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Journal of Food Engineering 79 (2007) 950–955

A thermostable a-amylase from a moderately thermophilicBacillus subtilis strain for starch processing

M. Asgher a,*, M. Javaid Asad a, S.U. Rahman b, R.L. Legge c

a Industrial Biotechnology Laboratory Department of Chemistry, University of Agriculture, Faisalabad, Pakistanb Department of Microbiology, University of Agriculture, Faisalabad, Pakistan

c Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1

Received 17 November 2004; received in revised form 27 October 2005; accepted 6 December 2005Available online 29 March 2006

Abstract

A newly isolated Bacillus subtilis JS-2004 strain was cultured in liquid media containing waste potato starch to produce a-amylase.The effect of calcium, yeast extract and glucose supplementation of the production medium on bacterial growth and enzyme productionwas studied. Maximum enzyme production 72 U/mL was achieved after 48 h cultivation at pH 7.0 and 50 �C. Addition of calcium andyeast extract enhanced microbial growth and enzyme production, where as glucose at 1.0% level showed a strong repression. Studies oncrude a-amylase characterization revealed that optimum activity was at pH 8.0 and 70 �C. The enzyme was quite stable for 1 h at 60 and70 �C, while at 80 and 90 �C, 12% and 48% of the original activities were lost, respectively. After incubation of crude enzyme solution for24 h at pH 8.0 at 70 �C, a decrease of about 6% of its original activity was observed. The enzyme was activated by Ca2+ (relative activity117%). It was strongly inhibited by Co2+, Cu2+, and Hg2+ but less affected by Mg2+, Zn2+, Ni2+, Fe2+, and Mn2+. The B. subtilis JS-2004strain produced high levels of thermostable a-amylase with characteristics suitable for application in starch processing and foodindustries.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Thermostable a-amylase; Bacillus subtilis JS-2004; Media optimization; Characterization; Starch processing

1. Introduction

Amylases are enzymes which hydrolyse starch moleculesto give diverse products including dextrins and progres-sively smaller polymers composed of glucose units (Win-dish & Mhatre, 1965). These enzymes have a greatsignificance with extensive biotechnological applicationsin bread and baking, food, textile, and paper industries(Pandey et al., 2000). Amylases having approximately25% of the enzyme market (Burhan et al., 2003; Rao,Tanksale, Gathe, & Deshpande, 1998; Sidhu, Sharma,Chakrabarti, & Gupta, 1997) have almost completely

0260-8774/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jfoodeng.2005.12.053

* Corresponding author. Tel.: +92 42 9200161x3309; fax: +92 419200764.

E-mail address: mabajwapk@yahoo.com (M. Asgher).

replaced chemical hydrolysis of starch in starch processingindustry (Pandey et al., 2000). Thermostable a-amylaseshave had extensive commercial applications in starch pro-cessing, brewing and sugar production (Leveque, Janecek,Haye, & Belarbi, 2000), desizing in textile industries (Hen-driksen, Pedersen, & Bisgard-Frantzen, 1999) and in deter-gent manufacturing processes (Hewitt & Solomons, 1996;Lin, Chyau, & Hsu, 1998). Thermostability is a desiredcharacteristic of most of the industrial enzymes. Thermo-stable a-amylases are available from the mesophile Bacillus

licheniformis (Morgan & Priest, 1981), Bacillus sp. ANT-6(Burhan et al., 2003) and Bacillus sp. ASMIA-2 (Teodoro& Martin, 2000).

Each application of a-amylase requires unique proper-ties with respect to specificity, stability, temperature andpH dependence (McTigue, Kelly, Doyle, & Fogarty,1995). Screening of microorganisms with higher a-amylase

M. Asgher et al. / Journal of Food Engineering 79 (2007) 950–955 951

activities could therefore, facilitate the discovery of novelamylases suitable to new industrial applications (Gupta,Gigras, Mohapatra, Goswami, & Chauhan, 2003; Wander-ley, Torres, Moraes, & Ulhoa, 2004). Thermophilic fermen-tation is also considered quite useful for technical andenvironmental purposes (Kristjanson, 1989; Sonnleitner& Fiechter, 1983). The advantages are, for instance, areduction in cooling costs, a better solubility of substrates,a lower viscosity allowing accelerated mixing and pump-ing, and reduced risk of microbial contamination.However, running a-amylase production processes athigher temperatures will require new process design andimproved knowledge of thermophilic bacteria (Levequeet al., 2000).

