Enzyme and Microbial Technology 37 (2005) 723–734

A novel raw starch digesting thermostable�-amylase fromBacillus sp.I-3 and its use in the direct hydrolysis of raw potato starch

Nidhi Goyal, J.K. Gupta, S.K. Soni∗

Department of Microbiology, Panjab University, Chandigarh 160014, India

Received 19 September 2004; received in revised form 19 April 2005; accepted 28 April 2005

Abstract

A new strain ofBacillus sp. I-3, isolated from natural soil samples, showed a high raw starch digesting activity towards potato starch.Upon optimization of various environmental and cultural conditions, the yield of�-amylase reached 642 U/mL. The kinetic characterizationof partially purified enzyme exhibited the maximum activity at 70◦C, pH 7.0 and revealed a high thermostability in the presence of 10 mMCaCl2·2H2O where it could retain more than 90% residual activity at 70◦C after 3.5 h. At 80, 90 and 100◦C, the enzyme retained 80, 59 and26% of its maximum activity after 2.5, 0.5 and 0.5 h, respectively. The enzyme preparation had a strong affinity towards raw potato starchgranules and was almost completely adsorbed onto it. It also hydrolyzed raw potato starch at a concentration of 12.5% significantly in a shortp©

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eriod of time of 12 h.2005 Elsevier Inc. All rights reserved.

eywords: Raw starch hydrolysis; Thermostable�-amylase;Bacillus sp.; Raw potato starch; Raw starch adsorption; Direct hydrolysis

. Introduction

Starch is the most abundant form of storage polysaccha-ides in plants and constitutes an inexpensive source for pro-uction of syrups containing glucose, fructose or maltose,hich are widely used in food industries[1]. In addition to

hat, the sugars produced can be fermented to produce bio-thanol[2]. In starch granules, the molecules are denselyacked in a polycrystalline state with inter and intramolecularonds and are hence insoluble in cold water and often resistant

o chemicals and enzymes[3]. In the course of conventionalnzymatic saccharification, a slurry containing 15% starch iselatinized where it is heated up to a temperature of 105◦C sos to open the crystalline structure for the enzyme action[4,5].his increases the viscosity of the slurry and poses prob-

ems with mixing and pumping[6]. The gelatinized starch ishen liquefied with high temperature�-amylase at the sameime followed by the saccharification with glucoamylase at auch lower temperature of 50–60◦C [5]. The whole process

equires a high-energy input, thus increasing the production

∗

cost of starch-based products. In view of energy costs, etive utilization of natural resources and viscosity probledirect hydrolysis of starch below gelatinization temperais desirable[7]. In recent years, the importance of enzymsaccharification of raw starch without heating has becwell recognized, mainly from the viewpoints of energy sings and effective utilization of the biomass thereby reduthe cost of starch processing[4,5,8]. This has generatedworld wide interest in the discovery of several raw starchgesting amylases which does not require the gelatinizand can directly hydrolyze the raw starch in a single stepthat too at moderate temperature much below the gelatition temperature[7,9].

It is more difficult for amylases to act on raw starch grules than on gelatinized starch. Previous studies indicthat the saccharification of raw starch by amylolytic enzymight be related to the extent of adsorption of enzyme tostarch granules. The microorganisms reported to be gooducers of amylase capable of digesting raw starch have mbeen fungi, such asAspergillus sp.,Rhizopus sp. andCorti-cium rolfsi [7–10], although there are reports of the possibof raw starch degradation by bacterial�- and�-amylases[4].

Corresponding author. Tel.: +91 172 2534149; fax: +91 172 2541409.

E-mail address: sonisk@pu.ac.in (S.K. Soni). Several amylases from molds and bacteria have been reported

141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2005.04.017

724 N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734

which digest the raw starch granules at ordinary temperatures(30–50◦C) involving mashes having 1% raw starch[11] inwhich significant saccharification is achieved after extendedperiod of incubation. Different studies demonstrated that thebetter hydrolysis of raw starch can be achieved by increasingthe incubation temperature approximately to 60◦C [12,13].Therefore, use of thermostable, raw starch digesting amy-lases is expected to give better hydrolysis of raw starch attemperatures ranging between 60 and 70◦C without loosingactivity during prolonged incubation.

Most raw starch digesting enzymes reported to date hardlydigest potato starch[6,7,13–15]. On the other hand, next tocorn, potato is the most important source of starch[6,16].Therefore, enzymes that are capable of digesting raw potatostarch are economically attractive for they can increase therange of starch sources for direct hydrolysis.

The purpose of this research was to isolate a microor-ganism capable of producing thermostable and efficient rawpotato starch digesting�-amylase for the direct hydrolysisof raw starch in a considerably less duration of time, at aconcentration of starch normally used in the starch industries(10–15%) at temperatures below the gelatinization tempera-ture of starch.

