulphate and hydrogen peroxide protect the starchÁ/iodine colour in the case of interference by these media components [9]. which also rely on the estimation of the reducing sugars are also.

3. by replacing a part of the flour with pre-gelatinised starch. Both cereal and fungal a-amylases are used to improve the fermentation of flour deficient in amylase activities. When the amylograph is used. This indicates that all the methods give an accurate and reliable measure of a-amylase activity and can be used as per the requirement. Physiology of a-amylase production The production of a-amylase by submerged fermentation (SmF) and solid state fermentation (SSF) has been thoroughly investigated and is affected by a variety of physicochemical factors. the thinner is the hot paste viscosity. It can successfully detect as low as 0 Á/50 ng of enzyme [20]. 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 blocking group (4. groups have been working on the development of a specific a-amylase determination method based on the use of new types of substrates. surface acting agents. 3. internationally standardised [22 Á/24] is accepted for assessing cereal aamylase activity in flour Á/enzyme preparations at 100 8C. Because fungal a-amylases have low thermostability. The assay is simple. temperature. values of 400 Á/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. Gupta et al. pH of the medium. / Process Biochemistry 38 (2003) 1599 Á/1616 1601 3.4. This method is simple and sensitive for aamylase determination. inoculum age. The reaction was monitored using the starch Á/iodine colour [21]. 3. they cannot be detected by the standard FN method at 100 8C [25]. This gives an insoluble network. This method has been modified and standardised [25] for measuring both cereal and fungal a-amylase activity at 300 8C. Thus the use of this method is restricted only to very specific tests and not for routine analysis. but even minute quantities of glucose might lead to erroneous results due to starch contamination by dextrin substrate [19]. Other novel substrates such as nitrophenyl derivatives of maltosaccharides have also been employed. phosphate.R.4-butanediol diglycide ether. Degradation of colour-complexed substrate For some years. Falling number (FN) method The falling number (FN) method. The higher the enzyme activity. including carbon and nitrogen source. Both these methods are commercially available as commercial kits. This is the result of formation of covalent bonds between starch and dye molecules. The use of nitrophenyl-maltosaccharides in conjunction with a specific yeast a-glucosidase can be used but these substrates are rapidly cleaved by glucoamylases commonly present in the culture broths. Physiochemical parameters The role of various physico-chemical parameters. Methods. 4. A falling number of about 400 indicates a normally malted flour.1.26]. . The use of nonreducing end blocked p-nitrophenyl maltoheptoside (BPNPG7) has also been described [21].1. pH and agitation have been studied.4. it is found that a-amylases exhibit lower affinity for low molecular weight substrates [18]. 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. which fall into this category.2. Amylograph/Farinograph test The milling and baking industries generally assess the diastatic activity of flours by means of an amylograph. Most notable among these are the composition of the growth medium. phosphate concentration. The enzymic hydrolysis of such insoluble starch derivatives yields soluble starch hydrolysates carrying the coloured marker. 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. which swells in water. a rapid and sensitive microassay based on dye cross linked starch for a-amylase detection has been reported. The synthesis of these substrates involves two major steps. Soluble starch is coloured under alkaline conditions using the dye. This method is also based on the relationship of peak viscosity of starch slurry and the enzyme activity level [23]. metal ions.3. temperature. aeration. The coloured starch is subsequently cross-linked by the addition of 1. carbon source and nitrogen source [5. reliable and accurate but is expensive as it involves the use of a synthetic substrate and specific enzymes. 4.6-O -benzylidene) prevents the hydrolysis of the substrate by the exo-acting enzymes and is thus specific for a-amylase. however. There was an excellent correlation between each of the assay procedures employed. Recently. The assay measures the release of free p -nitrophenyl groups. are the falling number test and the Amylograph or Farinograph test. 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 Á/4].

amyloliquefaciens where low levels of phosphate resulted in severely low cell density and no a-amylase production [63]. Similar findings were corroborated in B. peptone and com steep liquor supported maximum a-amylase production by bacterial strains [35. wheat bran has been used for the economic production of a-amylase by SSF [5]. However. a-Amylase production by A.36]. A-40-2. no conclusion can be drawn about the role of amino acids and vitamins in enhancing the a-amylase production in different microorganisms as the reports are highly variable. However. casitone [35. [44]. oryzae [55]. oryzae [57] and Vogel salts for A. b-Alanine. However. High a-amylase activities from A. 4. ammonium nitrate for A. 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]. Not only the carbon source. 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. Apart from maltose.2. high phosphate concentrations were . A number of other non-conventional substrates as lactose [34]. Nitrogen sources Organic nitrogen sources have been preferred for the production of a-amylase. the role of amino compounds was considered to be neither as nitrogen nor as a carbon source. Role of phosphate Phosphate plays an important regulatory role in the synthesis of primary and secondary metabolites in microorganisms [61. 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. There are reports that 5 days starved non-growing mycelia were the most appropriate for optimal induction by maltose. respectively. nidulans [29]. various inorganic salts such as ammonium sulphate for A. However. Yeast extract has been used in the production of a-amylase from Streptomyces sp. Yeast extract has also been used in conjunction with other nitrogen sources such as bactopeptone in the case of Bacillus sp. beef extract. in some strains. Most reports available on the induction of a-amylase in different strains of Aspergillus oryzae suggest that the general inducer molecule is maltose. subtilis [56]. oryzae DSM 63303 has been reported [29]. Apart from this. oryzae [30] and A.1. DL-nor valine and D-methionine were effective for the production of alkaline amylase by Bacillus sp.32]. a-Amylase production is also subjected to catabolite repression by glucose and other sugars. However. ammonium sulphate in the case of Bacillus subtilis [48]. but as stimulators of amylase synthesis and excretion [59]. oryzae when used as an additional nitrogen source than when ammonia was used as a sole source [50]. The carbon sources as glucose and maltose have been utilised for the production of a-amylase. CSL has also been used for the economical and efficient production of a-amylase from a mutant of B. like most other inducible enzymes [30. Yeast extract increased the productivity of a-amylase by 110Á/156% in A. has also been reported [40. / Process Biochemistry 38 (2003) 1599 Á/1616 4. A significant increase in enzyme production and conidiation in A.2 M phosphate levels has been reported [55]. but also the mycelial condition/age affect the synthesis of a-amylase by A. oryzae above 0. The use of wheat bran in liquid surface fermentation (LSF) for the production of a-amylase from Aspergillus fumigatus and from Clavatia gigantea. the role of glucose in the production of a-amylase in certain cases is controversial. It has been reported that only asparagine gave good enzyme yields [57] while the importance of arginine for a-amylase production from B. a-methyl-D-glycoside also served as inducers of a-amylase [28]. 