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

com/locate/procbio

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

1600

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

lases at length. a-Amylases are one of the most popular and important form of industrial amylases and the present review highlights the various aspects of microbial a-amylases.

2. Distribution of a-amylase among microorganisms a-Amylases are universally distributed throughout the animal, plant and microbial kingdoms. Over the past few decades, considerable research has been undertaken with the extracellular a-amylase being produced by a wide variety of microorganisms [1 /5]. The major advantage of using microorganisms for the production of amylases is the economical bulk production capacity and microbes are easy to manipulate to obtain enzymes of desired characteristics [5]. a-Amylase has been derived from several fungi, yeasts, bacteria and actinomycetes, however, enzymes from fungal and bacterial sources have dominated applications in industrial sectors [2].

peptone, corn steep liquor (CSL), etc. and thiol compounds with starch iodine complex. Copper sulphate and hydrogen peroxide protect the starch/iodine colour in the case of interference by these media components [9]. Further, zinc sulphate was found to be best for counteracting the interference of various metal ions. Various workers [10,11] have successfully used the original assay procedure in combination with flow injection analysis (FIA). The flow system comprised of an injection valve, a peristaltic pump, a photometer with a flow cell and 570 nm filter and a pen recorder. Samples are allowed to react with starch in a coil before iodine was added. Absorbance is then read at 570 nm. This method has many advantages including high sampling rates, fast response, flexibility and simple apparatus. 3.1.2. Sandstedt Kneen and Blish (SKB) method The SKB method [12], is one of the most widely adopted methods for determination of amylases used in the baking industry. The potency of most commercial amylases is described in terms of SKB [12] units. This method is used generally to express the diastatic strength of the malt and not for expressing a-amylase activity alone [13]. 3.1.3. Indian pharmacopoeia method As described in the Indian pharmacopoeia, this method is used to calculate a-amylase activity in terms of grams of starch digested by a given volume of enzyme [14]. This procedure involves incubation of the enzyme preparation in a range of dilutions in buffered starch substrate at 40 8C for 1 h. The solutions are then treated with iodine solution. The tube, which does not show any blue colour, is then used to calculate activity in terms of grams of starch digested. This method is usually employed for estimating a-amylase activity in cereals. 3.2. Increase in reducing sugars or dinitrosalicyclic acid (DNSA) method This method determines the increase in reducing sugars as a result of amylase action on starch [15]. The major defect in this assay is a slow loss in colour produced and destruction of glucose by constituents of the DNSA reagent. To overcome these limitations, a modified method for the estimation of reducing sugars was developed [16]. Rochelle salts were excluded and 0.05% sodium sulphate was added to prevent the oxidation of the reagent. Since then the modified method has been used extensively to measure reducing sugars without any further modifications in the procedure. Alternate methods, which also rely on the estimation of the reducing sugars are also, employed [17].

3. Determination of a-amylase activity a-Amylases are generally assayed using soluble starch or modified starch as the substrate. a-Amylase catalyses the hydrolysis of a-1,4 glycosidic linkages in starch to produce glucose, dextrins and limit dextrins. The reaction is monitored by an increase in the reducing sugar levels or decrease in the iodine colour of the treated substrate. Various methods are available for the determination of a-amylase activity [6]. These are based on decrease in starch/iodine colour intensity, increase in reducing sugars, degradation of colour-complexed substrate and decrease in viscosity of the starch suspension. 3.1. Decrease in starch /iodine colour intensity Starch forms a deep blue complex with iodine [7] and with progressive hydrolysis of the starch, it changes to red brown. Several procedures have been described for the quantitative determination of amylase based on this property. This method determines the dextrinising activity of a-amylase in terms of decrease in the iodine colour reaction. 3.1.1. Determination of dextrinising activity The dextrinising activity of a-amylases employs soluble starch as substrate and after terminating the reaction with dilute HCl, iodine solution is added. The decrease in absorbance at 620 nm is then measured against a substrate control. One percent decline in absorbance is considered as one unit of enzyme [8]. The major limitation of this assay is interference of media components including Luria broth, tryptone,

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

1601

3.3. Degradation of colour-complexed substrate For some years, groups have been working on the development of a specific a-amylase determination method based on the use of new types of substrates. These methods employ starch covalently complexed with blue dye such as Remazol brilliant Blue R [18] or Cibacron Blue F3 G-A [19] as an alternative substrate. The synthesis of these substrates involves two major steps. Soluble starch is coloured under alkaline conditions using the dye. This is the result of formation of covalent bonds between starch and dye molecules. The coloured starch is subsequently cross-linked by the addition of 1,4-butanediol diglycide ether. This gives an insoluble network, which swells in water. The enzymic hydrolysis of such insoluble starch derivatives yields soluble starch hydrolysates carrying the coloured marker. This method is simple and sensitive for aamylase determination, but even minute quantities of glucose might lead to erroneous results due to starch contamination by dextrin substrate [19]. Recently, a rapid and sensitive microassay based on dye cross linked starch for a-amylase detection has been reported. It can successfully detect as low as 0 /50 ng of enzyme [20]. Other novel substrates such as nitrophenyl derivatives of maltosaccharides have also been employed. The assay measures the release of free p -nitrophenyl groups. The use of nitrophenyl-maltosaccharides in conjunction with a specific yeast a-glucosidase can be used but these substrates are rapidly cleaved by glucoamylases commonly present in the culture broths. The use of nonreducing end blocked p-nitrophenyl maltoheptoside (BPNPG7) has also been described [21]. The blocking group (4,6-O -benzylidene) prevents the hydrolysis of the substrate by the exo-acting enzymes and is thus specific for a-amylase. The assay is simple, reliable and accurate but is expensive as it involves the use of a synthetic substrate and specific enzymes. Thus the use of this method is restricted only to very specific tests and not for routine analysis. A comparison was made for the use of end blocked p -nitrophenyl maltoheptoside (BPNPG7) with a number of accepted procedures that employ starch as the substrate. The reaction was monitored using the starch /iodine colour [21]. There was an excellent correlation between each of the assay procedures employed. This indicates that all the methods give an accurate and reliable measure of a-amylase activity and can be used as per the requirement. Both these methods are commercially available as commercial kits, however, it is found that a-amylases exhibit lower affinity for low molecular weight substrates [18]. 3.4. Decrease in viscosity of the starch suspension These methods are generally used in the bakery industry to assess the quality of the flour and not for

estimating a-amylase activity which are based on the determination of the rheological properties of the dough. Methods, which fall into this category, are the falling number test and the Amylograph or Farinograph test. 3.4.1. Falling number (FN) method The falling number (FN) method, internationally standardised [22 /24] is accepted for assessing cereal aamylase activity in flour /enzyme preparations at 100 8C. Both cereal and fungal a-amylases are used to improve the fermentation of flour deficient in amylase activities. Because fungal a-amylases have low thermostability, they cannot be detected by the standard FN method at 100 8C [25]. This method has been modified and standardised [25] for measuring both cereal and fungal a-amylase activity at 300 8C, by replacing a part of the flour with pre-gelatinised starch. A falling number of about 400 indicates a normally malted flour. 3.4.2. Amylograph/Farinograph test The milling and baking industries generally assess the diastatic activity of flours by means of an amylograph. This method is also based on the relationship of peak viscosity of starch slurry and the enzyme activity level [23]. The higher the enzyme activity, the thinner is the hot paste viscosity. When the amylograph is used, values of 400 /600 Brabender units of the Farinograph are considered optimal for bread baking flours (higher values indicate a lack and lower values indicate an excess of activity).

4. Physiology of a-amylase production The production of a-amylase by submerged fermentation (SmF) and solid state fermentation (SSF) has been thoroughly investigated and is affected by a variety of physicochemical factors. Most notable among these are the composition of the growth medium, pH of the medium, phosphate concentration, inoculum age, temperature, aeration, carbon source and nitrogen source [5,26]. Most reports among fungi have been limited to a few species of mesophilic fungi where attempts have been made to specify the cultural conditions and to select superior strains of the fungus to produce on a commercial scale [2 /4]. 4.1. Physiochemical parameters The role of various physico-chemical parameters, including carbon and nitrogen source, surface acting agents, phosphate, metal ions, temperature, pH and agitation have been studied.

1602

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

4.1.1. Substrate source: induction of a-amylase a-Amylase is an inducible enzyme and is generally induced in the presence of starch or its hydrolytic product, maltose [27 /30]. Most reports available on the induction of a-amylase in different strains of Aspergillus oryzae suggest that the general inducer molecule is maltose. There is a report of a 20-fold increase in enzyme activity when maltose and starch were used as inducers in A. oryzae (NRC 401013) [31]. Similarly strong a-amylase induction by starch and maltose in the case of A. oryzae DSM 63303 has been reported [29]. Apart from maltose, in some strains, other carbon sources as lactose, trehalose, a-methyl-D-glycoside also served as inducers of a-amylase [28]. Not only the carbon source, but also the mycelial condition/age affect the synthesis of a-amylase by A. oryzae M-13 [28]. There are reports that 5 days starved non-growing mycelia were the most appropriate for optimal induction by maltose. a-Amylase production is also subjected to catabolite repression by glucose and other sugars, like most other inducible enzymes [30,32]. However, the role of glucose in the production of a-amylase in certain cases is controversial. a-Amylase production by A. oryzae DSM 63303 was not repressed by glucose rather; a minimal level of the enzyme was induced in its presence [29]. However, xylose or fructose have been classified as strongly repressive although they supported good growth in Aspergillus nidulans [33]. The carbon sources as glucose and maltose have been utilised for the production of a-amylase. However, the use of starch remains promising and ubiquitous. A number of other non-conventional substrates as lactose [34], casitone [35,36], fructose [37], oilseed cakes [38] and starch processing waste water [39] have also been used for the production of a-amylase while the agro-processing byproduct, wheat bran has been used for the economic production of a-amylase by SSF [5]. The use of wheat bran in liquid surface fermentation (LSF) for the production of a-amylase from Aspergillus fumigatus and from Clavatia gigantea, respectively, has also been reported [40,41]. High a-amylase activities from A. fumigatus have also been reported using a-methyl-Dglycoside (a synthetic analogue of maltose) as substrate [42]. Use of low molecular weight dextran in combination with either Tween 80 or Triton X-100 for a-amylase production in the thermophilic fungus Thermomyces lanuginosus (ATCC 200065) has been reported [43]. Triton X-100 had no effect, whereas Tween 80 increases the a-amylase activity 27-fold. 4.1.2. Nitrogen sources Organic nitrogen sources have been preferred for the production of a-amylase. Yeast extract has been used in the production of a-amylase from Streptomyces sp. [44], Bacillus sp. IMD 435 [45] and Halomonas meridiana