2

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eref uidc ntedw n byc ran-u hy-d

2tes

t 0.2 gof ft entagw

A

A is the original�-amylase activity andB is the�-amylase ac-tivity in the supernatant after adsorption on raw potato starchgranules.

2.1.2. Determination of raw potato starch digestibilityThe reaction mixtures containing 500 U of�-amylase

preparation from different isolates and 100 mg of raw potatostarch in a final volume of 10 mL dispensed in 100 mL Er-lenmeyer flasks were incubated in rotary water bath shakerat 60◦C and 150 rpm. After a time interval of 6 h, the re-ducing liberated in the reaction mixtures were determined bydinitrosalicyclic acid method[18].

One strain, designated as I-3, giving the best adsorptionand hydolysis at 1% concentration of raw potato starch wasisolated and selected for further studies. It was identified onthe basis of various morphological, physicochemical and bio-chemical characteristics following the criteria laid down byBergey’s Manual of Systematic Bacteriology[19].

2.2. Profile of growth and extracellular α-amylaseproduction by Bacillus sp. I-3 in submerged fermentation

The basal medium used for the submerged cultures wascomposed of: peptone, 10.0 g/L; beef extract, 10.0 g/L; sol-uble starch, 10.0 g/L and sodium chloride, 5.0 g/L; pH 7.0.T kswc eso ewBT rif d ast

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m ith0 is-s archw -t 10%r ondi-t ions.

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thee ndi-v ns,v com-p fs ses ied

. Materials and methods

.1. Microorganism

To screen the raw potato starch digesting microorgannsoluble potato starch granules (SD Fine Chemicals, Interilized by washing in ethanol, were added to sterile nnt agar at 50◦C to a final concentration of 1%. The screenedium was dispensed in petridishes, which were inocuith soil samples collected from the vicinity of various floills of Chandigarh city and incubated for 48 h. Starchrolysis was assessed as clearing zones around the co

The colonies showing starch hydrolysis in the plates wurther evaluated by their enzyme productivity levels in liqulture, after 48 h, employing nutrient broth, supplemeith 1% raw (insoluble) potato starch granules and thehecking the enzyme affinity towards raw potato starch gles by determining the adsorption rate and ability torolyze 1% raw potato starch suspension at 60◦C in 6 h.

.1.1. Determination of raw potato starch adsorbabilityAffinity of the enzyme preparations from various isola

owards raw potato starch was studied by incubatingf raw potato granules with 1 mL of the enzyme at 37◦C

or 15 min. After centrifugation, the�-amylase activity ohe supernatant was measured and the adsorption percas calculated as follows:

dsorption (%)= A − B

A× 100

.

e

wenty milliliters of medium in 100 mL Erlenmeyer flasas inoculated with 2 mL of the inoculum (A600 0.8; cellount 2.5× 108/mL) obtained from 12 h old shake culturf Bacillus sp. I-3 in nutrient broth and incubated on nrunswick environmental shaker (150 rpm) for 72 h at 37◦C.he flasks were analyzed for growth (A600) and pH at regula

ntervals of time. The culture was centrifuged at 10,000× gor 20 min at 4◦C and the cell-free supernatant was usehe enzyme source.

.2.1. Enzyme assayThe activity of �-amylase was determined at 60◦C by

ixing 0.25 mL of appropriately diluted enzyme source w.25 mL of 0.2% (w/v) soluble starch (E. Merck, India) dolved in 0.1 M phosphate buffer, pH 7.0. The residual stas determined after 10 min[17]. One unit of�-amylase ac

ivity was defined as the amount of enzyme that causededuction in the starch–iodine color, under the assay cions. All values given are averages of three determinat

.3. Optimization of raw starch digesting α-amylaseroduction by Bacillus sp. I-3

The enzyme production was optimized by studyingffect of various cultural and environmental variables, iidually, in terms of various carbon, nitrogen, metal ioitamins and amino acids in the basal medium, whoseosition is described in Section2.2, after 48 h of incubation ohake cultures (150 rpm) at 37◦C, pH 7.0, unless, otherwitated (Table 1). The effect of carbon sources was stud

N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734 725

Table 1Effect of different cultural and environmental factors on�-amylase produc-tion by Bacillus sp. I-3 in submerged fermentation

Parameter Enzyme activity(U/mL)

Carbon sources (1%, w/v)Sugars

Fructose 219Galactose 324Glucose 51Glycerol 27Lactose 310Maltose 81Mannitol 19Sorbitol 53Sucrose 16Xylose 11

Gelatinised starchy sourcesSoluble starch 90Corn starch 81Potato starch 181Rice starch 51Wheat starch 218Rice flour 15Wheat bran 22Wheat flour 37