4.1. oilseed cakes [38] and starch processing waste water [39] have also been used for the production of a-amylase while the agro-processing byproduct. fructose [37]. However. Gupta et al. Similarly strong a-amylase induction by starch and maltose in the case of A. subtilis has also been well documented [60]. IMD 434 [47].1.3.1. other carbon sources as lactose. fumigatus have also been reported using a-methyl-Dglycoside (a synthetic analogue of maltose) as substrate [42]. Various other organic nitrogen sources have also been reported to support maximum a-amylase production by various bacteria and fungi. IMD 435 [45] and Halomonas meridiana [46]. organic nitrogen sources viz. oryzae (NRC 401013) [31]. the use of starch remains promising and ubiquitous.51 Á/54] soybean meal and casamino acids by A. Bacillus sp. ammonium sulphate and casein for C. In contrast. whereas Tween 80 increases the a-amylase activity 27-fold. Amino acids in conjunction with vitamins have also been reported to affect a-amylase production. a-Amylase production by Bacillus amyloliquefaciens ATCC 23350 increased by a factor of 300 in the presence of glycine [58].38. gigantea [40] and soybean flour and meat extract for A. trehalose. Triton X-100 had no effect. oryzae [49].62] and likewise it affects the growth of the organism and production of a-amylase. a minimal level of the enzyme was induced in its presence [29]. fumigatus [42] have been reported to support better a-amylase production in fungi. oryzae M-13 [28]. oryzae DSM 63303 was not repressed by glucose rather. xylose or fructose have been classified as strongly repressive although they supported good growth in Aspergillus nidulans [33].1602 R.41]. There is a report of a 20-fold increase in enzyme activity when maltose and starch were used as inducers in A. maltose [27 Á/30].

An inverse relationship between a-amylase production and growth rate was observed for Streptomyces sp.7. 4. Continuous and fed-batch cultures have been recognised as most effective for the production of the enzyme [60]and several groups have studied the effectiveness of these cultures. the buffering capacity of some media constituents sometimes eliminates the need for pH control [68]. / Process Biochemistry 38 (2003) 1599 Á/1616 1603 inhibitory to enzyme production by B. authors observed that at pH 3. Temperature The influence of temperature on amylase production is related to the growth of the organism. Fermentation studies on a-amylase production The effect of environmental conditions on the regulation of extracellular enzymes in batch cultures is well documented [77]. Fe2'. The production of a-amylase from B. However.6. 5. Gupta et al. Cl (. oryzae EI 212 [57] and 6.0 in A. morphology of A. oryzae in air-lift bioreactors and that pellet size decreased considerably as the air velocity increased [39]. Most of the Bacillus strains used commercially for the production of bacterial a-amylases by SmF have an optimum pH between 6. oryzae DAE 1679 [39]. In a series of batch experiments.0 Á/3.1. lanuginosus [17]. albeit with a reduction in enzyme yield.5. amyloliquefaciens [63]. oryzae . The pH change observed during the growth of the organism also affects product stability in the medium. both pellets and freely dispersed hyphal fragments were observed whereas at pH higher than 6 pellets were the only growth forms recorded. oryzae during batch cultivation has been done.2) [28]. In fungal processes. 4. temperatures as high as 80 8C have been used for amylase production from the hyperthermophile Thermococcus profundus [71]. This is also true of strains used in the production of the enzyme by SSF. Accordingly. the presence of Co2' enhancing the final biomass levels by 13-fold. Mn2'. 7. in the presence and absence of Co2' [66].79] have recorded similar observations for other strains of A. Agitation intensities of up to 300 rpm have normally been employed for the production of amylase from various microorganisms as reported in the literature. SO2( had no 4 effect while Ca2' was inhibitory to amylase production by A. It has been reported that a higher agitation speed is sometimes detrimental to mycelial growth and thus may decrease enzyme production. In the case of a-amylase production by Bacillus flavothermus in batch cultivation in a 20 l fermentor.5. Na '. amyloliquefaciens at 36 8C has been reported [67]. 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]. oryzae 557 accumulated aamylase in the mycelia when grown in phosphate or sulphate deficient medium and was released when the mycelia were replaced in a medium with alkaline pH (above 7.57]. Optimum yields of a-amylase were achieved at 30 Á/37 8C for A. A lot of work on the morphology and physiology of a-amylase production by A. The pH values also serves as a valuable indicator of the initiation and end of enzyme synthesis [69]. It was observed that the kinetics of enzyme synthesis was more of the growth associated than non-growth associated type [35].72Á/76]. a-Amylase has been produced at a much wider range of temperature among the bacteria. The optimum growth temperature was found to be 35 8C. It is demonstrated that when glucose was exhausted the biomass production stopped whereas the secretion of a-amylase increased rapidly [79]. a-Amylase production has also been reported at 55 8C by the thermophilic fungus Thermomonospora fusca [70] and at 50 8C by T. In most cases the pH used is not specified excepting pH 3.2 Á/4. Continuous production of amylase from B. amyloliquefaciens [58]. amyloliquefaciens MIR-41 [67].2 in the case of A. However. subtilis TN106 (pAT5) was enhanced substantially by extending batch cultivation with fed-batch operation . Agitation Agitation intensity influences the mixing and oxygen transfer rates in many fungal fermentations and thus influences mycelial morphology and product formation [69. oryzae EI 212 [57].1. amyloliquefaciens resulted in increased yield of a-amylase [65].1. pH Among the physical parameters. the pH of the growth medium plays an important role by inducing morphological change in the organism and in enzyme secretion.1. oryzae [55. Mo2'.4. Among the fungi.0 for growth and enzyme production. Role of other ions K '. One report states that inoculum quantity did not affect morphological changes in A.R. Mg2' played an important role and production was reduced to 50% when Mg2' was omitted from the medium. CRP strain [64]. In the pH range 4Á/5. most amylase production studies have been done with mesophilic fungi within the temperature range of 25 Á/37 8C. 4.0 and 7. Other groups [39. Na ' and Mg2' show coordinated stimulation of enzyme production by Bacillus sp. Similar findings were cited in another report with B. freely dispersed hyphal elements were formed. 4. Addition of zeolites to control ammonium ions in B.0 Á/8. a-amylase production and biomass peaked twice and highest activity was obtained after 24 h [34]. It is reported that A.8 for B. oryzae was critically affected by the growth pH [78].