[46]. Yeast extract has also been used in conjunction with other nitrogen sources such as bactopeptone in the case of Bacillus sp. IMD 434 [47], ammonium sulphate in the case of Bacillus subtilis [48], ammonium sulphate and casein for C. gigantea [40] and soybean flour and meat extract for A. oryzae [49]. Yeast extract increased the productivity of a-amylase by 110/156% in A. oryzae when used as an additional nitrogen source than when ammonia was used as a sole source [50]. Various other organic nitrogen sources have also been reported to support maximum a-amylase production by various bacteria and fungi. However, organic nitrogen sources viz. beef extract, peptone and com steep liquor supported maximum a-amylase production by bacterial strains [35,38,51 /54] soybean meal and casamino acids by A. oryzae [55]. CSL has also been used for the economical and efficient production of a-amylase from a mutant of B. subtilis [56]. Apart from this, various inorganic salts such as ammonium sulphate for A. oryzae [30] and A. nidulans [29], ammonium nitrate for A. oryzae [57] and Vogel salts for A. fumigatus [42] have been reported to support better a-amylase production in fungi. Amino acids in conjunction with vitamins have also been reported to affect a-amylase production. However, no conclusion can be drawn about the role of amino acids and vitamins in enhancing the a-amylase production in different microorganisms as the reports are highly variable. a-Amylase production by Bacillus amyloliquefaciens ATCC 23350 increased by a factor of 300 in the presence of glycine [58]. The effect of glycine was not only as a nitrogen source rather it affected a-amylase production by controlling pH and subsequently amylase production increased. b-Alanine, DL-nor valine and D-methionine were effective for the production of alkaline amylase by Bacillus sp. A-40-2. However, the role of amino compounds was considered to be neither as nitrogen nor as a carbon source, but as stimulators of amylase synthesis and excretion [59]. It has been reported that only asparagine gave good enzyme yields [57] while the importance of arginine for a-amylase production from B. subtilis has also been well documented [60].

4.1.3. Role of phosphate Phosphate plays an important regulatory role in the synthesis of primary and secondary metabolites in microorganisms [61,62] and likewise it affects the growth of the organism and production of a-amylase. A significant increase in enzyme production and conidiation in A. oryzae above 0.2 M phosphate levels has been reported [55]. Similar findings were corroborated in B. amyloliquefaciens where low levels of phosphate resulted in severely low cell density and no a-amylase production [63]. In contrast, high phosphate concentrations were

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

1603

inhibitory to enzyme production by B. amyloliquefaciens [58]. 4.1.4. Role of other ions K ', Na ', Fe2', Mn2', Mo2', Cl (, SO2( had no 4 effect while Ca2' was inhibitory to amylase production by A. oryzae EI 212 [57]. Mg2' played an important role and production was reduced to 50% when Mg2' was omitted from the medium. Na ' and Mg2' show coordinated stimulation of enzyme production by Bacillus sp. CRP strain [64]. Addition of zeolites to control ammonium ions in B. amyloliquefaciens resulted in increased yield of a-amylase [65]. An inverse relationship between a-amylase production and growth rate was observed for Streptomyces sp. in the presence and absence of Co2' [66], the presence of Co2' enhancing the final biomass levels by 13-fold, albeit with a reduction in enzyme yield. 4.1.5. pH Among the physical parameters, the pH of the growth medium plays an important role by inducing morphological change in the organism and in enzyme secretion. The pH change observed during the growth of the organism also affects product stability in the medium. Most of the Bacillus strains used commercially for the production of bacterial a-amylases by SmF have an optimum pH between 6.0 and 7.0 for growth and enzyme production. This is also true of strains used in the production of the enzyme by SSF. In most cases the pH used is not specified excepting pH 3.2 /4.2 in the case of A. oryzae DAE 1679 [39], 7.0 /8.0 in A. oryzae EI 212 [57] and 6.8 for B. amyloliquefaciens MIR-41 [67]. In fungal processes, the buffering capacity of some media constituents sometimes eliminates the need for pH control [68]. The pH values also serves as a valuable indicator of the initiation and end of enzyme synthesis [69]. It is reported that A. oryzae 557 accumulated aamylase in the mycelia when grown in phosphate or sulphate deficient medium and was released when the mycelia were replaced in a medium with alkaline pH (above 7.2) [28]. 4.1.6. Temperature The influence of temperature on amylase production is related to the growth of the organism. Among the fungi, most amylase production studies have been done with mesophilic fungi within the temperature range of 25 /37 8C. Optimum yields of a-amylase were achieved at 30 /37 8C for A. oryzae [55,57]. a-Amylase production has also been reported at 55 8C by the thermophilic fungus Thermomonospora fusca [70] and at 50 8C by T. lanuginosus [17]. a-Amylase has been produced at a much wider range of temperature among the bacteria. Continuous production of amylase from B. amyloliquefaciens at 36 8C has

been reported [67]. However, temperatures as high as 80 8C have been used for amylase production from the hyperthermophile Thermococcus profundus [71]. 4.1.7. Agitation Agitation intensity influences the mixing and oxygen transfer rates in many fungal fermentations and thus influences mycelial morphology and product formation [69,72/76]. It has been reported that a higher agitation speed is sometimes detrimental to mycelial growth and thus may decrease enzyme production. However, it is reported that the variations in mycelial morphology as a consequence of changes in agitation rate do not affect enzyme production at a constant specific growth rate [76]. Agitation intensities of up to 300 rpm have normally been employed for the production of amylase from various microorganisms as reported in the literature.

5. Fermentation studies on a-amylase production The effect of environmental conditions on the regulation of extracellular enzymes in batch cultures is well documented [77]. A lot of work on the morphology and physiology of a-amylase production by A. oryzae during batch cultivation has been done. Accordingly, morphology of A. oryzae was critically affected by the growth pH [78]. In a series of batch experiments, authors observed that at pH 3.0 /3.5, freely dispersed hyphal elements were formed. In the pH range 4/5, both pellets and freely dispersed hyphal fragments were observed whereas at pH higher than 6 pellets were the only growth forms recorded. Other groups [39,79] have recorded similar observations for other strains of A. oryzae . The optimum growth temperature was found to be 35 8C. It is demonstrated that when glucose was exhausted the biomass production stopped whereas the secretion of a-amylase increased rapidly [79]. One report states that inoculum quantity did not affect morphological changes in A. oryzae in air-lift bioreactors and that pellet size decreased considerably as the air velocity increased [39]. In the case of a-amylase production by Bacillus flavothermus in batch cultivation in a 20 l fermentor, a-amylase production and biomass peaked twice and highest activity was obtained after 24 h [34]. It was observed that the kinetics of enzyme synthesis was more of the growth associated than non-growth associated type [35]. Similar findings were cited in another report with B. amyloliquefaciens [63]. Continuous and fed-batch cultures have been recognised as most effective for the production of the enzyme [60]and several groups have studied the effectiveness of these cultures. The production of a-amylase from B. subtilis TN106 (pAT5) was enhanced substantially by extending batch cultivation with fed-batch operation

1604

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

[60]. The bulk enzyme activity was nearly 54% greater in a two-stage fed-batch operation at a feed rate of 31.65 ml h (1 of medium, than that attained in the single stage batch culture. The effects of controlled feeding of maltose at a feed rate of 1 /4 g h(1 for a-amylase and glucoamylase production from A. oryzae RIB 642 in a rotary draft tube fermentor (RTF) have been studied [49]. At a feed rate of 1 g h(1 the yields of a-amylase were twice than those obtained in batch cultures. When fed-batch cultivations were performed on a pilot scale RTF at a feed rate of 24 g h(1, the biomass and aamylase yields was higher than those obtained in a laboratory scale jar fermentor. A model to simulate the steady-state values for biomass yield, residual sugar concentration and specific rate of a-amylase production has been proposed which simulated experimental data very well [80]. Furthermore, it was found in chemostat experiments that the specific rate of a-amylase production decreased by up to 70% with increasing biomass concentration at a given dilution rate. Shifts in the dilution rate in continuous culture could be used to obtain different proportions of the enzymes, by the same strain [66]. It was further demonstrated that maximum production of a-amylase occurred in continuous culture at a dilution rate of 0.15 h(1 and amylase activity in the culture was low at dilution rates above 1.2 h (1. In contrast, in Bacillus sp. the switching of growth from batch to continuous cultivation resulted in the selection of a non a-amylase producing variant [63]. A decline in enzyme production was also accompanied by morphological and metabolic variations during continuous cultivation [81,82]. The industrial exploitation of SSF for enzyme production has been confined to processes involving fungi and it is generally believed that these techniques are not suitable for bacterial cultivation [5]. The use of SSF technique in a-amylase production and its specific advantages over other methods has been discussed extensively [5].

precipitation using ammonium sulphate or organic solvents such as chilled acetone. The crude enzyme is then subjected to chromatography, usually affinity, ion exchange and/or gel filtration. A number of reviews are available on purification and characterisation of aamylases from a range of microorganisms [1,2,4,26,83]. Table 1 summarises various purification strategies adopted for microbial a-amylases.

7. Biochemical properties of a-amylases The enzymic and physicochemical properties of aamylases from several microorganisms have been extensively studied and described [2 /4,83]. A summary is presented in Table 2. 7.1. Substrate specicity As holds true for the other enzymes, the substrate specificity of a-amylase varies from microorganism to microorganism. In general, a-amylases display highest specificity towards starch followed by amylose, amylopectin, cyclodextrin, glycogen and maltotriose. 7.2. pH optima and stability The pH optima of a-amylases vary from 2 to 12 [4]. aAmylases from most bacteria and fungi have pH optima in the acidic to neutral range [2]. a-Amylase from Alicyclobacillus acidocaldarius showed an acidic pH optima of 3 [84], in contrast to the alkaline amylase with optima of pH 9 /10.5 reported from an alkalophilic Bacillus sp. [85 /88]. Extremely alkalophilic a-amylase with pH optima of 11 /12 has been reported from Bacillus sp. GM8901 [89]. In some cases, the pH optimum was observed to be dependent upon temperature as in the case of Bacillus stearothermophilus DONK BS-1 [90] and on calcium as in the case of B. stearothermophilus [91]. a-Amylases are generally stable over a wide range of pH from 4 to 11 [3,4,45,47,85,92], however, a-amylases with stability in a narrow range have also been reported [46,86,93]. 7.3. Temperature optima and stability The temperature optimum for the activity of aamylase is related to the growth of the microorganism [4]. The lowest temperature optimum is reported to be 25 /30 8C for F. oxysporum amylase [94] and the highest of 100 and 130 8C from archaebacteria, Pyrococcus furiosus and Pyrococcus woesei , respectively [95 /97]. Temperature optima of enzymes from Micrococcus varians are calcium dependent [98] and that from H. meridiana is sodium chloride dependent [46].