Raw starchy sourcesCorn starch 35Potato starch 172Rice starch 53Wheat starch 55Rice flour 82Wheat bran 51Wheat flour 88

Nitrogen sources (1%, w/v)Beef extract 383Corn steep liquor 216Malt extract 5Bacto peptone 87Peptone M 28Peptone type I 309Proteose peptone 49Soyabean meal 417Soyapeptone 31Tryptone 6Yeast autolysate 33Yeast extract 18

Metal salts (10 mM)LiSO4 215MgSO4·7H2O 12CaCl2·2H2O 178FeCl3 30MnSO4·H2O 22CuSO4·5H2O 12HgCl2 9ZnSO4 7AgCl3 7

Vitamins (0.01%, w/v)Thiamine 100Riboflavin 74Pyridoxine 18Ca pantothenate 104Nicotinic acid 212

Table 1 (Continued )

Parameter Enzyme activity(U/mL)

B-complex 185

Amino acids (0.01%, w/v)Phenylalanine 176Threonine 165Glutamic acid 116Methionine 114Tryptophan 97Leucine 93Lysine 72Valine 68Cysteine 68Tyrosine 68Arginine 67Glycine 59Ornithine 59Histidine 54Aspartic acid 33Proline 31Alanine 22Serine 6

Inoculum size (%, v/v; cell count of 2.5× 108/mL)6 647 738 789 8210 9011 80

Incubation temperature (◦C)30 6737 9042 8450 8060 44

pH of the medium4.0 104.5 315.0 755.5 766.0 776.5 847.0 907.5 818.0 13

The basal medium having 1% soluble starch, 1% peptone, 1% beef extractand 0.5% NaCl with pH 7.0 was used at 37◦C as a control, which pro-duced 90 U/mL. The C, N and mineral components of the basal mediumwere replaced one after the other with other compounds keeping the otheringredients same while the role of vitamins, amino acids, inoculum size,temperature and pH was evaluated using the basal medium.

by replacing soluble starch with different sugars, gelatinizedand raw natural crude starch sources (1%, w/v). Similarly,the effect of nitrogen sources was studies by replacing pep-tone and beef extract with various nitrogen sources at a finalconcentration of 1% (w/v) while the effect of salts was eval-uated by substituting NaCl with various metal salts (10 mM).The inductive effect of various vitamins and amino acids onthe enzyme production was studied by supplementing them

726 N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734

separately at a level of 0.01% (w/v) in the basal medium.The optimal levels of various nutrients were determined sep-arately by varying their concentrations in the basal medium.The inoculum size was standardized by evaluating the ef-fect of varying inoculum size for seeding the basal medium.The effect of incubation temperature and initial pH on en-zyme production was studied by incubating the seeded basalmedium at different temperatures and initial pHs. The syner-gistic effect of all the factors was also studied by employingthe optimal concentrations of various nutrients giving the bestresults in the individual studies and an optimized medium wasworked out.

2.4. Partial purification of the α-amylase preparation

Since, the enzyme preparation was found to readily andstrongly bind to raw potato starch and digest it in signifi-cantly short time, raw starch adsorption-hydrolysis processwas attempted for the purification of�-amylase fromBacil-lus sp. I-3. The enzyme preparation was adsorbed onto theraw potato starch granules and was then subjected to hy-drolysis at 60◦C. The hydrolyzed sample was centrifuged toobtain the residual enzyme in the supernatant, which was di-alyzed against 0.1 mM phosphate buffer, pH 7.0. The enzymewas then precipitated with ammonium sulphate at a satura-t mMT

2p

o d pHv etali awp

2p

tud-i s int fer( byp with1 m5 itya

2pH

r en-t wasm min.T ndi-t

2.5.3. Effect of metal salts on amylase activityThe effect of various metal ions was studied by supple-

menting these, separately, at a concentration of 1 mM in thereaction mixture under normal assay.

2.6. Application of partially purified enzymepreparation from Bacillus sp. I-3 in the hydrolysis of rawpotato starch

A reaction mixture containing 10 mL of 1% raw potatostarch was incubated with 10 U of enzyme preparation permg of starch at temperatures ranging from 40 to 90◦C andthe degree of hydrolysis was determined after 3 and 5 h ofincubation. The starch granules recovered from the reactionmixture were analyzed for SEM studies to confirm the extentof degradation of raw potato starch granules. For SEM stud-ies, the samples, both the untreated and�-amylase treatedraw potato starch granules, were centrifuged at 10,000 rpmfor 5 min and the pellet was washed twice with pure ethanolthen witht-butyl alcohol followed by centrifugation and dry-ing. These were then attached to SEM stub with silver plate[20]. The mounted samples were spatter coated with goldusing fine coat, JEOL ion sputter, model JFC-100 and thenscanned at an operating voltage of 10 kV.