amylopectin. glycogen and maltotriose. cyclodextrin. The purification of a-amylases from microbial sources in most cases has involved classical purification methods.83]. At a feed rate of 1 g h(1 the yields of a-amylase were twice than those obtained in batch cultures. Substrate specificity As holds true for the other enzymes.86. a-Amylase from Alicyclobacillus acidocaldarius showed an acidic pH optima of 3 [84]. meridiana is sodium chloride dependent [46]. Temperature optima and stability The temperature optimum for the activity of aamylase is related to the growth of the microorganism [4]. ion exchange and/or gel filtration. selective concentration by .92]. GM8901 [89]. Furthermore. Biochemical properties of a-amylases The enzymic and physicochemical properties of aamylases from several microorganisms have been extensively studied and described [2 Á/4. than that attained in the single stage batch culture. 7. 7. Pyrococcus furiosus and Pyrococcus woesei . aAmylases from most bacteria and fungi have pH optima in the acidic to neutral range [2]. The lowest temperature optimum is reported to be 25 Á/30 8C for F. pH optima and stability The pH optima of a-amylases vary from 2 to 12 [4].3. [85 Á/88].4. 7. A summary is presented in Table 2. in Bacillus sp. Table 1 summarises various purification strategies adopted for microbial a-amylases.93]. the pH optimum was observed to be dependent upon temperature as in the case of Bacillus stearothermophilus DONK BS-1 [90] and on calcium as in the case of B. The industrial exploitation of SSF for enzyme production has been confined to processes involving fungi and it is generally believed that these techniques are not suitable for bacterial cultivation [5]. Shifts in the dilution rate in continuous culture could be used to obtain different proportions of the enzymes. stearothermophilus [91]. A number of reviews are available on purification and characterisation of aamylases from a range of microorganisms [1. usually affinity. oryzae RIB 642 in a rotary draft tube fermentor (RTF) have been studied [49].4.1604 R.82]. but enzyme applications in pharmaceutical and clinical sectors require high purity amylases. the substrate specificity of a-amylase varies from microorganism to microorganism. The commercial use of a-amylase generally does not require purification of the enzyme.65 ml h (1 of medium. a-amylases with stability in a narrow range have also been reported [46.45. residual sugar concentration and specific rate of a-amylase production has been proposed which simulated experimental data very well [80]. In contrast.26. In general. oxysporum amylase [94] and the highest of 100 and 130 8C from archaebacteria. The effects of controlled feeding of maltose at a feed rate of 1 Á/4 g h(1 for a-amylase and glucoamylase production from A. Extremely alkalophilic a-amylase with pH optima of 11 Á/12 has been reported from Bacillus sp. Temperature optima of enzymes from Micrococcus varians are calcium dependent [98] and that from H. it was found in chemostat experiments that the specific rate of a-amylase production decreased by up to 70% with increasing biomass concentration at a given dilution rate. respectively [95 Á/97]. Gupta et al. It was further demonstrated that maximum production of a-amylase occurred in continuous culture at a dilution rate of 0.85. The use of SSF technique in a-amylase production and its specific advantages over other methods has been discussed extensively [5]. in contrast to the alkaline amylase with optima of pH 9 Á/10. the biomass and aamylase yields was higher than those obtained in a laboratory scale jar fermentor. These methods involve separation of the culture from the fermentation broth.2. 7.1.15 h(1 and amylase activity in the culture was low at dilution rates above 1. precipitation using ammonium sulphate or organic solvents such as chilled acetone. the switching of growth from batch to continuous cultivation resulted in the selection of a non a-amylase producing variant [63].83]. When fed-batch cultivations were performed on a pilot scale RTF at a feed rate of 24 g h(1. A model to simulate the steady-state values for biomass yield.2. by the same strain [66]. A decline in enzyme production was also accompanied by morphological and metabolic variations during continuous cultivation [81.47.5 reported from an alkalophilic Bacillus sp. a-amylases display highest specificity towards starch followed by amylose. In some cases.2 h (1. 6. Purification of microbial a-amylases Industrial enzymes produced in bulk generally require little downstream processing and hence are relatively crude preparations. / Process Biochemistry 38 (2003) 1599 Á/1616 [60]. The crude enzyme is then subjected to chromatography. The bulk enzyme activity was nearly 54% greater in a two-stage fed-batch operation at a feed rate of 31. however. The enzyme in purified form is also a prerequisite in studies of structure Á/function relationships and biochemical properties. a-Amylases are generally stable over a wide range of pH from 4 to 11 [3.