6. Purication of microbial a-amylases Industrial enzymes produced in bulk generally require little downstream processing and hence are relatively crude preparations. The commercial use of a-amylase generally does not require purification of the enzyme, but enzyme applications in pharmaceutical and clinical sectors require high purity amylases. The enzyme in purified form is also a prerequisite in studies of structure /function relationships and biochemical properties. The purification of a-amylases from microbial sources in most cases has involved classical purification methods. These methods involve separation of the culture from the fermentation broth, selective concentration by

R. Gupta et al. / Process Biochemistry 38 (2003) 1599 /1616 Table 1 Purication strategies employed for a-amylase Microorganism Purification strategy Fold purification/ yield (%)

1605

Reference

Fungi and yeast A. oryzae NRC 401013 A. flavus LINK Cryptococcus sp. S-2 L. kononenkoae CBS5608 Saccharomyces cerevisiae YPB-G Schwanniomyces alluvius UCD-54-83 Thermomonospora curvata T. lanuginosus T. lanuginosus IISc91 Bacteria Bacillus sp. IMD435 Bacillus sp. IMD 434 Bacillus sp. WN 11 B. licheniformis CUMC 305 B. licheniformis NCIB 6346 B. stearothermophilus ATCC 12980 B. subtilis B. subtilis B. subtilis 65 Lactobacillus plantarum A6 Pseudomonas stutzeri Streptococcus bovis JB1 Thermomonospora curvata NCIMB 10081 T. profundus DT5432

DE52-Cellulose (pH 7.0), 70% (NH4)2SO4, Sephacryl S300, 70% (NH4)2SO4, DE52-Cellulose (pH 7.0) 50 /90% (NH4)2SO4, DEAE-Sephadex A50 (pH 6.5) Ultrafiltration, a-Cyclodextrin coupled with Sepharose 6B (pH 7.0) 60% (NH4)2SO4, crosslinked starch (pH 8.5), DEAE Bio-Gel A (pH 5.5) Ultrafiltration, b-Cyclodextrin linked Sepharose 6B (Epoxy activated, pH 4.5), Sephadex G-100 (pH 4.5) Ultrafiltration, DEAE-sephacel (pH 5.6), Sephadex G-150 (pH 5.6)

[31] 13.8/70 140/78 6000/52 5/2 10.8/17.1 [92] [152] [99] [153] [154] [155] [156] [17]

Ultrafiltration, 75% ethanol precipitation, Sephadex G-150 (pH 8.0), DEAE 66/9 Cellulose, ultrafiltration Ultrafiltration, DEAE-Trisacryl (pH 7.0), Phenyl-Sepharose (pH 7.0) Ultrafiltration, DEAE-Sephadex A50 (pH 5.0), ultrogel AcA54, DEAE-Sephadex 112/41 A50 (pH 8.0), Bio-Gel P-30 a-Cyclodextrin coupled Sepharose 6B (pH 6.0) Acetone precipitation, Resource Q (pH 7.0), Phenyl Sepharose CL-4B (pH 7.8) 60% (NH4)2SO4, DEAE Sepharose (pH 5.3), Sephadex G-75 65% (NH4)2S04, CM-Cellulose (pH 6.4) DEAE-Cellulose DE52 (pH 5.3) Adsorption on soluble starch (1%) in 10% (NH4)2SO4, washing with Aces (pH 7.5) and 10% (NH4)2SO4, DEAE chromatography (Zetaprep disk), ultrafiltration 60% (NH4)2SO4, Sephacryl-S200 HR (pH 8.0), 60% (NH4)2SO4, S-Sepharose Ultrafiltration Sephacryl S-300, CM Sephadex C-50 Ultrafiltration, 50 /80% (NH4)2SO4, ultrafiltration, DEAE-Cellulose Concentrated by drum humidifier, 25% (NH4)2SO4, 70% acetone 70% (NH4)2SO4, Sephadex G-25 (pH 7.5), Mono Q 85% (NH4)2S04, ultrafiltration, gel filtration (pH 6.0), DEAE-Sephacel (pH 8.O) 744/65 266/ / Amy I 65/13, Amy II 40.7/9.5 212/42 33/66 / 9/17 2.5/ / 30.85/24.8 20/35 1.036/ / 6.9/50 300/ /

[45] [47] [100] [86] [157] [158] [159] [83] [51] [160] [93] [161] [162] [71]

80% (NH4)2SO4, DEAE-Toyopearl 650 M (pH 7.5), Superdex 200 HR (pH 7.5) 816/26

Thermostabilities have not been estimated defactor in many studies. Thermostabilities as high as 4 h at 100 8C have been reported for Bacillus licheniformis CUMC 305 [86]. Many factors affect thermostability. These include the presence of calcium, substrate and other stabilisers [4]. The stabilising effect of starch was observed in a-amylases from B. licheniformis CUMC 305 [85], Lipomyces kononenkoae [98] and Bacillus sp. WN 11 [100]. Thermal stabilisation of the enzyme in the presence of calcium has also been reported from time to time [100 /102]. 7.4. Molecular weight Molecular weights of a-amylases vary from about 10 to 210 kDa. The lowest value, 10 kDa for Bacillus caldolyticus [103] and the highest of 210 kDa for Chloroflexus aurantiacus has been reported [104]. Molecular weights of microbial a-amylases are usually 50/ 60 kDa as shown directly by analysis of cloned aamylase genes and deduced amino acid sequences [4].

Carbohydrate moieties raise the molecular weight of some a-amylases. Glycoproteins have been detected in A. oryzae [105,106], L . kononenkoae [98], B. stearothermophilus [107] and B. subtilis strains [108,109]. Glycosylation of bacterial proteins is rare. A carbohydrate content as high as 56% has been reported in S. castelii [110] whereas this is about 10% for other a-amylases [4]. 7.5. Inhibitors Many metal cations, especially heavy metal ions, sulphydryl group reagents, N -bromosuccinimide, 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' to a-amylase is much stronger than that of other ions. The amount of bound calcium varies from one to ten. Crystalline Taka-

1606

Table 2 Properties of some microbial amylases Source pI Molecular weight (kDa) pH optima/stabi- Temperature oplity tima/stability Inhibitors Stabilisers Additional properties Reference

Fungi and yeast A. oryzae A. flavus LINK

/ 3.5

/ 52.5

65.4/5.0 /9.0 6.0/6.0 /10.0

50 8C/50 8C (30 min) 55 8C/50 8C (1 h)

/ Ag2' , Hg2'

/ Ca2'

Km (0.13%)

[28]

A. foetidus ATCC 10254 A. awamori A. awamori ATCC 22342 A. chevalieri NSPRI 105 A. flavus A. fumigatus A. hennebergi Blochweitz A. niger

/ / 4.2 / / / / 3.44

41.5 / 54.0 68.0 / / 50.0 58.0 61.0 / 56.23

5.0/ / 5.0/6.0 /7.0 4.8 /5.0/3.5 /6.5 (24 h) 5.5/ / 5.25/5.0 /8.0 6.0/ / 5.5/ / 4.0 /5.0/2.2 /7.0 5.0 /6.0/5.0 /8.5 5.0/4.0 /6.0 5.0; 6.0/5.2 /6.0 ('/Ca); 5.8 /7.0 ((/Ca) 5.0/6.0 /8.0 4.8 /6.6/ / 5.0 /5.9/5.8 /7.2 (over a year, 10 8C); 5.0 /8.2 (37 8C, 30 min) 5.0 /6.0/ / 3.0 /5.5/ /

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) //60 8C (15

(60 (10 (60 (15 (10 (40 (15 min)

Km (0.5 g l (1); Vmax (108.67 [92] mM reducing sugar mg(1 protein min (1 Km 0/2.19 mg ml (1 [163] R. Gupta et al. / Process Biochemistry 38 (2003) 1599 /1616 [164] Km 0/1 mg ml (1 [32] [165] [164] / / Acid stable [41] [166] [167 / 171] [172] [173 / 175] [164] [176] [177 / 180]

Ag ' , Cu2' , Fe3' , Hg2' , halides Substrate Hg2' , Pb2' , maltose EDTA, DNP Ag ' , Cu2' , Hg2' , halides / / / / / Ag ' , Al3' , Cu2' , Hg2' , Pb2' , Zn2' , EDTA Ca2' , Mg2' Substrate / / Ca2' Ca2' / Ca2'

Km 0/0.19 mg ml (1

3.75 A. niger ATCC 13469 / A. niger van Tieghem / CFTRI 1105 A. oryzae A. oryzae A. oryzae / / /

//40 8C (15 min) 50 8C/ B/60 8C 60 8C/65 8C (10 min) 40 8C/55 8C (10 min) 35 /37 8C/ / //60 8C (90 min, '/Ca) 50 8C (30 min, (/Ca) 30 /40 8C/ / 60 /70 8C/ /

/ NaF and MgSO4 stimulation / Km 0/7.13, 4.35, 3.12 mM Taka Diastase, TakaAmylase A, Km 0/ 29, 2.4%, 4.7, 10.2, 2.4 mM Km 0/4.16 mg ml (1 Higher thermal stability than commercial Takaamylase Km (0.13%)

/ / 53.0

Ag ' , Cu2' , Fe3' , Hg2' , halides Substrate / PCMB / Ca2'

A. oryzae 245 (ATCC / 9376) A. usamii /

/ 54.0

/ /

/ /

[181,182] [183]

A. oryzae M13

4.0

52.0

5.4/5.0 /9.0

50 8C/ 5/50 8C (min)

[28]

Table 2 (Continued ) Source pI Molecular weight (kDa) 66.0 pH optima/stabi- Temperature oplity tima/stability Inhibitors Stabilisers Additional properties Reference

Cryptococcus S-2

4.2

6.0/ /

50 /60 8C/90 8C (CaCl2) 45 /50 8C/50 8C (30 min) 70 8C/ /

Hg2' , Ag2' , Cu2' , Zn2'

Fusarium vasinfectum Atk L. kononenkoae CBS 5608

/ 3.5

/ 76.0

4.4 /5.0:5.8:7.8 / 8.0/3.8 /10.0 4.5 /5.0/5.0 /7.0 (1 h)

Cu2' , Mn2' , Zn2' DTT, Cu2' , Ag2'

/ Starch

Raw starch digesting en[152] zyme; end products, G1, G2, G3, G4 / [184] Km (0.8 g l (1); Kcat (622 s (1); insensitive to Ca2' ; end products, G3, G4, G5, G6 / End products, G1, G2, G3 [99]