Optimization of the raw starch hydrolysis by the enzymepreparation was also attempted by varying the starch concen-t ffecto yingi on-td ationw

12 ho er-t tog-r r( ace-t

ary-i ther atos nw nt ofh

3

toA y-l udiess terial� eds ound1 tures( sing

ion value of 60% and was again dialyzed against 0.1ris–HCl buffer.

.5. Characterization of partially purified α-amylasereparation

The kinetics studies on the partially purified�-amylasebtained was carried out in terms of the temperature anersus activity and stability profiles, effect of various mons on activity and by studying the affinity of it towards rotato starch granules.

.5.1. Activity versus temperature and thermostabilityrofiles

The effect of temperature on amylase activity was sed by assaying the enzyme at different temperaturehe range of 37–100◦C at pH 7.0 using phosphate buf0.1 M). The thermostability of the enzyme was testedre-incubating the enzyme preparation, without and0 mM CaCl2·2H2O at varying temperatures ranging fro0 to 100◦C for 3.5 h and determining the residual activt regular intervals of 0.5 h.

.5.2. Activity versus pH and stability profilespH activity and stability profiles were studied in a

ange of 4.0–8.0 using different buffers at 0.1 M concrations. For stability studies, one volume of enzymeixed with respective buffers and incubated up to 60he residual activity was determined at normal assay co

ions.

ration and the enzyme dose in the reaction mixture. Ef raw potato starch concentration was studied by var

ts concentration from 1 to 15% in the reaction mixture caining 10 mL of 1000 U enzyme preparation at 70◦C. Toetermine the extent of starch hydrolysis, glucose estimas done after 12 h of incubation.The end products of raw potato starch hydrolysis after

f incubation at 70◦C were withdrawn and identified to ascain the extent of hydrolysis by ascending paper chromaaphy with the solvent system ofn-butanol–pyridine–wate6:4:3) and a detection reagent comprising 100 mL ofone, 0.66 g of phthalic acid and 930 mL of acetone[21].

Effect of the enzyme concentration was studied was vng its concentration from 0.5 to 2 U/mg raw starch ineaction mixture containing 10 mL of 12.5% of raw pottarch granules at 70◦C. Similarly, the glucose estimatioas done after 24 h of incubation to determine the exteydrolysis of starch.

. Results and discussion

Although most of the reports indicate fungi belongingspergillus andRhizopus to be the good producers of am

ases capable of digesting raw starches, few recent stuggest the possibility of raw starch degradation by bac-amylases too[4,7]. Further, most of the reports publisho far indicate the digestion of raw starch granules at ar% concentration by these enzymes at ordinary tempera30–50◦C) and at a very slow rate. As the starch proces

N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734 727

Fig. 1. Pattern of growth, pH and extracellular�-amylase production duringsubmerged fermentation withBacillus sp. I-3 at 37◦C, pH 7.0. The basalmedium used for enzyme production had 1% soluble starch, 1% peptone,1% beef extract and 0.5% NaCl with pH 7.0.

industry employs the mashes containing around 15% starchfor the conventional conversions, there is, thus, an urgent needto explore thermostable raw starch digesting amylases whichcan be used effectively at around 70◦C for the efficient hy-drolysis of much higher concentrated mashes of raw starchesquickly. The bacterial strain ofBacillus sp. I-3 used in thepresent study was isolated from local soil. It was a mesophilehaving a growth temperature in the range of 30–60◦C withan optima at 37◦C and pH 5.0–7.5 with an optima at pH7.0. It produced 90 U/mL of thermostable�-amylase, whichshowed very high affinity towards raw potato starch granules(94% adsorption) and brought about 90% hydrolysis of 1%solution of the starch, at 60◦C after 6 h.

3.1. Pattern of growth and α-amylase production

The enzyme production by most of the organisms isgrowth associated and is induced by the presence of the sub-strate in the medium. Its levels generally increase during theexponential phase and a peak is attained towards the end ofthis phase or during the stationary phase. After the peak, theenzyme levels tend to decline either due to the increase inthe glucose concentration resulting from the degradation ofstarch, or due the rise of protease levels in the environmentor due to the drastic change in pH levels[22–24]. Bacillussp. I-3 grew lograthimatically from 2–24 h and then entered as eringt int laset h andt , int e re-l ringt uba-t L att clinef

3.2. Optimization of α-amylase production during SmF

Amylase production is known to be induced by a variety ofcarbohydrates, nitrogen compounds and minerals[25]. In or-der to achieve high enzyme yield, efforts are made to developa suitable medium and to work out the favorable environmen-tal conditions for the proper growth and maximum secretionof enzyme. The medium is generally selected on the basisof economically feasible components. Development of suchmedium requires the right selection of cheaper and readilyavailable components. The growth and enzyme production ofany organism are greatly influenced by both environmentalconditions and the nutrients available in the growth medium.Some nutrients favor the growth while other induces betteryields of amylase.