0). These include the presence of calcium. Mono Q 85% (NH4)2S04. ultrogel AcA54.8 20/35 1.7/9. b-Cyclodextrin linked Sepharose 6B (Epoxy activated. 50 Á/80% (NH4)2SO4. S-2 L. Glycoproteins have been detected in A. / Process Biochemistry 38 (2003) 1599 Á/1616 Table 1 Purification strategies employed for a-amylase Microorganism Purification strategy Fold purification/ yield (%) 1605 Reference Fungi and yeast A. Calcium and stability of a-amylase a-Amylase is a metalloenzyme. CM-Cellulose (pH 6.5).5) Ultrafiltration.8/17.5. 70% (NH4)2SO4.036/ Á/ 6. Sephadex G-25 (pH 7. ultrafiltration. IMD435 Bacillus sp. ultrafiltration 60% (NH4)2SO4. subtilis B. stearothermophilus ATCC 12980 B. The stabilising effect of starch was observed in a-amylases from B. DEAE-Sephacel (pH 8. CM Sephadex C-50 Ultrafiltration. iodoacetate. Carbohydrate moieties raise the molecular weight of some a-amylases. DE52-Cellulose (pH 7.5) and 10% (NH4)2SO4.0). DEAE Bio-Gel A (pH 5. DEAE-Trisacryl (pH 7.8/70 140/78 6000/52 5/2 10. DEAE-Toyopearl 650 M (pH 7. Bio-Gel P-30 a-Cyclodextrin coupled Sepharose 6B (pH 6. p hydroxyl mercuribenzoic acid. ultrafiltration Ultrafiltration.0) Acetone precipitation. flavus LINK Cryptococcus sp.8) 60% (NH4)2SO4. licheniformis CUMC 305 B.O) 744/65 266/ Á/ Amy I 65/13.5/ Á/ 30. Many factors affect thermostability. N -bromosuccinimide. DEAE-Cellulose Concentrated by drum humidifier. L . 25% (NH4)2SO4.0). 7.0).6. 7.109]. Sephadex G-100 (pH 4.4. Phenyl Sepharose CL-4B (pH 7. profundus DT5432 DE52-Cellulose (pH 7. Resource Q (pH 7.1 [92] [152] [99] [153] [154] [155] [156] [17] Ultrafiltration. DEAE Sepharose (pH 5. Gupta et al. washing with Aces (pH 7. 10 kDa for Bacillus caldolyticus [103] and the highest of 210 kDa for Chloroflexus aurantiacus has been reported [104].0). ultrafiltration.5).3). gel filtration (pH 6.4) DEAE-Cellulose DE52 (pH 5. Superdex 200 HR (pH 7.6). 75% ethanol precipitation. stearothermophilus [107] and B.R. lanuginosus T.106]. The affinity of Ca2' to a-amylase is much stronger than that of other ions. Inhibitors Many metal cations. castelii [110] whereas this is about 10% for other a-amylases [4].5) 816/26 Thermostabilities have not been estimated defactor in many studies. oryzae NRC 401013 A. DEAE-Sephadex A50 (pH 5. 7.5 212/42 33/66 Á/ 9/17 2. WN 11 B. DEAE 66/9 Cellulose.0).5). kononenkoae [98]. Molecular weights of microbial a-amylases are usually 50Á/ 60 kDa as shown directly by analysis of cloned aamylase genes and deduced amino acid sequences [4]. Sephadex G-75 65% (NH4)2S04. pH 4. 70% acetone 70% (NH4)2SO4. The amount of bound calcium varies from one to ten.0) Ultrafiltration. Sephadex G-150 (pH 5.6) [31] 13.9/50 300/ Á/ [45] [47] [100] [86] [157] [158] [159] [83] [51] [160] [93] [161] [162] [71] 80% (NH4)2SO4. subtilis strains [108. BSA. DEAE-sephacel (pH 5.5) Ultrafiltration. Sephacryl-S200 HR (pH 8. DEAE chromatography (Zetaprep disk). WN 11 [100].0). B.5) Ultrafiltration. Amy II 40.5). S-Sepharose Ultrafiltration Sephacryl S-300.0) 50 Á/90% (NH4)2SO4. subtilis B. a-Cyclodextrin coupled with Sepharose 6B (pH 7. sulphydryl group reagents. The lowest value. Thermostabilities as high as 4 h at 100 8C have been reported for Bacillus licheniformis CUMC 305 [86]. Sephacryl S300. Crystalline Taka- . kononenkoae CBS5608 Saccharomyces cerevisiae YPB-G Schwanniomyces alluvius UCD-54-83 Thermomonospora curvata T.0) 60% (NH4)2SO4. licheniformis NCIB 6346 B.3) Adsorption on soluble starch (1%) in 10% (NH4)2SO4. Glycosylation of bacterial proteins is rare. subtilis 65 Lactobacillus plantarum A6 Pseudomonas stutzeri Streptococcus bovis JB1 Thermomonospora curvata NCIMB 10081 T. Sephadex G-150 (pH 8. Phenyl-Sepharose (pH 7. DEAE-Sephadex 112/41 A50 (pH 8. Lipomyces kononenkoae [98] and Bacillus sp. licheniformis CUMC 305 [85]. lanuginosus IISc91 Bacteria Bacillus sp. crosslinked starch (pH 8. 60% (NH4)2SO4. especially heavy metal ions. which contains at least one Ca2' ion [111]. Thermal stabilisation of the enzyme in the presence of calcium has also been reported from time to time [100 Á/102].0). EDTA and EGTA inhibit a-amylases. IMD 434 Bacillus sp. substrate and other stabilisers [4]. 70% (NH4)2SO4. A carbohydrate content as high as 56% has been reported in S. Molecular weight Molecular weights of a-amylases vary from about 10 to 210 kDa.85/24. oryzae [105. DEAE-Sephadex A50 (pH 6.