Paecilomyces sp. ATCC 46889 Saccharomyces cerevisiae Schwanniomyces alluvius UCD 5483 T. lanuginosus IISc 91 Trichoderma viride

/ / / / /

69.0 54.1 61.9 42.0 / / 22.5 28.0

4.0/4.0 /9.0 5.0/ / 6.3/4.5 /7.5 5.6/ / 5.0 /5.5/4.0 /7.0 6.0/4.5 /9.0 9.0/6.0 /11.0 9.0/7.0 /9.0

45 8C/45 8C (10 min, '/Ca) 50 8C/ / 40 8C/ 5/40 8C 65 8C/50 8C ( /7 h) //60 8C (10 min) 45 /55 8C/ / 76 8C/ B/60 8C 90 8C/60 8C (3 h), 100 8C (4 h) in presence of soluble starch 70 /90 8C/85 8C (1 h) 55 /70 8C/ /

/ / / / / / / Hg2' , Cu2' , Ni2' , Zn2' , Ag2' , Fe2' , Co2' , Cd2' , Al3' , Mn2' , p- chloromercuribenzoic acid, sodium iodoacetate, EDTA / EDTA

Ca2' / / Ca2' / / / Na2' , Ca2' Mg2' , azide, F ( , SO2( , SO2( , S2O2( , MoO2( , 3 4 3 4 WO2( , cysteine, glutathione, 4 thiourea, b-mercaptoethanol, sod. glycerophosphate / Ca2'

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

[185] [153]

Km (0.364 mg ml (1); end [154] product, G1 A.E. (44 kJ mol (1; Km (2.5 [17] mg ml (1); end product, G2 / [186] / End product, G5 E.A. (5.1)/105 J mol (1); Km (1.274 mg ml (1); Vmax (0.738 mg glucose ml (1 min(1 End products, G1, G2, G3, G5 Higher affinity for branched chain substrate; E.A. (14 kcal); extremely resistant to heat inactivation; effect of EDTA reversed by Ca2' Km 0/14 mg ml (1; enzyme active after acetone and ethanol treatment Ca2' enhances thermostability Km (3.845 mg ml (1); Vmax (585.1 mg); end product, G2 End products, G1, G2 [187] [85] [86]

Bacteria B. brevis HPD 31 / B. licheniformis / B. licheniformis / CUMC 305 licheniformis CUMC 305

B. licheniformis NCIB / 6346 B. stearothermophilus 4.82

62 /65 /

7.0/7.0 /10.0 4.6 /5.1/ /

[157] [101]

B. stearothermophilus ATCC 12980 B. stearothermophilus MFF4 B. subtilis B. subtilis 65

8.8

59.0

5.0 /6.0/6.0 /7.5 (1 h, 80 8C) 5.5 /6.0/ / 6.5/5/7.0 6.0/6.0 /9.0

/ / /

/ 48.0 68.0

70 /80 8C/(5 days) 70 8C or (45 min) 90 8C 70 /75 8C/half life 5.1 h at 80 8C 50 8C/ 5/50 8C 60 8C/60 8C (5 min)

Cd2' , Cu2' , Hg2' , Pb2' , Zn2' , Ca2' , Na2' , B.S.A. denaturation by 6 M urea / Hg2' , Fe3' , Al3' / Mn2' , Co2'

[4]

[102] [159] [51] 1607

Cu2' , Fe3' , Mn2' , Hg2' , Zn2' , Ca2' Pb2' , Al3' , Cd2' , Ag2' , EDTA

1608

Table 2 (Continued ) Source pI Molecular weight (kDa) 56.0 pH optima/stabi- Temperature oplity tima/stability Inhibitors Stabilisers Additional properties Reference

B. licheniformis M27

Bacillus sp. IMD 435 Bacillus sp. IMD 434

5.6 5.9

63.0 69.2

6.5 /7.0 and 8.5 / 85 /90 8C/ / 9.0/5/7.0 and ]/ 90 8C 7.5 6.0 and 6.5 / 6.0/4.0 /9.0 65 8C/40 8C (1 h)

Ca2'

/ N -Bromosuccinimide, p -hydroxymercuribenzoic 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

Bacillus sp. XAL 601

Amy 176.0, Amy 2-53.0 /

5.5/5.5 /9.0 (1 h) 75 /80 8C/80 8C (4 h) 9.0/ / 70 8C/ /

Fe3' , Hg2' , Cu2'

E.A. (25 kJ mol (1); thermostability dependent upon pH stability End products, G1, G2, G3, G4 End products, G1, G2; specificity for raw starch; Km ( 1.9 mm) Half-life increases to 110 8C in presence of 20% (w/v) substrate No requirement for Ca2' ; starch increases temperature stability End products, G1, G2, G3, G4 Adsorbs to raw starch or cellulose hydrolysis products, G2 and G4 / End products, G2, G3; showed activity in 30% salts Km (2.38 g l (1); A.E. (30.9 kJ mol (1) / End product, G1 E.A. (13 400 and 5200 cal mol (1; end product, G4 Km (0.88 mg ml (1); Kcat (2510 mmol reducing sugar mg(1 protein); end products, G2, G3, G4 End products, G1, G3; Km (8.0 /8.2 mM) End products, G2, G3; Km (0.23%) End product, G2; low affinity for G3

[188]

[45] [47]

[189] R. Gupta et al. / Process Biochemistry 38 (2003) 1599 /1616

[100]

[190]

[87]

Escherichia coli H. meridiana DSM 5425 L. plantarum A6

/ /

48.0 / 50.0

6.5/5/7.0 7.0/5.0 /7.0

50 8C/ B/70 8C 37 8C/ /

Hg2' , Fe3' , Al3' /

Mn2' , Co2' Ca2'

[159] [46] [160]

5.5//3.0 / B/8.0 65 8C/ /

Micromonospora melanosporea M. melanosporea Pseudomonas stutzeri Streptococcus bovis JB1

7.6 7.6 / 4.5

45.0 45.0 12.5 77.0

7.0/ / 7.0/6.0 /12.0 8.0/7.0 /9.5 5.0 /6.0/5.5 /8.5

55 8C/40 8C (pH 11 /12, 40 min) 55 8C/ / 47 8C/40 8C (1 h) //50 8C (1 h)

/ N -bromosuccinimide, iodine, acetic acid, Hg2' , dimethyl aminobenzaldehyde / / / / Hg2' , p -chloromercuribenzoic acid (both reversible by DTT) / Ca2' /

[191] [191] [93] [161]

Streptomyces sp. IMD 8.9(1), 2679 8.7(2), 7.2(3) T. profundus DT5432 /

47.8

5.5/ /

60 8C/ /, 60 / 65 8C/ /, 65 8C/ /

[44]

42.0

5.5 /6.0/5.9 /9.8

Thermomonospora curvata

6.2

60.9

6.0/ /

80 8C/80 8C (3 h), Iodoacetic acid, N -bromosuccinic 90 8C (15 min) acid, SDS, guanidine hydrochloride 65 8C/ / /

Ca2'

[71]

[162]

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

1609

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

Starch, Ca2'

Stabilisers

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 reactivated 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 thermostable than without it [4,115]. a-Amylase from A. oryzae EI 212 is inactivated in the presence of Ca2', but retains activity after EDTA treatment [116]. There are also reports where Ca2' did not have any effect on the enzyme [117].

End products, G4, G5; Km [155] (0.3 mg ml (1) End products, G3, G4, G6; [70] Km (3.3 mg ml (1); E.A. (59 kJ mol (1)

Additional properties

8. Industrial applications of a-amylase Amylases are among the most important hydrolytic enzymes for all starch based industries, and the commercialisation of amylases is oldest with first use in 1984, as a pharmaceutical aid for the treatment of digestive disorders. In the present day scenario, amylases find application in all the industrial processes such as in food, detergents, textiles and in paper industry, for the hydrolysis of starch. In this light, microbial amylases have completely replaced chemical hydrolysis in the starch processing industry. They can also be of potential use in the pharmaceutical and fine chemical industries. Today, amylases have the major world market share of enzymes [118]. Several different amylase preparations are available with various enzyme manufacturers for specific use in varied industries. A comprehensive account on commercial applications of a-amylases is quoted by Godfrey and West [119]. Various applications of a-amylase are dealt here in brief. 8.1. Bread and baking industry and as an antistaling agent The baking industry has made use of these enzymes for hundreds of years to manufacture a wide variety of high quality products. For decades, enzymes such as malt and microbial a-amylases have been widely used in the baking industry [120,121]. These enzymes were used in bread and rolls to give these products a higher volume, better colour and a softer crumb. It is the malt preparation that has led the way and opened the opportunities for many enzymes to be used commercially in baking. Today, many enzyme preparations such as proteases, lipases, xylanases, pullulanases, pentosanases, cellullases, glucose oxidases, lipoxygenases etc.

Inhibitors

B.S.A. 62.0 / T. curvata

pH optima/stabi- Temperature oplity tima/stability

Molecular weight (kDa)

pI

Table 2 (Continued )

T. fusca YX

Source

5.5 /6.0/activated 65 8C/ / at pH 7.0 /8.0 6.0/ / 60 8C/ B/65 8C

1610

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

are being used in the bread industry for varied purposes [13,99,121 /123], but none had been able to replace aamylases. Till date, the a-amylases used in baking have been cereal enzymes from barley malt and microbial enzymes from fungi and bacteria [124,125]. Fungal a-amylases have been permitted as bread additives since 1955 in the US and in 1963 in UK after confirmation of their GRAS status [126]. Presently they are used all over the world to different extents. Supplementation of flour with exogenous fungal a-amylase having higher activities is common in the present day modern and continuous baking process [126]. a-Amylase supplementation in flour not only enhances the rate of fermentation and reduces the viscosity of dough (resulting in improvements in the volume and texture of the product, but also generates additional sugar in the dough, which improves the taste, crust colour and toasting qualities of the bread [127]. One of the new applications of a-amylase in the industry has been in retarding the staling of baked products, which reduces the shelf life of these products. Upon storage the crumb becomes dry and firm, the crust loses its crispness and the flavour of the bread deteriorates. All these undesirable changes in the bread are together known as staling. The importance of retrogradation of starch fraction in bread staling has been emphasised [128]. A loss of more than US $1 billion is incurred in USA alone every year due to the staling of bread. Conventionally various additives are used to prevent staling and improve the texture and flavour of baked products. Additives include chemicals, small sugars, enzymes/their combinations, milk powder; emulsifiers, monoglycerides/diglycerides, sugar esters, lecithin, etc; granulated fat, anti-oxidant (ascorbic acid or potassium borate), sugars/salts [129]. Recently emphasis has been given to the use of enzymes in dough improvement/as anti-staling agents, e.g. a-amylase [130,131], branching enzymes [132] and debranching enzymes [133], maltogenic amylases [134], b-amylases [135] amyloglucosidases [136]. Pullulanases and a-amylase combination are used for efficient antistaling property [133]. However, a slight excess of a-amylases was also used which is undesirable as it causes stickiness in bread [134]. Therefore, a recent trend is to use intermediate temperature stable (ITS) a-amylases [13,124,125,137]. They are active after starch gelatinisation and become inactive much before the completion of the baking process. Further, the dextrin with 4/9 degree of polymerisation produced by these shows the anti-staling properties. Although a wide variety of microbial aamylases is known, a-amylase with ITS property has been reported from only a few microorganisms [99,123,138,139].