In this article the production of thermostable a-amylaseby a moderate thermophilic Bacillus subtilis strain JS-2004isolated in Pakistan is reported.

2. Materials and methods

2.1. Selection and isolation of bacterial strain

B. subtilis JS-2004, a moderate thermophilic bacteriumwas isolated in the Department of Microbiology, Univer-sity of Agriculture, Faisalabad, Pakistan. Two samplesfrom positive growth on nutrient agar medium I consistingof 2% peptone, 1% yeast extract, 1% NaCl, and 2% agar atpH 7.0 were selected. Culture suspensions in sterilizedwater were spread on medium I. The plates were incubatedat 50 �C for 48 h. The bacterial colonies appearing on plateI were transferred to medium II containing 1% solublestarch, 0.2% yeast extract, 0.5% peptone, 0.1% MgSO4,0.1% NaCl, 0.02% CaCl2, and 2% agar at pH 7.0. Thesecultures were incubated at 50 �C for 48 h. Typical culturaland morphological characteristics were observed for Bacil-

lus species. Amylase producing colonies were selected byflooding the media II plates with iodine solution.

2.2. Enzyme production medium

The enzyme production was carried out in the basalmedium of the following composition (%): 0.1 KH2PO4,0.25 Na2HPO4, 0.1 NaCl, 0.2 (NH4)2SO4, 0.005MgSO4 Æ 7H2O, 0.005 CaCl2, 0.2 tryptone, and one wastepotato starch powder. The initial pH of the medium wasadjusted to 7.0 unless otherwise mentioned. The basal med-ium was sterilized by autoclaving at 121 �C for 15 min.Erlenmeyer flasks (500 ml) containing 100 ml of culturemedium were inoculated with 1 ml of an overnight cultureand incubated at 40 �C (except temperature optimizationexperiment) in a rotary shaker at 150 rpm for 144 h. At reg-ular interval (12 h), the triplicate samples were harvestedand the cells were separated by centrifugation (10,000 · g20 min) at 4 �C in a refrigerated centrifuge (EYLA-2001Japan). The supernatant was used for enzyme assay andcharacterization studies.

2.3. Optimization of medium and culture conditions

Initially, the organism was grown in the liquid mediumfor 24–96 h at pH 7.0 and 40 �C and then, supplementedwith calcium (10 mM), yeast extract (0.5%), and glucose(0.5%) to study the effect of these nutrients on growthand enzyme production by B. subtilis JS-2004. The effectof varying pH values (5–10) and temperatures (30–70 �C)on a-amylase production by the bacterium was alsoinvestigated.

2.4. a-Amylase assay

The activity of a-amylase was assayed by incubating0.5 ml enzyme with 0.5 ml soluble starch (1%, w/v) pre-pared in 0.1 M sodium phosphate buffer (pH 7.0). Afterincubation at 37 �C for 60 min the reaction was stoppedby the addition of 2 ml of 3-5-dinitrosalicylic acid reagent(Bernfeld, 1955) and absorbance was measured in a UV/Vis spectrophotometer (Hitachi). One unit (U) is definedas the amount of enzyme which releases 1 lmol of reducingend groups per minute in 0.1 M sodium phosphate buffer(pH 7.0) with 0.5% (w/v) soluble starch as substrate at37 �C.

2.5. Effect of pH on enzyme activity and stability

The pH optimum of the enzyme was determined byvarying the pH of the assay reaction mixture using thefollowing buffers (0.1 M): sodium acetate (pH 5.0–5.5), sodium phosphate (pH 6.0–7.0), Tris–HCl (pH7.5–8) and glycine–NaOH buffer (pH 9–10). To deter-mine the stability of a-amylase, the enzyme was pre-incubated in different buffers (pH 5–10) for 60 min. Theresidual enzyme activity was determined as describedearlier.

2.6. Effect of temperature on enzyme activity and stability

The temperature optimum of the enzyme was evaluatedby measuring the a-amylase activity at different tempera-tures (40–100 �C) in 0.1 M sodium phosphate buffer (pH7.0). The effect of temperature on amylase stability wasdetermined by measuring the residual activity after 1 and24 h of pre-incubation in 0.1 M sodium phosphate buffer(pH 7.0), at temperatures ranging from 40 to 100 �C.