Of the various carbon sources used in the present study,galactose at the concentration of 1% proved to be the bestinducer for�-amylase production (324 U/mL) byBacillussp. I-3 (Table 1; Fig. 2a) while soyabean meal at 3% concen-tration proved to be the best nitrogen source for enzyme pro-duction, revealing an activity of 471 U/mL (Table 1; Fig. 2b).Strains ofB. stearothermophilus andB. amylolyticus secretemaximum in a medium supplemented with 1% peptone, 0.5%yeast extract and 0.5% maltose under vigorous shaking con-ditions [26]. Hamilton et al.[3] noted maximum raw starchdigesting�-amylase yield inBacillus sp. in a medium con-t as then beanm on inSm[

sedL umr fs zymep %c ymep noa ninea f1 n thep

or-t uallys ed att( tante h mi-c h ano e pHrs d byt wass d6

tationary phase, of a duration of nearly 30 h, before enthe decline phase (Fig. 1). The amylase production patternhis organism also indicates that the induction of the amyook place during the log phase in the presence of starche maximum yield is obtained, after 48 h of incubationhe mid stationary growth phase which is probably due thease of the all the intracellular fractions of the enzyme duhe stationary phase because of cell lysis. On further incion, the enzyme yield declined gradually to give 40 U/mhe end of 72 h. The pH of the culture broth showed a derom 7.0 to 4.85 after 48 h.

aining lactose as the carbon source and yeast extractitrogen source. Several earlier reports also indicate soyaeal as the best nitrogen source for amylase producti

acharomycopsis capsularis [23], Botryodiploidia theovro-ae [27], Bacillus sp. SN-1[25] and Bacillus sp. AS-1

28].The enzyme yield exhibited a gradual rise with increa

iSO4 concentration up to 20 mM in the production medievealing 246 U/mL (Table 1; Fig. 2e). The requirement oome vitamins has already been observed for the enroduction in different organisms. Nicotonic acid at 0.01oncentration proved to be the most favorable for the enzroduction (Table 1; Fig. 2c). Also, among the various amicids supplemented in the production medium, phenylalat a concentration of 0.02% (Fig. 2d) and inoculum size o0% proved to be the best for the amylase production iresent study (Table 1).

Role of temperature in the SmF growth is very impant and productions of enzymes or metabolites are usensitive to the temperature. Enzyme synthesis occurremperature between 30 and 60◦C with an optimum of 37◦CTable 1). pH is one another among the other most impornvironmental factors for any fermentation process. Eacroorganism has a pH range for its growth and activity witptimum pH range. Enzyme synthesis occurred at a widange from 4.0 to 8.0 with an optimum of 7.0 (Table 1). Whenynergistic effect of the exogenous compounds requirehe organism grown at the optimum temperature and pHtudied in the optimized media, the�-amylase yield touche42 U/mL (Fig. 3).

728 N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734

Fig. 2. Effect of varying concentrations of different nutrients on�-amylase production byBacillus sp. I-3. The basal medium used for optimization had 1%soluble starch, 1% peptone, 1% beef extract and 0.5% NaCl with pH 7.0 and was incubated at 37◦C for 48 h as shake cultures. (a) Soluble starch was replacedwith varying concentrations of galactose; (b) peptone and beef extract were replaced with varying concentrations of soyabean meal; (c) basal mediumwassupplemented with varying concentrations of nicotinic acid; (d) basal medium was supplemented with varying concentrations of phenylalanine; and (e) NaClwas replaced with varying concentrations of lithium sulphate.

3.3. Partial purification and characterization ofα-amylase preparation

As the raw starch digesting amylases have a lot of po-tential in the bulk processing of starch at the industrial scalewhere crude enzymes are generally exploited taking into con-sideration the high cost of enzyme purification. However, itis significant to obtain enzymes with higher specific activityfor their kinetic characterization. Traditionally, the purifica-tion of amylases from fermentation media has been done inseveral steps which include centrifugation of culture filtrate

after extraction from solid media, selective concentration ofthe supernatant by ultrafiltration and selective precipitationof the enzyme by ammonium sulphate or organic solvent suchas ethanol, in the cold, followed by affinity or ion-exchangechromatography and gel filtration[29]. Several one-stepor two-step rapid purification methods including acetoneprecipitation[30], ammonium sulphate precipitation[31],hydrophobic interaction and ion-exchange chromatography[32,33] have been developed for the partial purification ofamylases, but a different protocol has been used in the presentstudy to partially purify the enzyme preparation. Since, the