8.2. Starch liquefaction and saccharication The major market for a-amylases lies in the production of starch hydrolysates such as glucose and fructose. Starch is converted into high fructose corn syrups (HFCS). Because of their high sweetening property, these are used in huge quantities in the beverage industry as sweeteners for soft drinks. The process requires the use of a highly thermostable a-amylase for starch liquefaction. The use of enzyme in starch liquefaction is well established and has been extensively reviewed [2,140]. 8.3. Textile desizing Modern production processes for textiles introduce a considerable strain on the warp during weaving. The yarn must, therefore, be prevented from breaking. For this purpose a removable protective layer is applied to the threads. The materials that are used for this size layer are quite different. Starch is a very attractive size, because it is cheap, easily available in most regions of the world, and it can be removed quite easily. Good desizing of starch sized textiles is achieved by the application of a-amylases, which selectively remove the size and do not attack the fibres. It also randomly cleaves the starch into dextrins that are water soluble and can be removed by washing. The use of a-amylases in warp sizing of textile fibres for manufacturing fibres with great strength has been reported [141]. 8.4. Paper industry The use of a-amylase for the production of low viscosity, high molecular weight starch for coating of paper is reported [142]. The use of amylases in the pulp and paper industry is in the modification of starches for coated paper. As for textiles, sizing of paper is performed to protect the paper against mechanical damage during processing. It also improves the quality of the finished paper. The size enhances the stiffness and strength in paper. It also improves the erasibilty and is a good coating for the paper. Starch is also a good sizing agent for the finishing of paper. Starch is added to the paper in the size press and paper picks up the starch by passing through two rollers that transfer the starch slurry. The temperature of this process lies in the range of 45 /60 8C. A constant viscosity of the starch is required for reproducible results at this stage. The mill also has the flexibility of varying the starch viscosity for different paper grades. The viscosity of the natural starch is too high for paper sizing and is adjusted by partially degrading the polymer with a-amylases in a batch or continuous processes. The conditions depend upon the source of starch and the a-amylase used [143]. A number of amylases exist for use in the paper

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

1611

industry, which include Amizyme (PMP Fermentation Products, Peoria, USA), Termamyl , Fungamyl, BAN (Novozymes, Denmark) and a-amylase G9995 (Enzyme Biosystems, USA). 8.5. Detergent applications Enzymes now comprise as one of the ingredients of modern compact detergents. The main advantage of enzyme application in detergents is due to much milder conditions than with enzyme free detergents. The early automatic dishwashing detergents were very harsh, caused injury when ingested and were not compatible with delicate china and wooden dishware. This forced the detergent industries to search for milder and more efficient solutions [144]. Enzymes also allow lowering of washing temperatures. a-Amylases have been used in powder laundry detergents since 1975. Nowadays, 90% of all liquid detergents contain a-amylase [145] and the demand for a-amylases for automatic dishwashing detergents is growing. One of the limitations of aamylases in detergents is that the enzyme shows sensitivity to calcium and stability is severely compromised in a low calcium environment. In addition, most wild-type a-amylases are sensitive to oxidants which are generally a component of detergent formulations. Stability against oxidants in household detergents was achieved by utilising successful strategies followed with other enzymes such as protease. Recently scientists from the two major detergent enzyme suppliers Novozymes and Genencore International have used protein engineering to improve the bleach stability of the amylases [146 /148]. They independently replaced oxidation sensitive amino acids with other amino acids. The replacement of met at position 197 by leu in B. licheniformis amylase resulted in an amylase with improved resistance against oxidative compounds. This improved oxidation stability resulted in better storage stability and performance of the mutant enzyme in the bleach containing detergent formulations. Genencore International and Novozyme have introduced these new products in the market under the trade names Purafect OxAm and Duramyl , respectively. 8.6. Analysis in medicinal and clinical chemistry With the advent of new frontiers in biotechnology, the spectrum of amylase applications has expanded into many other fields, such as clinical, medicinal and analytical chemistry. There are several processes in the medicinal and clinical areas that involve the application of amylases. The application of a liquid stable reagent, based on a-amylase for the Ciba Corning Express clinical chemistry system has been described [149]. A process for the detection of higher oligosaccharides, which involved the application of amylase was also

developed [96]. This method was claimed to be more efficient than the silver nitrate test. Biosensors with an electrolyte isolator semiconductor capacitor (EIS-CAP) transducer for process monitoring were also developed [150].

9. Conclusions As evident from the foregoing review, amylases are among the most important enzymes used in industrial processes. Although, the use of amylases, a-amylases in particular, in starch liquefaction and other starch based industries has been prevalent for many decades and a number of microbial sources exist for the efficient production of this enzyme, the commercial production of this enzyme has been limited to only a few selected strains of fungi and bacteria. Moreover, the demand for these enzymes is further limited with specific applications as in the food industry, wherein fungal a-amylases are preferred over other microbial sources due to their more accepted GRAS status. Structural conformation plays an important role on amylase activity [151]. Further there arises a need for more efficient a-amylases in various sectors, which can be achieved either by chemical modification of the existing enzymes or through protein engineering. In the light of modern biotechnology, a-amylases are now gaining importance in biopharmaceutical applications. Still, their application in food and starch based industries is the major market and thus the demand of a-amylases would always be high in these sectors.

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 extracellular enzymes. In: Wiseman A, editor. Topics in enzyme and fermentation biotechnology, vol. 3. New York: Wiley, 1979:45 / 108. [4] Vihinen M, Mantsala P. Microbial amylolytic enzymes. Crit Rev Biochem Mol Biol 1989;24:329 /418. [5] Lonsane BK, Ramesh MV. Production of bacterial thermostable a-amylase by solid state fermentation: a potential tool for achieving economy in enzyme production and starch hydrolysis. In: Advances in applied microbiology, vol. 35. San Diego: California Academic Press, 1990:1 /56. [6] Priest FG. Extracellular enzyme synthesis in the genus Bacillus . Bacteriol Rev 1977;41:711 /53. [7] Hollo J, Szeitli J. The reaction of starch with iodine. In: Rodley JA, editor. Starch and its derivatives, 4th ed. Chapman & Hall, 1968:203 /46.

1612

R. Gupta et al. / Process Biochemistry 38 (2003) 1599 /1616 [31] Eratt JA, Douglas PE, Moranelli F, Seligy VL. The induction of a-amylase by starch in Aspergillus oryzae : evidence for controlled mRNA expression. Can J Biochem Cell Biol 1984;62:678 /90. [32] Bhella RS, Altosaar I. Purication and some properties of the extracellular a-amylase from Aspergillus awamori . Can J Microbiol 1985;31:149. [33] Arst HN, Bailey CR. The regulation of carbon metabolism in Aspergillus nidulans . In: Smith JE, Pateman JA, editors. Genetics and physiology of Aspergillus . New York: Academic Press, 1977:131 /46. [34] Kelly CT, Bolton DJ, Fogarty WM. Biphasic production of aamylase of Bacillus avothermus in batch fermentation. Biotechnol Lett 1997;19:75 /7. [35] Emanuilova EI, Toda K. a-Amylase production in batch and continuous cultures by Bacillus caldolyticus . Appl Microbiol Biotechnol 1984;19:301 /5. [36] Cheng CY, Yabe I, Toda K. Selective growth of a mutant in continuous culture of Bacillus caldolyticus for production of aamylase. Appl Microbiol Biotechnol 1989;30:125 /9. [37] Welker NE, Campbell LL. Effect of carbon sources on formation of a-amylase by Bacillus stearothermophilus . J Bacteriol 1963;86:681 /6. [38] Krishnan T, Chandra AK. Effect of oilseed cakes on a-amylase production by Bacillus licheniformis CUMC-305. Appl Environ Microbiol 1982;44:270 /4. [39] Jin B, Van Leeuwen JH, Patel B. Mycelial morphology and fungal protein production from starch processing wastewater in submerged cultures of Aspergillus oryzae . Process Biochem 1999;34:335 /40. [40] Kekos D, Galiotou-Panayotou M, Macris BJ. Some nutritional factors affecting a-amylase production by Calvatia gigantea . Appl Microbiol Biotechnol 1987;26:527 /30. [41] Domingues CM, Peralta RM. Production of amylase by soil fungi and partial biochemical characterization of amylase of a selected strain (Aspergillus fumigatus fresenius ), Can J Microbiol 1993;39:681 /5. [42] Goto CE, Barbosa EP, Kistner LCL, Moreira FG, Lenartoviez V, Peralta RM. Production of amylase by Aspergillus fumigatus utilizing a-methyl D-glucoside, a synthetic analog of maltose as substrate. FEMS Microbiol Lett 1998;167:139 /43. [43] Arnesen S, Eriksen SH, Olsen J, Jensen B. Increased production of alpha amylase from Thermomyces lanuginosus by the addition of Tween-80. Enzyme Microb Technol 1998;23:249 /52. [44] McMahon HEM, Kelly CT, Fogarty WM. High maltose producing amylolytic system of a Streptomyces sp. Biotechnol Lett 1999;21:23 /6. [45] Hamilton LM, Kelly CT, Fogarty WM. Production and properties of the raw starch-digesting a-amylase of Bacillus sp. IMD 435. Process Biochem 1999;35:27 /31. [46] Coronado MJ, Vargas C, Hofemeister J, Ventosa A, Nieto JJ. Production and biochemical characterization of an a-amylase from the moderate halophile Halomonas meridiana . FEMS Microbiol Lett 2000;183:67 /71. [47] Hamilton LM, Kelly CT, Fogarty WM. Purication and properties of the raw starch degrading a-amylase of Bacillus sp. IMD434. Biotechol Lett 1999;21:111 /5. [48] Dercova K, Augustin J, Krajcova D. Cell growth and a-amylase production characteristics of Bacillus subtilis . Folia Microbiol 1992;37:17 /23. [49] Imai Y, Suzuki M, Masamoto M, Nagayasu K. Amylase production by Aspergillus oryzae in a new kind of fermentor with a rotary draft tube. J Ferment Bioeng 1993;76:459 /64. [50] Pedersen H, Nielsen J. The inuence of nitrogen sources on the a-amylase productivity of Aspergillus oryzae in continuous cultures. Appl Microbiol Biotechnol 2000;53:278 /81.