2.7. Effect of metal ions on enzyme activity

For determining the effect of metal ions on amylaseactivity, enzyme assay was performed after pre-incubation,at 60 �C (optimum) for 60 min, of the enzyme with variousmetal ions each at a concentration of 2 mM. The enzymeassay was carried out in the presence of CaCl2 Æ 2H2O,MgSO4 Æ 7H2O, FeSO4, CoCl2, MnSO4 Æ 4H2O, ZnSO4 Æ7H2O, HgCl2, NiCl2, Pb(NO3)2, and CuSO4.

952 M. Asgher et al. / Journal of Food Engineering 79 (2007) 950–955

3. Results and discussion

3.1. Characterization of bacterial strain

The B. subtilis JS-2004 strain was gram positive, positiveon Voges proskauer test by development of pink color in2–3 min. It was motile with 1.5 · 0.5–0.8 lm rods. Thespores were central, greenish in color and were non-bulgingwith rounded ends. Catalase was positive on nutrient agar.Yellow color in methyl red test indicated negative reactionand indol was not formed. Glucose and manitol fermenta-tion tests were positive by color change from red to yellowfor acid production and absence of gas. The pH for growthwas 7.0 and optimum temperature for growth was 50 �C.The strain B. subtilis JS-2004 possessed the ability to pro-duce a-amylase and hydrolyze starch.

3.2. Amylase production

The results on the time-course studies on a-amylase pro-duction and cell growth of B. subtilis JS-2004 grown inbasal medium supplemented with 1% waste potato as indu-cer substrate are shown in Fig. 1. a-Amylase productionpeaked (44.84 U/ml) at 48 h and was found to decline grad-ually up to 96 h (20.12 U/ml). It was observed that maxi-mum a-amylase production by B. subtilis JS-2004occurred when cell population reached the peak. The kinet-ics of enzyme synthesis was more of the growth associatedthan non-growth associated type. Effective induction maynot occur until the stationary phase has been reachedand the readily available carbon source was depleted(Huang, Ridway, Gu, & Moo-Young, 2003; Wanderleyet al., 2004). In the case of a-amylase production by Bacil-

lus flavothermus, enzyme production and biomass peakedtwice and highest activity was obtained after 24 h (Kelly,Bolton, & Fogarty, 1997). Similar findings have beenreported on Bacillus amyloliquefaciens (Hillier, Wase,Emery, & Solomons, 1997) and Bacillus sp. ANT-6 (Bur-han et al., 2003).

0

5

10

15

20

25

30

35

40

45

50

12 24 36 48 60 72 84 96

Incubation time (hours)

Enzy

me

activ

ity (U

/ml)

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Cell dry w

t. (mg/m

l)

Enzyme activity Cell dry wt.

Fig. 1. Time-course studies on growth and a-amylase production byBacillus subtilis JS-2004.

The supplementation of liquid culture medium with10 mM calcium stimulated bacterial growth and enhanceda-amylase production (Fig. 2). The positive effect of cal-cium was observed at all time intervals but the maximumenzyme activity was still after 48 h. a-Amylase is knownto be a calcium metalloenzyme having at least one calciumion associated with its molecule. Enhanced bacterialgrowth and enzyme activity may be the result of increasedavailability of calcium ions (Hewitt & Solomons, 1996).

The addition of yeast extract (1.0%) to the liquid med-ium along with 10 mM calcium increased both the celldry weight and the enzyme synthesis at all incubation peri-ods. The increase in this case was more pronounced than incase of calcium. The result suggests that growth and syn-thesis of a-amylase by B. subtilis JS-2004 is favored byyeast extract. The amylase synthesis by several microorgan-isms has been correlated to the presence or absence of dif-ferent nitrogen sources and various amino acids in thegrowth medium. Organic sources like yeast extract, pep-tone usually have stimulating effects (Forgaty & Kelly,1980; Hamilton, Kelly, & Fogarty, 1999; Hewitt & Solo-mons, 1996).

A decrease in cell growth and enzyme production wasobserved when glucose was added to the fermentation med-ium. The addition of 1.0% glucose to the culture mediumalong with 1% waste potato starch was found to repressthe growth of B. subtilis JS-2004 and synthesis of a-amylase(Fig. 3). Synthesis of amylases in most species of the genusBacillus is repressed by readily metabolizable substratessuch as glucose (Lin et al., 1998). Haseltine, Rolfsmeier,and Blum (1996) reported a repression of a-amylase syn-thesis by glucose addition in Sulfolobus solfataricus anddescribed that glucose prevents a-amylase gene expressionand not merely secretion of preformed enzyme.