0/ Á/ 3. Hg2' . Hg2' .0 Á/6.4 mM Km 0/4. Cu2' .35.2 Á/7.0 Á/7.0 Á/8.0/5.6/ Á/ 5.0/5.5/ Á/ 5.2 Á/6.1606 Table 2 Properties of some microbial amylases Source pI Molecular weight (kDa) pH optima/stabi.0 50 8C/50 8C (30 min) 55 8C/50 8C (1 h) Á/ Ag2' .2 (37 8C. 2.0 6.0 Á/8.5 Á/6.5 (24 h) 5.0 61.19 mg ml (1 3.0/ Á/ 5.0 Á/9.5 g l (1).0 ((/Ca) 5.75 A.13.16 mg ml (1 Higher thermal stability than commercial Takaamylase Km (0. Pb2' .0 4.25/5. Al3' .0 Á/ Á/ Á/ Á/ [181. (/Ca) 30 Á/40 8C/ Á/ 60 Á/70 8C/ Á/ Á/ NaF and MgSO4 stimulation Á/ Km 0/7. '/Ca) 50 8C (30 min.9/5. flavus A. usamii Á/ Á/ 54.0/2. oryzae Á/ Á/ Á/ Á//40 8C (15 min) 50 8C/ B/60 8C 60 8C/65 8C (10 min) 40 8C/55 8C (10 min) 35 Á/37 8C/ Á/ Á//60 8C (90 min.8 Á/6. ‘‘TakaAmylase A’’. 30 min) 5. Cu2' .67 [92] mM reducing sugar mg(1 protein min (1 Km 0/2.0 Ag ' . / Process Biochemistry 38 (2003) 1599 Á/1616 [164] Km 0/1 mg ml (1 [32] [165] [164] Á/ Á/ Acid stable [41] [166] [167 Á/ 171] [172] [173 Á/ 175] [164] [176] [177 Á/ 180] Ag ' . chevalieri NSPRI 105 A. maltose EDTA.0 Á/5. niger Á/ Á/ 4.0/ Á/ 5.8 Á/5. Zn2' .2 (over a year. awamori ATCC 22342 A.0/6.182] [183] A. 6.2 Á/ Á/ Á/ Á/ 3. 3.0 52. EDTA Ca2' .0 4. Gupta et al.8 Á/7.0/6.0/4.0 5. fumigatus A.13%) [28] A. 4. 2. hennebergi Blochweitz A.7.0 Á/8. niger ATCC 13469 Á/ A. oryzae A. DNP Ag ' .0 Á/8.0 6. Fe3' . Pb2' . oryzae M13 4.0/6.0 68.0 Á/5.12 mM ‘‘Taka Diastase’’. oryzae A.5 65. niger van Tieghem Á/ CFTRI 1105 A.5/ Á/ 45 8C/35 8C min) 40 8C/55 8C min) 50 8C/40 8C min) 40 8C/60 8C min) 50 8C/55 8C min) 50 8C/60 8C min) 50 8C/40 8C min) Á//60 8C (15 (60 (10 (60 (15 (10 (40 (15 min) Á/ Á/ Km (0.0/3. halides Á/ Á/ Á/ Á/ Á/ Ag ' .44 41.23 5. 10.0 58. halides Substrate Á/ PCMB Á/ Ca2' A.0 Á/5. 4.0 Á/10.0 Á/ Á/ 50. oryzae A. awamori A.5 Á/ 52. Vmax (108.0 5.19 mg ml (1 [163] R.5 Á/ 54.4/5.8 Á/7.0 50 8C/ 5/50 8C (min) Á/ Á/ [28] . flavus LINK Á/ 3. Cu2' . 10 8C).13%) Á/ Á/ 53. Hg2' Á/ Ca2' Km (0. Mg2' Substrate Á/ Á/ Ca2' Ca2' Á/ Ca2' Km 0/0.0 Á/9. Km 0/ 29. oryzae 245 (ATCC Á/ 9376) A. 5.4%. Hg2' .5/ Á/ 4.4/5.2.Temperature oplity tima/stability Inhibitors Stabilisers Additional properties Reference Fungi and yeast A. foetidus ATCC 10254 A. 5.0. Fe3' .0 ('/Ca). halides Substrate Hg2' .0 5. Hg2' . Cu2' .5 5.0 Á/6.0 Á/6.0 Á/ 56.