[8] Fuwa H. A new method for microdetermination of amylase activity by the use of amylose as substrate. J Biochem 1954;41:583 /603. [9] Manonmani HK, Kunhi AAM. Interference of thiol compounds with dextrinizing activity assay of a-amylase by starch /iodine color reaction: modication of the method to eliminate this interference. World J Microbiol Biotechnol 1999;15:485 /7. [10] Hansen PW. Determination of fungal a-amylase by ow injection analysis. Anal Chim Acta 1984;158:375 /7. [11] Carlsen M, Marcher J, Nielsen J. An improved FIA-method for measuring a-amylase in cultivation media. Biotechnol Tech 1994;8:479 /82. [12] Sandstedt RM, Kneen E, Blish MJ. A standardised Wohlgemuth procedure for a-amylase activity. Cereal Chem 1939;16:712 /23. [13] Kulp K. Enzymes as dough improvers. In: Kamel BS, Stauffer CE, editors. Advances in baking technology. New York: Blackie Academic and Professional, VCH Publishers, 1993:152 /78. [14] Indian Standard. Colorimetric determination of alpha-amylase activity in cereals and cereal products. IS 10524. Indian Standards Institution, 1982. New Delhi. [15] Bernfeld P. Amylases, a and b. Methods Enzymol 1955;1:149 / 58. [16] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426 /8. [17] Mishra RS, Maheshwari R. Amylases of the thermophilic fungus Thermomyces lanuginosus : their purication, properties, action on starch and response to heat. J Biosci 1996;21:653 /72. [18] Ceska M, Hultman E, Ingelman BGA. A new method for determination of a-amylase. Experentia 1969;25:555 /6. [19] Dhawale MR, Wilson JJ, Khachatourians GG, Ingledew WM. Improved method for detection of starch hydrolysis. Appl Environ Microbiol 1982;44:747 /50. [20] Wong DWS, Batt SB, Robertson GH. Microassay for rapid screening of alpha-amylase activity. J Agric Food Chem Washington, DC: Am Chem Soc 2000;48:4540 /3. [21] Sheehan H, McCleary BV. A new procedure for the measurement of fungal and bacterial a-amylase. Biotechnol Tech 1988;2:289 /92. [22] International Association for Cereal Chemistry. Standard methods of the ICC. 1968: Method 104, approved 1960; Method 105, approved 1980; Method 107, approved 1968; Method 108, approved 1968. Verlag Moritz Schafer, Detmold, West Germany. [23] American Association of Cereal Chemists. Approved methods of the AACC.1972: 7th ed. Method 56-81B, approved November 1972. The Association, St. Paul, MN. [24] International Organization for Standardization. International standard ISO 3093, approved 1974. The Secretriat: Magyar Szabvanyugyi Hivatal, 1450, Budapest 9, Pf 24, Hungary. [25] Perten H. A modied falling number method suitable for measuring both cereal and fungal a-amylase activity. Cereal Chem 1984;61:108 /11. [26] Fogarty WM, Kelly CT. Starch degrading enzymes of microbial origin. Prog Ind Microbiol 1979;15:87 /150. [27] Tonomura K, Suzuki H, Nakamura N, Kuraya K, Tanabe O. On the inducers of a-amylase formation in Aspergillus oryzae . Agric Biol Chem 1961;25:1 /6. [28] Yabuki M, Ono N, Hoshino K, Fukui S. Rapid induction of aamylase by non-growing mycelia of Aspergillus oryzae . Appl Environ Microbiol 1977;34:1 /6. [29] Lachmund A, Urmann U, Minol K, Wirsel S, Ruttkowski E. Regulation of a-amylase formation in Aspergillus oryzae and Aspergillus nidulans transformants. Curr Microbiol 1993;26:47 / 51. [30] Morkeberg R, Carlsen M, Neilsen J. Induction and repression of a-amylase production in batch and continuous cultures of Aspergillus oryzae . Microbiology 1995;141:2449 /54.

R. Gupta et al. / Process Biochemistry 38 (2003) 1599 /1616 [51] Hayashida S, Teramoto Y, Inoue T. Production and characteristics of raw potato starch digesting a-amylase from Bacillus subtilis 65. Appl Environ Microbiol 1988;54:1516 /22. [52] Rukhaiyar R, Srivastava SK. Effect of various carbon substrate on a-amylase production from Bacillus sp. J Microb Biotechnol 1995;10:76 /82. [53] Yoshigi N, Chikano T, Kamimura M. Production of an extracellular a-amylase from Bacillus cereus NY-14. J Jpn Soc Starch Sci 1985;32:217 /21. [54] Cheng CY, Yabe I, Toda K. Predominant growth of a-amylase regulation mutant in continuous culture of Bacillus caldolyticus . J Ferment Bioeng 1989;67:176 /81. [55] Ueno S, Miyama M, Ohashi Y, Izumiya M, Kusaka I. Secretory enzyme production and conidiation of Aspergillus oryzae in submerged liquid culture. Appl Microbiol Biotechnol 1987;26:273 /6. [56] Shah NK, Upadhyay CM, Nehete PN, Kothari RM, Hegde MV. An economical, upgraded, stabilized and efcient preparation of a-amylase. J Biotechnol 1990;16:97 /108. [57] Kundu AK, Das S, Gupta TK. Inuence of culture and nutritional conditions on the production of amylase by the submerged culture of Aspergillus oryzae . J Ferment Technol 1973;51:142 /50. [58] Zhang Q, Tsukagoshi N, Miyashiro S, Udaka S. Increased production of a-amylase by Bacillus amyloliquefaciens in the presence of glycine. Appl Environ Microbiol 1983;46:293 /5. [59] Ikura Y, Horikoshi K. Effect of amino compounds on alkaline amylase production by alkalophilic Bacillus sp. J Ferment Technol 1987;65:707 /9. [60] Lee J, Parulekar SJ. Enhanced production of a-amylase in fedbatch cultures of Bacillus subtilis TN 106[pAT5]. Biotechnol Bioeng 1993;42:1142 /50. [61] Dean ACR. Inuence of environment on the control of enzyme synthesis. J Appl Chem Biotechnol 1972;22:245 /59. [62] Mertz FP, Doolin LE. The effect of phosphate on the biosynthesis of vanilomycin. Can J Microbiol 1973;19:263 /70. [63] Hillier P, Wase DAJ, Emery AN, Solomons GL. Instability of aamylase production and morphological variation in continuous culture of Bacillus amyloliquefaciens is associated with plasmid loss. Process Biochem 1997;32:51 /9. [64] Wu WX, Mabinadji J, Betrand TF, Wu WX. Effect of culture conditions on the production of an extracellular thermostable alpha-amylase from an isolate of Bacillus sp. J Zhejiang Univ Agric Life Sci 1999;25:404 /8. [65] Pazlarova J, Votruba J. Use of zeolite to control ammonium in Bacillus amyloliquifaciens a-amylase fermentations. Appl Microbiol Biotechnol 1996;45:314 /8. [66] McMahon HEM, Kelly CT, Fogarty WM. Effect of growth rate on a-amylase production by Streptomyces sp. IMD 2679. Appl Microbiol Biotechnol 1997;48:504 /9. [67] Castro PML, Hayter PM, Ison AP, Bull AT. Application of statistical design to the optimization of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells. Appl Microbiol Biotechnol 1992;38:84 /90. [68] Chahal DS. Growth characteristics of microorganisms in solid state fermentation for upgrading of protein values of lignocelluloses and cellulase production. In: Blanch HW, Papoutsakis ET, Stephanopoulos G, editors. Foundations of biochemical engineering kinetics and thermodynamics in biological systems. American Chemical Society, Washington DC. ACS symposium series, No. 207, 1983:421 /42. [69] Friedrich J, Cimerman A, Steiner W. Submerged production of pectinolytic enzymes by Aspergillus niger : effect of different aeration/agitation regimes. Appl Microbiol Biotechnol 1989;31:490 /4. [70] Busch JE, Stutzenberger FJ. Amylolytic activity of Thermomonospora fusca . World J Microbiol Biotechnol 1997;13:637 /42.