N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734 729

Fig. 3. �-Amylase yields by submerged cultures ofBacillus sp. I-3 on var-ious media at 37◦C, pH 7.0. Basal medium containing 1% soluble starch,1% peptone, 1% beef extract and 0.5% NaCl with pH 7.0 was incubated at37◦C for 48 h as shake cultures. Optimized medium containing 1% galac-tose, 3% soyabean meal, 0.02% phenylalanine, 0.01% nicotinic acid and20 mM lithium sulphate with pH 7.0 was incubated at 37◦C for 48 h asshake cultures.

enzyme preparation was found to readily and strongly bind toraw potato starch and digest it in significantly short time, rawstarch adsorption–desorption process was attempted for thepurification of�-amylase fromBacillus sp. I-3. The enzymewas allowed to get adsorbed onto the raw potato granules.Desorption of the adsorbed enzyme was tried by using vari-ous elutants (0.1 M phosphate buffer pH 5.0, 5.5, 6.0, 6.5, 7.0and 7.5 and sodium chloride 5, 10, 15 and 20 mM), but noneof these could remove the enzyme adsorbed on the raw potatostarch granules. In a similar study, Saha and Shen[34] triedthe elution of the adsorbed enzyme from raw starch by usingvarious buffers of different pH including raising temperatureand changing pH. These conditions, however, had no signifi-cant effect on elution of the enzyme. On the other hand, Lin etal. [35] purified a raw starch degrading amylase from culturesupernatant ofBacillus sp. TS-23 by adding raw corn starchand the enzyme was eluted from the raw starch with an elu-tion solution containing maltose in Tris–HCl buffer. In orderto elute the adsorbed enzyme, hydrolysis of the starch gran-ules was carried out at 60◦C and the mixture was dialyzedto remove the glucose from it. It was then concentrated byprecipitation with ammonium sulphate and again dialyzed.

When defining the proposed unit of activity for any en-zyme, the International Unit of Biochemistry stated that reac-tion conditions should be specified and optimal. This impliesthat enzymes activities are only valid within a range of physi-c cingm ivitya fors esigno f en-z d pHa nt,

Fig. 4. Temperature and pH vs. activity profiles of�-amylase fromBacil-lus sp. I-3. Optimized medium containing 1% galactose, 3% soyabean meal,0.02% phenylalanine, 0.01% nicotinic acid and 20 mM lithium sulphate withpH 7.0 was incubated at 37◦C for 48 h as shake cultures for enzyme pro-duction. For studying the effect of temperature on enzyme activity, the assaypH was kept constant at 7.0 and for studying the effect of pH on enzymeactivity, the assay temperature was kept constant at 60◦C.

divalent or polyvalent also exert a significant influence on theactivity of an enzyme.

As most of the industrial processes involving�-amylasesoperate at high temperatures exceeding 50◦C, due to higherreaction rates at these temperatures, the thermostable rawstarch digesting amylases are, thus, of great importancefor starch hydrolysis. In this regard, the�-amylase fromBacillus sp. I-3, which was optimally active at 70◦C anddisplayed 97, 93 and 41% of its peak activity at 80, 90 and100◦C (Fig. 4), could be a good candidate for the efficientand quick hydrolysis of raw starches. The enzyme revealeda high thermostability, retaining about 94% residual activityat 70◦C in presence of 10 mM CaCl2·2H2O even after 3.5 h(Fig. 5). The half lives at 80 and 90◦C improved signifi-cantly to 2.5 and 0.5 h, respectively, with supplementation of10 mM CaCl2·2H2O (Fig. 5). The stabilizing effect of Ca2+

on thermostability of the enzyme can be explained due to thesalting out of hydrophobic residues by Ca2+ in the protein,thus causing the adoption of a compact structure[33] andhas also been reported in other organisms[32,36,37].

Many industrial processes involving enzymatic processesdo not have an adequate pH control and the pH usually fluc-tuates around the required value. Thus, pH optima data deter-mined under the controlled conditions of the laboratory needto be applied carefully in processes. It is important to knowthe pH activity curve in quantitative detail for the particular

al properties. Therefore, optimum conditions for produaximum enzyme activities need to be determined. Actnd stability data of enzyme is also useful in screeninguitable new enzymes for a particular process, in the df multi-enzyme processes and in the characterization oyme. Every enzyme has an optimum temperature ant which it has maximum activity. Metal ions, monovale

730 N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734

Fig. 5. Thermostability profiles of�-amylase fromBacillus sp. I-3 at different temperatures. Optimized medium containing 1% galactose, 3% soyabean meal,0.02% phenylalanine, 0.01% nicotinic acid and 20 mM lithium sulphate with pH 7.0 was incubated at 37◦C for 48 h as shake cultures for enzyme production.The enzyme assays were carried out at 60◦C, pH 7.0.