5 (1 h. p.chloromercuribenzoic acid.5 Á/5. Cd2' . end products.0/5.0 6. end product. G1.E. MoO2( . ATCC 46889 Saccharomyces cerevisiae Schwanniomyces alluvius UCD 5483 T. Zn2' .0/6. Pb2' .5 28. (5.845 mg ml (1).0 4. brevis HPD 31 Á/ B. G2 End products.1 mg).6/ Á/ 5. Cu2' . licheniformis Á/ B.0 Á/6. G5 Higher affinity for branched chain substrate. Al3' Á/ Mn2' . Mn2' .0 5. G6 Á/ End products.0 Á/7.5/5/7.0/7.6 Á/5. insensitive to Ca2' . licheniformis Á/ CUMC 305 licheniformis CUMC 305 B. Km (2. 80 8C) 5. glycerophosphate Á/ Ca2' R. (44 kJ mol (1. Zn2' Á/ Fusarium vasinfectum Atk L. Mn2' . G2.8 Á/ 8. G2 Á/ [186] Á/ End product.0 Á/9. G4 Á/ [184] Km (0.0 Á/ Á/ Á/ Á/ 48.0/6. sod. SO2( .364 mg ml (1). effect of EDTA reversed by Ca2' Km 0/14 mg ml (1. F ( . licheniformis NCIB Á/ 6346 B.3/4.8:7.0 pH optima/stabi. Fe2' .0:5.1/ Á/ [157] [101] B. Vmax (0. Cu2' . Ca2' Pb2' .4 Á/5. G4. G1.0/3. stearothermophilus 4. G2.0 6.0 5.0 Á/9. end product.A. subtilis B.0/6.0 Á/7. Ag2' Á/ Starch Raw starch digesting en[152] zyme. SO2( . 4 thiourea.5 Á/ 76. lanuginosus IISc 91 Trichoderma viride Á/ Á/ Á/ Á/ Á/ 69.1 61.S. G5 E.0/4. Co2' [4] [102] [159] [51] 1607 Cu2' . stearothermophilus MFF4 B. stearothermophilus ATCC 12980 B.0/ Á/ 6.274 mg ml (1). Cd2' .1 h at 80 8C 50 8C/ 5/50 8C 60 8C/60 8C (5 min) Cd2' . sodium iodoacetate. G3. G3.0/4.5 Á/6.0 45 8C/45 8C (10 min. Ca2' Mg2' . Ni2' . 3 4 3 4 WO2( . S2O2( .0 (1 h) Cu2' . Km (1. subtilis 65 8. G1. Gupta et al.0/ Á/ 50 Á/60 8C/90 8C (CaCl2) 45 Á/50 8C/50 8C (30 min) 70 8C/ Á/ Hg2' .5/4. G1 A.0 54. Mn2' . Zn2' .0 9. azide.0 4. EDTA . Ca2' .2 6. Fe3' . cysteine. extremely resistant to heat inactivation.8 g l (1). Ag2' . Ag2' . denaturation by 6 M urea Á/ Hg2' . end [154] product.1)/105 J mol (1). G1.9 42. Al3' .0 70 Á/80 8C/(5 days) 70 8C or (45 min) 90 8C 70 Á/75 8C/half life 5. G5. EDTA Á/ EDTA Ca2' Á/ Á/ Ca2' Á/ Á/ Á/ Na2' . Fe3' .0 9. kononenkoae CBS 5608 Á/ 3. B. enzyme active after acetone and ethanol treatment Ca2' enhances thermostability Km (3. '/Ca) 50 8C/ Á/ 40 8C/ 5/40 8C 65 8C/50 8C ( /7 h) Á//60 8C (10 min) 45 Á/55 8C/ Á/ 76 8C/ B/60 8C 90 8C/60 8C (3 h). (14 kcal). Cu2' .A.0 Á/11.82 62 Á/65 Á/ 7. G2.5 Á/7.Temperature oplity tima/stability Inhibitors Stabilisers Additional properties Reference Cryptococcus S-2 4. end products.8 59. G3.0/ Á/ 6. Ag2' .0 4. 100 8C (4 h) in presence of soluble starch 70 Á/90 8C/85 8C (1 h) 55 Á/70 8C/ Á/ Á/ Á/ Á/ Á/ Á/ Á/ Á/ Hg2' . Na2' . Cu2' . Al3' .0 Á/9. Zn2' DTT. Hg2' . b-mercaptoethanol.A.0 4. Zn2' . G2 [187] [85] [86] Bacteria B. glutathione. Kcat (622 s (1).5 Á/9. Vmax (585.0 Á/10. Co2' .0 68.8 Á/10. G3 [99] Paecilomyces sp.738 mg glucose ml (1 min(1 End products.Table 2 (Continued ) Source pI Molecular weight (kDa) 66.0 Á/7. / Process Biochemistry 38 (2003) 1599 Á/1616 [185] [153] Km (0.5 5.5 [17] mg ml (1). Hg2' .0 Á/5.0/7.0 Á/ Á/ 22. E.

guanidine hydrochloride 65 8C/ Á/ Á/ Ca2' [71] Á/ [162] .0 82 8C/90 Á/95 8C Starch.8 5.0/ Á/ 80 8C/80 8C (3 h).0 45. Amy 2-53. Kcat (2510 mmol reducing sugar mg(1 protein). meridiana DSM 5425 L. IMD 434 5. G3.0 pH optima/stabi.0/5.0/ Á/ 70 8C/ Á/ Fe3' . plantarum A6 Á/ Á/ 48.6/4.0 Á/8. G3. G2.0 7. G2. Hg2' .0 Á/7.9 6. 40 min) 55 8C/ Á/ 47 8C/40 8C (1 h) Á//50 8C (1 h) Á/ N -bromosuccinimide. 60 Á/ 65 8C/ Á/.0 Á/12. Iodoacetic acid.E.0/5. G1. Km (8. 7. G1. G4 End products.0/5. G2. N -bromosuccinic 90 8C (15 min) acid.5//3. WN 11 Á/ Bacillus sp. G3. G1.0 8. Hg2' .0 7.0 Á/ 5.5 55 8C/40 8C (pH 11 Á/12.6 Á/ 4.0 65 8C/ Á/ Micromonospora melanosporea M.5 77.9(1).23%) End product. Fe3' .0 Á/ B/8. 2679 8.0/6.0/5/7.8 Thermomonospora curvata 6.5 Á/7.5/ Á/ 60 8C/ Á/.A. thermostability dependent upon pH stability End products.5 Á/8.A. acetic acid. G4 Km (0. G4 End products. 