The organism showed poor growth in the culture mediaadjusted to pH 5.0, 6.0, and 10.0 (Fig. 4). There was a stim-ulation of enzyme synthesis with an increase in pH from 5to 7 and higher enzyme synthesis at pH 7.0 is a result ofenhanced bacterial growth. Among the physical parame-ters, the pH of the growth medium plays an important role

0

10

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70

12 24 36 48 60 72 84 96Incubation time (hours)

Enzy

me

activ

ity (U

/ml)

0

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1

1.5

2

2.5

3

3.5

4

4.5

5

Cell dry w

t. (mg/m

l)

Enzyme activity Cell dry wt.

Fig. 2. Effect of 10 mM calcium on growth and a-amylase production byBacillus subtilis JS-2004.

0

10

20

30

40

50

60

70

12 24 36 48 60 72Incubation time (hours)

Enzy

me

activ

ity (U

/ml)

Potato starch Potato starch+Glucose

Fig. 3. Effect of 1.0% glucose addition to the medium on a-amylaseproduction by Bacillus subtilis JS-2004.

0

10

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40

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60

70

5 6 7 8 9 10pH

Enzy

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ity (U

/ml)

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1.5

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2.5

3

3.5

4

4.5

5

Cell dry w

t. (mg/m

l)

Enzyme activity Cell dry wt

Fig. 4. Effect of pH on growth and a-amylase production by Bacillus

subtilis JS-2004.

0

20

40

60

80

100

30 35 40 45 50 55 60 65 70

Incubation temperature (°C)

Enzy

me

activ

ity (U

/ml)

0

1

2

3

4

5

6

Cell dry w

t. (mg/m

l)

Enzyme activity Cell dry wt

Fig. 5. Effect of temperature on cell growth and a-amylase production byBacillus subtilis JS-2004.

Enzy

me

activ

ity (U

/ml)

0

20

40

60

80

100

120

5 7 9 10pH

Activity Stability after 24 h

6 8

Fig. 6. Effect of pH on activity and stability of Bacillus subtilis JS-2004 a-amylase.

M. Asgher et al. / Journal of Food Engineering 79 (2007) 950–955 953

by inducing morphological change in the organism and inenzyme secretion. The pH change observed during thegrowth of the organism also affects product stability inthe medium. Most of the Bacillus strains used commerciallyfor the production of a-amylases by SmF have an optimumpH between 6.0 and 9.0 for growth and enzyme production(Burhan et al., 2003; Castro, Hayter, Ison, & Bull, 1992;Jin, Van-Leeuwen, & Patel, 1999). In our study B. subtilis

JS-2004 strain showed optimum growth and maximum a-amylase yield at pH 7.0.

Enzyme synthesis occurred at temperatures between 30and 50 �C. The bacterium could grow satisfactorily at alltemperatures tested but the maximal a-amylase activity inthe growth medium was achieved at 50 �C (Fig. 5). Areduction in enzyme activity was observed at temperaturesabove 50 �C. The influence of temperature on amylase pro-duction is related to the growth of the organism. A widerange of temperature (35–80 �C) has been reported foroptimum growth and a-amylase production in bacteria(Bajpai & Bajpai, 1989; Burhan et al., 2003; Castro et al.,1992; Lin et al., 1998). Very recently Konsula and Liako-poulou-Kyriakides (2004) reported that a thermophilic B.

subtilis strain, isolated from fresh sheep’s milk, producedmaximum extracellular thermostable a-amylase at 40 �Cin a medium containing low starch concentration.

3.3. Characterization of crude a-amylase

3.3.1. Effect of pH on activity and stability of a-amylase

The effect of pH on a-amylase activity and stability isshown in Fig. 6. The a-amylase of B. subtilis JS-2004 strainwas found to be active in very broad pH range. The opti-mum pH was found to be 8.0. The enzyme activity at pH5.5 and 10.0 were 68% and 45% of that at pH 8.0, respec-tively. After incubation of crude enzyme solution for 24 hat pH 8.0, a decrease of about 6% of its original activityat pH 8.0 was observed. At pH 10.0, the enzyme lost54% of its activity. Regarding to Bacillus genus a-amylaseswith optimum activities at pH values as low as 3.5 or ashigh as 12 have been reported (Hayashi, Abiba, & Hori-kosh, 1988; Horikoshi, 1971; Kim, Gu, Jeong, Byun, &Shin, 1995; Konsula & Liakopoulou-Kyriakides, 2004).