enzyme substrate system under consideration, including theother prevailing physical conditions under which the reactionis to take place so that the maximum potential of the enzymecan be attained. Further, in most of the cases,�-amylasesand amyloglucosidases are used together for the completehydrolysis of the starch as�-amylases generally do not bringabout the complete hydrolysis of starch, the same may betrue for raw starch digesting�-amylases. To exploit the op-portunity of synergism, the temperature and pH optima of the�-amylases and AMGs should be compatible. Thermostable�-amylases currently in use are not stable at lower pH val-ues where saccharification of liquefied starch is carried out[38]. As a result, up to now, starch is liquefied at a pH around7.0. The use of liquefying amylases that are active and sta-

ble around the saccharification pH is economically attractivefor it could avoid or reduce the use of acid to lower the pHfrom liquefying to saccharifying range[38]. This again willminimize the use of ion-exchange media used during down-stream processing[39]. Moreover, the use of�-amylases thatoperate at lower pH values reduce the formation of unwantedproducts, such as maltulose, which is usually produced athigher operation pH[40]. The�-amylase ofBacillus sp. I-3 showed pH optima at pH 7.0 and displayed 96, 97, and98% of its peak activity at pH 5.5, 6.0 and 6.5, respectively(Fig. 4) where most of the amyloglucosidases are also active[40]. The enzyme was also found to be almost 80% stableat pH range of 5.0–5.5 and almost 90% stable at pH rangeof 6.0–7.0 (Fig. 6) and, thus, can also be a potential candi-

N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734 731

Fig. 6. pH stability profiles of�-amylase fromBacillus sp. I-3. Optimizedmedium containing 1% galactose, 3% soyabean meal, 0.02% phenylalanine,0.01% nicotinic acid and 20 mM lithium sulphate with pH 7.0 was incubatedat 37◦C for 48 h as shake cultures for enzyme production. The enzyme assayswere carried out at 60◦C, pH 7.0.

date for synergistic use along with amyloglucosidase for thecomplete hydrolyisis of raw starchy mashes.

The activity of various enzymes is influenced by the pres-ence of metal ions. Some require certain metal ions as cofac-tors and thus called metalloproteins while others are inhib-ited by their presence in the reaction mixture. The enzymeactivity of �-amylase fromBacillus sp. I-3 got significantlypromoted in the presence of FeSO4, CuSO4 and MnSO4 sug-gesting the enzyme to be a metalloprotein needing a cofactorfor its maximum activity (Fig. 7). This is also corroboratedby the inhibitory effect of EDTA on the enzyme activity.

Binding of the raw starch digesting amylases to the starchgranules is usually affected by a C-terminal domain withinthe enzyme, which has been shown to be necessary for degra-dation of granular starch by mould glucoamylases[26]. How-ever, in case of bacterial�-amylases, binding to starch gran-ules is not an obligatory requirement for the hydrolysis ofthe same. Kelly et al.[7] found that raw starch digesting�-amylase ofBacillus sp. IMD 370 showed no adsorbability toany kind of raw starch, at any pH, therefore, showing that ad-sorbability was apparently not necessary for raw starch diges-tion by bacterial enzyme. However, Itkor et al.[4] observedthat the amylase preparation was more than 98% adsorbedonto both raw corn and potato starches. Similar results werealso observed by Saha et al.[41] and Gautam and Gupta[42] where the bacterial enzyme preparation could adsorbo ymep chg affin-i rawp ss oft ules.

Fig. 7. Effect of various metal salts and EDTA on the activity of�-amylasepreparation fromBacillus sp. I-3. Optimized medium containing 1% galac-tose, 3% soyabean meal, 0.02% phenylalanine, 0.01% nicotinic acid and20 mM lithium sulphate with pH 7.0 was incubated at 37◦C for 48 h asshake cultures for enzyme production. The enzyme assays were carried outat 60◦C, pH 7.0.

3.4. Applications of amylase preparation from Bacillussp. I-3 in the hydrolysis of raw potato starch

Raw potato starch granules are the most resistant to en-zymatic hydrolysis because of the larger size of these gran-ules[26]. The potential application of raw starch digesting�-amylase preparation fromBacillus sp. I-3 was evaluated

F mB diumc 0.01%n d at3

nto raw corn starches. Study on the affinities of the enzreparation fromBacillus sp. I-3 towards raw potato starranules revealed that the enzyme had a very strong

ty for the same. It showed 94% adsorption on to theotato starch granules indicating the potential usefulne

he enzyme for the hydrolysis of raw potato starch gran

ig. 8. Pattern of 1% raw starch hydrolysis with�-amylase preparation froacillus sp. I-3 after 3 and 5 h at different temperatures. Optimized meontaining 1% galactose, 3% soyabean meal, 0.02% phenylalanine,icotinic acid and 20 mM lithium sulphate with pH 7.0 was incubate7◦C for 48 h as shake cultures for enzyme production.