65 8C/ Á/ Á/ Á/ [44] 42.0 6. iodine.2(3) T.2 60.5 Á/6. dimethyl aminobenzaldehyde Á/ Á/ Á/ Á/ Hg2' .0 Á/9.0/7. G2.5/5.5 Á/8. XAL 601 Á/ Amy 176. G2.0 50 8C/ B/70 8C 37 8C/ Á/ Hg2' .88 mg ml (1).0 Á/8. IMD 435 Bacillus sp. low affinity for G3 [188] [45] [47] [189] R.2 mM) End products. DTT Bacillus sp. (25 kJ mol (1).0 and ]/ 90 8C 7.5 45. Gupta et al.38 g l (1). starch increases temperature stability End products. (13 400 and 5200 cal mol (1. G2. G1.2 6. A. licheniformis M27 Á/ Bacillus sp.1608 Table 2 (Continued ) Source pI Molecular weight (kDa) 56. showed activity in 30% salts Km (2.9 Á/9.9 mm) Half-life increases to 110 8C in presence of 20% (w/v) substrate No requirement for Ca2' .5 5.0/ Á/ 7.0 12.5 Á/ 85 Á/90 8C/ / 9. Cu2' Á/ E. G3. G2.0 Á/9.0 5.0 65 8C/40 8C (1 h) Á/ Ca2' Á/ N -Bromosuccinimide. melanosporea Pseudomonas stutzeri Streptococcus bovis JB1 7.5 6. WN 11 Á/ Á/ 5.0 Á/6.6 7. G1 E.0 Á/ 50.0 and 8. end product.6 5.0 (1 h) 75 Á/80 8C/80 8C (4 h) 9. Ca2' Bacillus sp. (30.0 and 6. SDS. p -chloromercuribenzoic acid (both reversible by DTT) Á/ Ca2' Á/ [191] [191] [93] [161] Streptomyces sp.0. p -hydroxymercuribenzoic acid Á/ Á/ Cysteine. Km (0. / Process Biochemistry 38 (2003) 1599 Á/1616 [100] [190] Á/ Á/ [87] Escherichia coli H. G3. Co2' Ca2' [159] [46] [160] 5. end products.9 kJ mol (1) Á/ End product.0/ Á/ 75 Á/80 8C/ Á/ Á/ Á/ Bacillus sp. specificity for raw starch. IMD 8.5 Á/9.Temperature oplity tima/stability Inhibitors Stabilisers Additional properties Reference B. G2 and G4 Á/ End products. US 100 Á/ Á/ 5.5/5/7. Km ( 1.7(2).5 Á/ 6. G3.0/4.0 69. G4 Adsorbs to raw starch or cellulose hydrolysis products. profundus DT5432 Á/ 47. Al3' Á/ Mn2' .9 63.

R. glucose.0 Á/8. enzymes such as malt and microbial a-amylases have been widely used in the baking industry [120.3 mg ml (1). microbial amylases have completely replaced chemical hydrolysis in the starch processing industry. pentosanases. 8.0 Á/ T. amylases have the major world market share of enzymes [118].A. They can also be of potential use in the pharmaceutical and fine chemical industries. E. Inhibitors B. oryzae EI 212 is inactivated in the presence of Ca2'. G3. These enzymes were used in bread and rolls to give these products a higher volume. Various applications of a-amylase are dealt here in brief. Industrial applications of a-amylase Amylases are among the most important hydrolytic enzymes for all starch based industries. caldolyticus amylase [113]. E. curvata Á/ pH optima/stabi.0/ Á/ 60 8C/ B/65 8C Á/ . 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]. It is the malt preparation that has led the way and opened the opportunities for many enzymes to be used commercially in baking. A comprehensive account on commercial applications of a-amylases is quoted by Godfrey and West [119]. Several different amylase preparations are available with various enzyme manufacturers for specific use in varied industries. for the hydrolysis of starch.5 Á/6. maltotetraose. a-amylases are much more thermostable than without it [4.A. kJ.Temperature oplity tima/stability Molecular weight (kDa) pI Table 2 (Continued ) T. 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. Today. G2. kilo joules. (59 kJ mol (1) Additional properties 8. G5. maltopentaose. maltose. Km [155] (0. textiles and in paper industry. / Process Biochemistry 38 (2003) 1599 Á/1616 Reference 1609 G1. In the present day scenario. For decades. pullulanases. kilo calories.121]. enzyme activation energy. many enzyme preparations such as proteases.0 6. In other systems usually one Ca2' ion is sufficient to stabilise the enzyme. cellullases. a-Amylase from A. G5. EDTA inactivated TAA can be reactivated by Sr2'. lipoxygenases etc. G4.115]. detergents. G6. Gupta et al.A. as a pharmaceutical aid for the treatment of digestive disorders. [70] Km (3.S. 62. In this light. End products. In the presence of Ca2'. and the commercialisation of amylases is oldest with first use in 1984. Ca2' Stabilisers amylase A (TAA) contains ten Ca2' ions but only one is tightly bound [112].. Starch. kcal.1. Today. fusca YX Source Á/ Á/ 5. Mg2' and Ba2' [114]. xylanases. but retains activity after EDTA treatment [116]. better colour and a softer crumb. Some studies have been carried out on the ability of other ions to replace Ca2' as Sr2' in B. Calcium free enzymes can be reactivated by adding Ca2' ions. G4. G4. glucose oxidases.3 mg ml (1) End products. Ca2' can be removed from amylases by dialysis against EDTA or by electrodialysis. maltotriose. amylases find application in all the industrial processes such as in food.0/activated 65 8C/ Á/ at pH 7. There are also reports where Ca2' did not have any effect on the enzyme [117]. G3. lipases.