3.3.2. Effect of temperature on activity and stability

The supernatant amylolytic activities were assayed atdifferent temperatures ranging from 40 to 100 �C at opti-mum pH (Fig. 7). Enzyme activity increased with tempera-ture within the range of 40–70 �C. A reduction in enzymeactivity was observed at temperatures above 70 �C. Theresidual activity of crude a-amylase incubated at differenttemperatures for a period of 1 h and 24 h was estimatedunder standard assay conditions. The enzyme retained100% of its activity at 80 �C when incubated for 1 h. Afterincubation for 24 h at 50 and 60 �C the enzyme retained 72and 68% of the original activities, respectively. Optimum

0

20

40

60

80

100

120

40 50 60 70 80 90 100

Temperature (ºC)

Rel

ativ

e ac

tivity

(%)

ActivityStability after 1hStability after 24h

Fig. 7. Effect of temperature on activity and stability of Bacillus subtilis

JS-2004 a-amylase.

Table 1Effect of metal ions on activity of a-amylase produced by Bacillus subtilis

JS-2004-1 under optimum conditions

Metal ions (2 mM) Enzyme activity (%) of maximum (86 U/ml)

Ca2+ 117Mg2+ 88Ni2+ 80Fe2+ 70Mn2+ 91Zn2+ 85Co2+ 46Cu2+ 32Hg2+ 27

954 M. Asgher et al. / Journal of Food Engineering 79 (2007) 950–955

temperature of B. subtilis JS-2004 a-amylase was 70 �C,which is comparable to that described for other Bacillus

a-amylases (Bajpai & Bajpai, 1989; Burhan et al., 2003;Dobreva et al., 1998; Jana & Pati, 1997; Lin et al., 1998).Thermostability for 4 h at 100 �C have been reported fora-amylase from B. licheniformis CUMC 305 (Krishnan &Chandra, 1983). Bacillus sp. ANT-6 a-amylase was stableafter overnight (85.5%) and 24 h (55%) incubation at100 �C and pH 10.5 (Burhan et al., 2003). Similar resultswere also reported by Jana, Chattopadhyay, and Pati(1997). A novel strain of Bacillus stearothermophilus iso-lated from the samples of a potato processing industryhad a highly thermostable a-amylase. The temperatureoptimum for the activity of this enzyme was 70 �C butpH optimum for activity was relatively low, in the range5.5–6.0 (Wind, Buitelaar, Egglink, Huizing, & DijKhuized,1994). a-Amylases from Bacillus genus are heat stable andthis is a desirable property for industrial starchliquefaction.

3.3.3. Effect of metal ions on activity and stability

B. subtilis JS-2004 a-amylases was activated by 2 mMCa2+ but inhibited by all other metal ions to a variableextent. Results suggest that a-amylase did not requireany ions for catalytic activity except Ca2+ and was acti-vated (relative activity 117%) by calcium. a-Amylases con-tain at least one Ca2+ ion and affinity of ca is muchstronger than that of other ions (Gupta et al., 2003) slightactivity inhibition was observed by (Mg2+, Ni2+, Fe2+,Mn2+, and Zn2+) since the relative activity was higher than70% (Table 1). A stronger inhibitory effect was observed incase of Co2+, Cu2+, and Hg2+. Konsula and Liakopoulou-Kyriakides (2004) also found that thermostability of an a-amylase from a B. subtilis strain isolated from sheep’s milkwas enhanced in the presence of 8 mM calcium. The inhibi-tion by Hg2+ may indicate the importance of indole aminoacid residues in enzyme function as has been demonstratedfor other microbial a-amylases (Gupta et al., 2003). Theinhibition of B. subtilis JS-2004 a-amylase by Co2+,Cu2+, and Ba2+ ions could be due to competition betweenthe exogenous cations and the protein-associated cations,resulting in decreased metalloenzyme activity (Leveque

et al., 2000). The effects of metal ions on the activity ofa-amylase in Bacillus sp. strain KSM-1378, was investi-gated by Igarashi et al. (1988). Ni2+, Cd2+, Zn2+, andHg2+ ions strongly inhibited the enzymatic activity by 82,91, 100, and 100%, respectively. On the other hand, inBacillus sp. TS-23, Ni2+ and Cd2+ slightly inhibited amy-lase activity.

4. Conclusions

The B. subtilis JS-2004 strain produced high levels ofthermostable a-amylase with characteristics suitable forapplication in starch processing and other food industries.The production process can be commercialized after fur-ther optimization for enhanced enzyme production.

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