732 N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734

Plate 1. Scanning electron micrograph showing the smooth and intact sur-face of untreated raw potato starch granules.

by studying the extent of hydrolysis of raw potato starchgranules. It was observed that the enzyme preparation couldsubstantially hydrolyze the raw starch granules in a short du-ration of time in a temperature range of 60–90◦C indicatingthermostable nature of the enzyme. Maximum hydrolysis ofthe raw potato starch granules at 1% concentration occurredat 70◦C with 90 and 89% conversion to glucose was achievedafter 5 and 3 h, respectively, indicating that most of the hy-drolysis occurred during the earlier 3 h (Fig. 8). This is incontrast to the various published reports where it was seenthat the potato starch is least susceptible to hydrolysis evenafter prolonged incubation of days[26].

The scanning electron microscopy of the treated starchgranules indicates that the granules were more or less com-pletely damaged, thus revealing that the granules were almostcompletely hydrolyzed by the enzyme preparation.Plate 1shows that the surface of untreated starch granules in the rawform was smooth, whereasPlate 2shows that the pores ofthe hydrolyzed raw starch potato granules were randomlydistributed due to the breakdown of the granules by the en-

P pturedr mB

Fig. 9. Hydrolysis of: (a) different concentrations of raw potato starch with100 U/mL of�-amylase preparation fromBacillus sp. I-3; and (b) 12.5% rawpotato starch with varying doses of enzyme preparation. Optimized mediumcontaining 1% galactose, 3% soyabean meal, 0.02% phenylalanine, 0.01%nicotinic acid and 20 mM lithium sulphate with pH 7.0 was incubated at37◦C for 48 h as shake cultures for enzyme production. Hydrolysis in boththe cases was carried out at 70◦C.

zyme. This strongly supports the efficient action of amylasepreparation towards raw potato starch granules.

With a constant enzyme dose, as the concentration ofstarch was increased, conversion efficiency to glucosedecreased. With an increase in starch concentration, thehydrolysis remained almost constant (80–90%) up to 12.5%starch concentration. At 15% concentration, the glucoseformation dropped to 64% after 12 h of incubation (Fig. 9a).

When the enzyme dose was varied from 0.5 to 2 U/mgand incubated with 12.5% concentration of raw potatostarch, almost similar pattern of hydrolysis was observedat all the enzyme doses. With an enzyme dose of 0.5, 1.0and 2 U/mg, 80, 82 and 82% hydrolysis was observed,respectively (Fig. 9b). This strongly suggests that at such alow concentration of enzyme dose (0.5 U/mg) is sufficient

late 2. Scanning electron micrograph showing the deformed and ruaw potato starch granules after treatment with�-amylase preparation froacillus sp. I-3.

N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734 733

Plate 3. Two dimensional paper chromatogram of the end products of rawpotato starch after hydrolysis with�-amylase preparation fromBacillus sp.I-3.

to bring about an appreciable hydrolysis of raw potatostarch at a reasonably high concentration of 12.5%. Paperchromatography results revealed that the main end productsof hydrolysis carried out in the present study were a mixtureof glucose, maltose and maltotriose (Plate 3).

In conclusion, the present study has yielded a highlythermostable bacterial raw starch digesting�-amylase bysubmerged fermentation with a natural isolate ofBacillus sp.I-3. The enzyme was active over a broad temperature rangeof 50–100◦C with optima at 70◦C and a relative activityof 93% at 90◦C. This also displayed a reasonably goodthermostability profile with 94% residual activity after 3.5 hof incubation at 60 or 70◦C. The enzyme revealed a highaffinity towards raw potato starch and got almost completelyadsorbed onto it leading to good degree of hydrolysis atvarious temperatures. Almost all raw starch digesting fungalglucoamylases known to date adsorb to raw starch. On theother hand, raw starch digesting bacterial�-amylases greatlyvary in their ability to bind to starch granules[12,21,43]. Theamylase fromBacillus sp. I-3 completely got adsorbed toraw potato starch granules. This is a very interesting propertyfor it could offer the opportunity of developing an affinityprocedure for the isolation and concentration of the enzymedirectly from the culture broth. In view of these properties,the �-amylase fromBacillus sp. I-3 appears to a good can-didate for the direct hydrolysis of potato starches, omittinga ationr , i.e.0 ns.

Since this study was purely on a laboratory scale at 100 mLErlenmeyer flask level, there is a need to scale-up theproduction and application trials of the enzyme before anyconclusion can be drawn for the commercial utilization ofthis enzyme. It is further pointed out that though the rawpotato starch digesting�-amylase fromBacillus sp. I-3 canalone bring about significant hydrolysis of potato starch,it may also be tried in synergism with some glucoamylasepreparation to bring about a complete hydrolysis.

Acknowledgement

The financial support provided by the Department ofBiotechnology (DBT), Government of India, is highly ac-knowledged.

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