1610 R. Additives include chemicals. A number of amylases exist for use in the paper . The conditions depend upon the source of starch and the a-amylase used [143]. 8.121 Á/123].4. Starch is converted into high fructose corn syrups (HFCS). 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. Upon storage the crumb becomes dry and firm. which improves the taste. Paper industry The use of a-amylase for the production of low viscosity.140]. be prevented from breaking. the a-amylases used in baking have been cereal enzymes from barley malt and microbial enzymes from fungi and bacteria [124.2. Starch is a very attractive size. a-amylase with ‘ITS’ property has been reported from only a few microorganisms [99. sugar esters.99. 8. All these undesirable changes in the bread are together known as staling. which selectively remove the size and do not attack the fibres.131].125. Good desizing of starch sized textiles is achieved by the application of a-amylases. the dextrin with 4Á/9 degree of polymerisation produced by these shows the anti-staling properties. high molecular weight starch for coating of paper is reported [142].g. The size enhances the stiffness and strength in paper. sizing of paper is performed to protect the paper against mechanical damage during processing. small sugars. The use of enzyme in starch liquefaction is well established and has been extensively reviewed [2.123. The materials that are used for this size layer are quite different.124. therefore. milk powder. branching enzymes [132] and debranching enzymes [133]. but also generates additional sugar in the dough.137]. It also improves the quality of the finished paper. For this purpose a removable protective layer is applied to the threads. e. a recent trend is to use intermediate temperature stable (ITS) a-amylases [13. the crust loses its crispness and the flavour of the bread deteriorates. sugars/salts [129]. They are active after starch gelatinisation and become inactive much before the completion of the baking process. Although a wide variety of microbial aamylases is known. Because of their high sweetening property. Textile desizing Modern production processes for textiles introduce a considerable strain on the warp during weaving. enzymes/their combinations. One of the new applications of a-amylase in the industry has been in retarding the staling of baked products. Supplementation of flour with exogenous fungal a-amylase having higher activities is common in the present day modern and continuous baking process [126]. lecithin. emulsifiers. crust colour and toasting qualities of the bread [127]. and it can be removed quite easily. The yarn must. The importance of retrogradation of starch fraction in bread staling has been emphasised [128]. As for textiles. Till date. Recently emphasis has been given to the use of enzymes in dough improvement/as anti-staling agents. The mill also has the flexibility of varying the starch viscosity for different paper grades. maltogenic amylases [134]. b-amylases [135] amyloglucosidases [136]. The use of a-amylases in warp sizing of textile fibres for manufacturing fibres with great strength has been reported [141]. granulated fat. The process requires the use of a highly thermostable a-amylase for starch liquefaction. Starch liquefaction and saccharification The major market for a-amylases lies in the production of starch hydrolysates such as glucose and fructose. A constant viscosity of the starch is required for reproducible results at this stage. However. a slight excess of a-amylases was also used which is undesirable as it causes stickiness in bread [134]. Therefore. Pullulanases and a-amylase combination are used for efficient antistaling property [133]. / Process Biochemistry 38 (2003) 1599 Á/1616 are being used in the bread industry for varied purposes [13. The temperature of this process lies in the range of 45 Á/60 8C. Further. It also randomly cleaves the starch into dextrins that are water soluble and can be removed by washing. these are used in huge quantities in the beverage industry as sweeteners for soft drinks. Conventionally various additives are used to prevent staling and improve the texture and flavour of baked products. but none had been able to replace aamylases. Starch is added to the paper in the size press and paper picks up the starch by passing through two rollers that transfer the starch slurry. anti-oxidant (ascorbic acid or potassium borate).125].138. because it is cheap. Presently they are used all over the world to different extents.139].3. Gupta et al. easily available in most regions of the world. A loss of more than US $1 billion is incurred in USA alone every year due to the staling of bread. 8. monoglycerides/diglycerides. Starch is also a good sizing agent for the finishing of paper. 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]. It also improves the erasibilty and is a good coating for the paper. a-amylase [130. etc. which reduces the shelf life of these products. a-Amylase supplementation in flour not only enhances the rate of fermentation and reduces the viscosity of dough (resulting in improvements in the volume and texture of the product. The use of amylases in the pulp and paper industry is in the modification of starches for coated paper.

24:329 Á/418. References [1] Windish WW. Starch and its derivatives. editor.5. which involved the application of amylase was also developed [96]. USA). the commercial production of this enzyme has been limited to only a few selected strains of fungi and bacteria. amylases are among the most important enzymes used in industrial processes. Soccol VT. 9. San Diego: California Academic Press. 90% of all liquid detergents contain a-amylase [145] and the demand for a-amylases for automatic dishwashing detergents is growing. They independently replaced oxidation sensitive amino acids with other amino acids. Detergent applications Enzymes now comprise as one of the ingredients of modern compact detergents. 1968:203 Á/46. 8. [5] Lonsane BK. 35. the spectrum of amylase applications has expanded into many other fields. Szeitli J. which include Amizyme† (PMP Fermentation Products. vol. Fungamyl. The application of a liquid stable reagent. [3] Fogarty WM. wherein fungal a-amylases are preferred over other microbial sources due to their more accepted GRAS status. most wild-type a-amylases are sensitive to oxidants which are generally a component of detergent formulations. In: Wiseman A. Stability against oxidants in household detergents was achieved by utilising successful strategies followed with other enzymes such as protease. In: Wayne WU. There are several processes in the medicinal and clinical areas that involve the application of amylases. Analysis in medicinal and clinical chemistry With the advent of new frontiers in biotechnology. Conclusions As evident from the foregoing review. the use of amylases. Bacteriol Rev 1977. Mhatre NS. Denmark) and a-amylase G9995† (Enzyme Biosystems. Enzymes also allow lowering of washing temperatures. One of the limitations of aamylases in detergents is that the enzyme shows sensitivity to calcium and stability is severely compromised in a low calcium environment. a-Amylases have been used in powder laundry detergents since 1975. This improved oxidation stability resulted in better storage stability and performance of the mutant enzyme in the bleach containing detergent formulations. Nowadays. 3. Chapman & Hall. In addition. The reaction of starch with iodine. In: Advances in applied microbiology. Biosensors with an electrolyte isolator semiconductor capacitor (EIS-CAP) transducer for process monitoring were also developed [150]. BAN† (Novozymes. a-amylases in particular. editor. 4th ed. This forced the detergent industries to search for milder and more efficient solutions [144]. Developments in microbial extracellular enzymes. [7] Hollo J. based on a-amylase for the Ciba Corning Express clinical chemistry system has been described [149]. 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