1613

[71] Chung YC, Kobayashi T, Kanai H, Akiba T, Kudo T. Purication and properties of extracellular amylase from the hyperthermophilic archeon Thermococcus profundus DT5432. Appl Environ Microbiol 1995;61:1502 /6. [72] Bhavaraju SM, Blanch HW. A model for pellet breakup in fungal fermentations. J Ferment Technol 1976;54:466 /8. [73] Justen P, Paul GC, Nienow AW, Thomas CR. Dependence of mycelial morphology on impeller type and agitation intensity. Biotechnol Bioeng 1996;52:672 /84. [74] Bocking SP, Wiebe MG, Robson GD, Hansen K, Christiansen LH, Trinci APJ. Effect of branch frequency in Aspergillus oryzae on protein secretion and culture viscosity. Biotechnol Bioeng 1999;65:638 /48. [75] Cui YQ, Van der Lans RGJM, Luyben KCAM. Effect of agitation intensities on fungal morphology of submerged fermentation. Biotechnol Bioeng 1997;55:715 /26. [76] Amanullah A, Blair R, Nienow AW, Thomas CR. Effects of agitation intensity on mycelial morphology and protein production in chemostat cultures of recombinant Aspergillus oryzae . Biotechnol Bioeng 1999;62:434 /46. [77] Priest FG. Products and applications. In: Harwood CR, editor. Biotechnology handbooks, Bacillus . New York, London: Plenum Press, 1989:293 /320. [78] Carlsen M, Nielsen J, Villadser J. Growth and a-amylase production by Aspergillus oryzae during continuous cultivations. J Biotechnol 1996;45:81 /93. [79] Spohr A, Carlsen M, Nielsen J, Villadsen J. Morphological characterization of recombinant strains of Aspergillus oryzae producing a-amylase during batch cultivations. Biotechnol Lett 1997;19:257 /61. [80] Agger T, Spohr AB, Carlsen M, Nielsen J. Growth and product formation of Aspergillus oryzae during submerged cultivations: verication of a morphologically structured model using uorescent probes. Biotechnol Bioeng 1998;57:321 /9. [81] Heineken FG, OConner RJ. Continuous culture studies on the biosynthesis of alkaline protease, neutral protease and a-amylase by Bacillus subtilis NRRL-B3411. J Gen Microbiol 1972;73:35 / 44. [82] Delgado G, Topete M, Galindo E. Interaction of cultural conditions and end product distribution in Bacillus subtilis grown in shake asks. J Microbiol Biotechnol 1989;31:288 /92. [83] Bohdziewicz J. Ultraltration of technical amylolytic enzymes. Process Biochem 1996;31:185 /91. [84] Schwermann B, Pfau K, Liliensiek B, Schleyer M, Fischer T, Bakker EP. Purication, properties and structural aspects of a thermoacidophilic a-amylase from Alicyclobacillus acidocaldarius ATCC 27009. Insight into acidostability of proteins. Eur J Biochem 1994;226:981 /91. [85] Saito NA. Thermophilic extracellular a-amylase from Bacillus licheniformis . Arch Biochem Biophys 1973;155:290 /8. [86] Krishnan T, Chandra AK. Purication and characterization of a-amylase from Bacillus licheniformis CUMC 305. Appl Environ Microbiol 1983;46:430 /7. [87] Lee SP, Morikawa M, Takagi M, Imanaka T. Cloning of the aap T gene and characterization of its product, a-amylase / pullulanase (AapT), from thermophilic and alkaliphilic Bacillus sp. Strain XAL601. Appl Environ Microbiol 1994;60:3764 /73. [88] Shinke R, Aoki K, Murokam S, Inoue T, Babat T. Alkaline and thermophilic amylases of industrial use. In: Dordick JS, Russell AJ, editors. Enzyme engineering XIII, vol. 799. New York: Annals of the New York Academy of Sciences, 1996:332 /40. [89] Kim TU, Gu BG, Jeong JY, Byun SM, Shin YC. Purication and characterization of a maltotetraose forming alkaline aamylase from an alkalophilic Bacillus sp. GM8901. Appl Environ Microbiol 1995;61:3105 /12. [90] Ogasahara K, Imanishi A, Isemura T. Studies on thermophilic aamylase from Bacillus stearothermophilus . I. Some general and

1614

R. Gupta et al. / Process Biochemistry 38 (2003) 1599 /1616 physico-chemical properties of thermophilic a-amylase. J Biochem 1970;67:65. Pfueller SL, Elliott WH. The extracellular a-amylase of Bacillus stearothermophilus . J Biol Chem 1969;244:48. Khoo SL, Amirul A-A, Kamaruzaman M, Nazalan N, Azizan MN. Purication and characterization of a-amylase from Aspergillus avus . Folia Microbiol 1994;39:392 /8. Robyt J, Ackerman RJ. Isolation, purication and characterization of a maltotetraose producing amylase from Pseudomonas stutzeri . Arch Biochem Biophys 1971;145:105 /14. Chary SJ, Reddy SM. Starch degrading enzymes of two species of Fusarium . Folia Microbiol 1985;30:452. Fogarty WM, Kelly CT. Economic microbiology. In: Rose AH, editor. Microbial enzymes and bioconversions, vol. 5. London: Academic Press, 1980:115 /70. Giri NY, Mohan AR, Rao LV, Rao CP. Immobilization of aamylase complex in detection of higher oligosaccharides on paper. Curr Sci 1990;59:1339 /40. Ray RR, Jana DC, Nanda G. Immobilization of b-amylase from Bacillus megaterium B6 into gelatin lm by cross-linking. J Appl Bacteriol 1995;79:157 /62. Kobayashi T, Kamekura M, Kanlayakrit W, Ohnishi H. Production, purication and characterization of an amylase from the moderate halophile Micrococcus varians subspecies halophilus . Microbios 1986;46:165. Prieto JA, Bort BR, Martinez J, Randez-Gil F, Buesa C, Sanz P. Purication and characterization of a new a-amylase of intermediate thermal stability from the yeast Lipomyces kononenkoae . Biochem Cell Biol 1995;73:41 /9. Mamo G, Gashe BA, Gessesse A. A highly thermostable amylase from a newly isolated thermophillic Bacillus sp. WN11. J Appl Microbiol 1999;86:557 /60. Manning GB, Campbell LL. Thermostable a-amylase of Bacillus stearothermophilus . I. Crystallization and some general properties. J Biol Chem 1961;236:2952 /7. Wind RD, Buitelaar RM, Eggink G, Huizing HJ, Dijkhuizen L. Characterization of a new Bacillus stearothermophilus isolate: a highly thermostable a-amylase producing strain. Appl Microbiol Biotechnol 1994;41:155 /62. Grootegoed JA, Lauwers AM, Heinen W. Separation and partial purication of extracellular amylase and protease from Bacillus caldolyticus . Arch Microbiol 1973;90:223. Ratanakhanokchai K, Kaneko J, Kamio Y, Izaki K. Purication and properties of a maltotetraose and maltotriose producing amylase from Chloroexus aurantiacus . Appl Environ Microbiol 1992;58:2490 /4. McKelvy J, Lee YC. Microheterogeneity of the carbohydrate group of Aspergillus oryzae a-amylase. Arch Biochem Biophys 1969;132:99 /110. Eriksen SH, Jensen B, Olsen J. Effect of N -linked glycosylation on secretion, activity and stability of a-amylase from Aspergillus oryzae . Curr Microbiol 1998;37:117 /22. Srivastava RAK. Studies on extracellular and intracellular puried amylases from a thermophilic Bacillus stearothermophilus . Enzyme Microb Technol 1984;6:422. Yamane K, Yamaguchi K, Maruo B. Purication and properties of a cross-reacting material related to a-amylase and biochemical comparison with parental a-amylase. Biochim Biophys Acta 1973;295:323. Matsuzaki H, Yamane K, Yamaguchi K, Nagata Y, Maruo B. Hybrid a-amylase produced by transformants of Bacillus subtilis I. Immunological and chemical properties of a-amylases produced by the parental strain and the transformants. Biochim Biophys Acta 1974;365:235 /47. Sills AM, Sauder ME, Stewart GG. Isolation and characterization of the amylolytic system of Schwanniomyces castelli . J Inst Brew 1984;90:311. [111] Vallee BL, Stein EA, Summerwell WM, Fischer EM. Metal content of a-amylases of various origins. J Biol Chem 1959;231:2901 /5. [112] Oikawa A, Maeda A. The role of calcium in Taka amylase A. J Biochem 1957;44:745. [113] Heinen W, Lauwers AM. Amylase activity and stability at high and low temperature depending on calcium and other divalent cations. Experientia 1975;26:77. [114] Oikawa A. The role of calcium in Taka amylase A II. The exchange reaction. Can J Biochem 1959;46:463. [115] Robyt J, French D. Action pattern and specicity of an amylase from Bacillus subtilis . Arch Biochem Biophys 1963;100:451 /67. [116] Kundu AK, Das S. Production of amylase in liquid culture by a strain of Aspergillus oryzae . Appl Microbiol 1970;19:598. [117] Laderman KA, Davis BR, Krutzsch HC, Lewis MS, Griko YV, Privalov PL, Annsen CB. The purication and characterization of an extremely thermostable a-amylase from hypothermophilic archaebacterium Pyrococcus furiosus . J Biol Chem 1993;268:24394 /401. [118] Aehle W, Misset O. Enzymes for industrial applications. In: Rehm HJ, Reed G, editors. Biotechnology, 2nd ed. Germany: Wiley-VCH, 1999:189 /216. [119] Godfrey T, West S. In: Godfrey T, West S, editors. Industrial enzymology. 2nd ed. New York: Stockton Press, 1996. p. 91,105 /31,192,339 /56,361 /71. [120] Hamer RJ. Enzymes in the baking industry. In: Tucker GA, Woods LFJ, editors. Enzymes in food processing. Galsgow: Blackie Academic and Professional, 1995:190 /222. [121] Si JQ. Enzymes, baking, bread making. In: Flickinger MC, Drew SW, editors. Encyclopedia of bioprocess technology: fermentation, biocatalysis and bioseparation, vol. 2. Wiley, 1999:947 /58. [122] Pintauro ND. Bread and baked goods. In: Pintauro ND, editor. Food processing enzymes */recent developments. Food technology review No. 52. Park Ridge, NJ, USA: Noyes Data Corporation, 1979. [123] Monfort A, Blasco A, Preito JA, Sanz P. Combined expression of Aspergillus nidulans endoxylanase X-24 and Aspergillus oryzae a-amylase in industrial bakers yeast and their use in bread making. Appl Environ Microbiol 1996;62:3712 /5. [124] Hebeda RE, Bowles LK, Teague WM. Developments in enzymes for retarding staling of baked goods. Cereal Foods World 1990;35:453 /7. [125] Hebeda RE, Bowles LK, Teague WM. Use of intermediate temperature stability enzymes for retarding staling in baked goods. Cereal Foods World 1991;36:618 /24. [126] Pritchard PA. Studies on the bread improving mechanisms of fungal a-amylase. J Biol Educ 1992;26:12 /8. [127] Van Dam HW, Hille JDR. Yeast and enzymes in bread making. Cereal Foods World 1992;37:245 /52. [128] Kulp K, Ponte GJ. Staling white pan bread: fundamental causes. Crit Rev Food Sci Nutr 1981;15:1 /48. [129] Spendler T, Jorgensen O. Use of a branching enzyme in baking. 1997. Patent Application WO97/41736. [130] De Stefanis VA, Turner EW. Modied enzyme system to inhibit bread rming method for preparing same and use of same in bread and other bakery products. 1981. Patent Application US4299848. [131] Cole MS. Antistaling baking composition. 1982. Patent Application US4320151. [132] Okada S, Kitahata S, Yoshikawa S, Sugimoto T, Sugimoto K. Process for the production of branching enzyme and a method for improving the qualities of food products therewith. 1984. Patent Application US4454161. [133] Carroll JO, Boyce COL, Wong TM, Starace CA. Bread antistaling method. 1987. Patent Application US4654216. [134] Olesen T. Antistaling process and agent. 1991. Patent Application WO9104669.

[91] [92]

[93]

[94] [95]

[96]

[97]

[98]

[99]

[100]

[101]

[102]

[103]

[104]

[105]

[106]

[107]

[108]

[109]

[110]

R. Gupta et al. / Process Biochemistry 38 (2003) 1599 /1616 [135] Wursch P, Gumy D. Inhibition of amylopectin retrogradation by partial beta amylolysis. Carbohydr Res 1994;256:129 /37. [136] Vidal FD, Gerrity AB. Antistaling agent for bakery products. 1979. Patent application US4160848. [137] Ahuja A, Gupta R, Saxena RK, Gigras P. An antistaling enzyme from microbes for baked products. In: Crowther JS, Marthi B, editors. Proceedings of the microbiological safety of processed foods. New Delhi: Oxford and IBH Publishing Co. Pvt. Ltd, 1998:127. [138] Kraus JK, Hebeda RE. Method for retarding staling of baked goods. 1993. US patent 5,209,938. [139] Gigras P, Sahai V, Gupta R. Statistical media optimization and production of ITS alpha amylase from A. oryzae in a bioreactor. Curr Microbiol 2002;45(3):203 /8. [140] Van der Maarel MJEC, Van der Veen B, Uitdehaag JCM, Leemhuis H, Dijkhuizen L. Properties and applications of starch converting enzymes of alpha amylase family. J Biotechnol 2002;94:137 /55. [141] Hendriksen HV, Pedersen S, Bisgard-Frantzen H. A process for textile warp sizing using enzymatically modied starches. 1999. Patent Application WO 99/35325. [142] Bruinenberg PM, Hulst AC, Faber A, Voogd RH. A process for surface sizing or coating of paper. 1996. European Patent Application EP 0,690,170 A1. [143] Tolan JS. Pulp and paper. In: Godfrey T, West S, editors. Industrial enzymology, 2nd ed. New York: Stockton Press, 1996:327 /38. [144] Van Ee JH, van Rijswijk WC, Bollier M. Enzymatic automated dishwash detergents. Chim Oggi 1992;10:21 /4. [145] Kottwitz B, Upadek H, Carrer G. Applications and benets of enzymes in detergent. Chim Oggi 1994;12:21 /4. [146] Svendsen A, Bisgaard-Frantzen H. PCT patent publication. WO 94/0, 1994. [147] Tierny L, Danko S, Dauberman J, Vaha-Vahe P, Winetzky D. Performance advantages of novel a-amylases in automatic dishwashing. Am Oil Chem Soc 86th San Antonio Annual meeting, 1995. [148] Bisgaard-Frantzen H, Borchert T, Svendsen A, Thellersen MH, Van Der Zee P. PCT Patent Application. WO 95/10603, 1995. [149] Becks S, Bielawaski C, Henton D, Padala R, Burrows K, Slaby R. Application of a liquid stable amylase reagent on the Ciba Corning Express clinical chemistry system. Clin Chem 1995;41:S186. [150] Menzel C, Lerch T, Schneider K, Weidemann R, Tollnick C, Kretymer G, Scheper T, Schugert K. Application of biosensors with an electrolyte isolator semiconductor capacitor (EIS-CAP) transducer for process monitoring. Process Biochem 1998;33:175 /80. [151] Mac Gregor EA, Janecek S, Svensson B. Relationship of sequence and structure to specicity in the alpha amylase family of enzymes. Biochim Biophys Acta 2001;1546:1 /20. [152] Iefuji P, Chino M, Kato M, Iimura Y. Raw-starch-digesting and thermostable a-amylase from the yeast Cryptococcus sp. S-2: purication, characterization, cloning and sequencing. Biochem J 1996;318:989 /96. [153] De Moraes LMP, Astol-Filho S, Ulhao CJ. Purication and some properties of an a-amylase glucoamylase fusion protein from Saccharomyces cerevisiae . World J Microbiol Biotechnol 1999;15:561 /4. [154] Wilson JJ, Ingledew M. Isolation and characterization of Schwanniomyces alluvius amylolytic enzymes, Appl Environ Mirobiol 1982;44:301 /7. [155] Glymph JL, Stutzenberger FJ. Production, purication and characterization of a-amylase from Thermomonospora curvata . Appl Environ Microbiol 1977;34:391 /7.

1615

[156] Jenson B, Olsen J, Allermann K. Purication of extracellular amylolytic enzymes from the thermophilic fungus Thermomyces lanuginosus . Can J Microbiol 1988;34:218 /23. [157] Morgan FJ, Priest FG. Characterization of a thermostable aamylase from Bacillus licheniformis NCIB 6346. J Appl Bacteriol 1981;50:107 /14. [158] Vihinen M, Mantsala P. Characterization of a thermostable Bacillus stearothermophilus a-amylase. Biotechnol Appl Biochem 1990;12:427 /35. [159] Marco JL, Bataus LA, Valencia FF, Ulho CJ, Astol-Filho S, Felix CR. Purication and characterization of a truncated Bacillus subtilis a-amylase produced by Escherichia coli . Appl Microbiol Biotechnol 1996;44:746 /52. [160] Giraud E, Gosselin L, Marin B, Parada JL, Raimbault M. Purication and characterization of an extracellular amylase from Lactobacillus plantarum strain A6. J Appl Bacteriol 1993;75:276 /82. [161] Freer SN. Purication and characterization of the extracellular a-amylase from Streptococcus bovis JB 1. Appl Environ Microbiol 1993;59:1398 /402. [162] Collins BS, Kelly CT, Fogarty WM, Doyle EM. The high maltose producing a-amylase of the thermophilic actinomycete, Thermomonospora curvata . Appl Microbiol Biotechnol 1993;39:31 /5. [163] Michelena VV, Castillo FJ. Production of amylase by Aspergillus foetidus on rice our medium and characterization of the enzyme. J Appl Bacteriol 1984;56:395. [164] Perevozchenko II, Tsyperovich AS. Comparative investigation of the properties of a-amylases of mold fungi of the genus Aspergillus . Appl Biochem Microbiol 1972;8:7. [165] Olutiola PO. a-Amylolytic activity of Aspergillus chevalieri from mouldy maize seeds. Indian Phytopathol 1982;35:428. [166] Alazard D, Baldensperger JF. Amylolytic enzymes from Aspergellus hennebergi (A. niger group): purication and characterization of amylases from solid and liquid cultures. Carbohydr Res 1982;107:231. [167] Minoda Y, Yamada K. Acid-stable a-amylase of black Aspergilli . I. Detection and purication of acid-stable dextrinizing amylase. Agric Biol Chem 1963;27:806. [168] Minoda Y, Arai M, Torigoe Y, Yamada K. Acid-stable aamylase of black Aspergilli . III. Separation of acid-stable aamylase and acid-unstable a-amylase from the same mold amylase preparation. Agric Biol Chem 1968;32:110 /4. [169] Minoda Y, Arai M, Yamada K. Acid-stable a-amylase of black Aspergilli . V. Amino acid composition and amino-terminal amino acid. Agric Biol Chem 1969;33:572. [170] Arai M, Koyano T, Ozawa H, Minoda Y, Yamada K. Acidstable a-amylase of black Aspergilli . IV. Some physicochemical properties. Agric Biol Chem 1968;32:507. [171] Arai M, Minoda Y, Yamada K. Acid-stable a-amylase of black Aspergilli . VI. Carbohydrate and metal content. Agric Biol Chem 1969;33:922. [172] Bhumibhamon O. Production of amyloglucosidase by submerged culture. Thailand J Agric Sci 1983;16:173. [173] Ramachandran N, Skreekantiah KR, Murthy VS. Studies on the thermophilic amylolytic enzymes of a strain of Aspergillus niger . Starch 1978;30:272. [174] Ramachandran N, Skreekantiah KR, Murthy VS. Inuence of media composition on the production of a-amylase and amyloglucosidase by a strain of Aspergillus niger . Starch 1979;31:134. [175] Ramasesh N, Skreekantiah KR, Murthy VS. Purication and characterization of a thermophilic a-amylase of Aspergillus niger van Tieghem. Starch 1982;34:274. [176] Jodal I, Kandra L, Harangi J, Nanasi P, Szejtli J. Hydrolysis of cyclodextrin by Aspergillus oryzae a-amylase. Starch 1984;36:140.

1616

R. Gupta et al. / Process Biochemistry 38 (2003) 1599 /1616 [186] Schellart JA, Visser FMW, Zandstva T, Middlehover WJ. Starch degradation by the mold Trichoderma viride I. The mechanism of degradation. Antonie Van Leeuwenhock J Microbiol Serol 1976;42:229. [187] Ebisu S, Mori M, Takagi H, Kadowaki K, Yamagata H, Tsukagoshi N, Udaka S. Production of a fungal protein, Taka amylase A, by protein-producing Bacillus brevis HPD31. J Ind Microbiol 1993;11:83 /8. [188] Ramesh MV, Lonsane BK. Characteristics and novel features of thermostable a-amylase produced by Bacillus licheniformis M-27 under solid state fermentation. Starch 1990;42:233 /8. [189] Ali MB, Mezghani M, Bejar S. A thermostable alpha amylase producing maltohexose from a newly isolated Bacillus sp . US100: study of activity and molecular cloning of the corresponding gene. Enzyme Microb Technol 1999;24:584 /9. [190] Mamo G, Gessesse A. Purication and charcterization of two raw starch degrading thermostable a-amylase from a thermophillic Bacillus . Enzyme Microb Technol 1999;25:433 /8. [191] Kelly TT, Collins BS, Fogarty WM, Doyle EM. Mechanisms of action of the a-amylase of Micromonospora melanosporea . Appl Microbiol Biotechnol 1993;39:599 /603.

[177] Hanrahan VM, Caldwell ML. A study of action of Taka amylase. J Am Chem Soc 1953;75:2191. [178] Hanrahan VM, Caldwell ML. Additional studies of the properties of Taka amylase. J Am Chem Soc 1953;75:4030. [179] Minoda Y, Koyano T, Arai M, Yamada K. Acid-stable aamylase of black Aspergilli. II. Some general properties. Agric Biol Chem 1968;32:104 /9. [180] Suetsugu N, Koyama S, Takeo KI, Kuge T. Kinetic studies on the hydrolyses of a, b- and g-cyclodextrins by Taka amylase A. J Biochem 1974;76:57. [181] Toda H, Kondo K, Narita K. The complete amino acid sequence of Taka-amylase A. Proc Jpn Acad 1982;58:208. [182] Sills AM, Sauder ME, Steward GG. Amylase activity in certain yeasts and a fungal species. Dev Ind Microbiol 1983;24:295. [183] Suganuma T, Tahara N, Kitahara K, Nagahama T, Inuzuka K. N-terminal sequence of amino acids and some properties of an acid stable a-amylase from citric acid Koji (Aspergillus usamii var.). Biosci Biotech Biochem 1996;60:177 /9. [184] Narayanan AS, Shanmugasundaram ERB. Studies on amylase of Fusarium vasinfectum . Arch Biochem Biophys 1967;118:317. [185] Zenin CT, Park YK. Purication and characterization of acid aamylase from Paecilomyces sp . J Ferment Technol 1983;61:109.