Biotechnology and Bioprocess Engineering 15: 000-000 (2010)

DOI 10.1007/s12257-009-0072-5

Purification and Characterization of Highly Thermostable α-amylase from Thermophilic Alicyclobacillus acidocaldarius

G. Satheesh kumar, M. Subhosh Chandra, K. V. Mallaiah, P. Sreenivasulu, and Yong-Lark Choi

Received: 6 April 2009 / Revised: 22 October 2009 / Accepted: 23 November 2009

© The Korean Society for Biotechnology and Bioengineering and Springer 2010

Abstract In this study, the production of extracellular

thermostable α-amylase by newly isolated thermophilic

Alicyclobacillus acidocaldarius was detected on LB agar

plates containing 1.0% soluble potato starch and incubated

at 60oC. This extracellular α-amylase was purified to

homogeneity by ammonium sulphate precipitation follow-

ed by Sephadex and ion-exchange chromatography. The

α-amylase was purified to 8.138 fold homogeneity with a

final recovery of 58% and a specific activity of 3,239 U/mg

proteins. The purified α-amylase appeared as a single

protein band on SDS-PAGE with a molecular mass of

94.5 kDa. Non-denaturing PAGE analysis showed one

major band associated with enzyme activity, indicating the

absence of isoenzymes. A TLC analysis showed maltose as

major end product of the enzyme. The optimum assay

temperature and pH for enzyme activity were 60oC and 6.0

respectively; however, the enzyme activity was stable over

a wide range of pH and temperatures. The α-amylase

retained its activity in the presence of the denaturing

agents - SDS, Triton X-100, Tween-20, Tween-80, and was

significantly inhibited by EDTA and urea. Calcium ions

increased the enzyme activity, while Hg2+, Zn2+, and Co2+

had inhibitory effects. The Km and Vmax values were found

to be 2.9 mg/mL and 7936 U/mL respectively.

Keywords: Thermophilic Alicyclobacillus acidocaldarius,

α-amylase, denaturing SDS-PAGE, non denaturing PAGE,

characterization, stability

1. Introduction

Thermophilic microorganisms are of special interest for

production of thermostable enzymes. The α-amylases (EC

3.2.1.1) are exo-enzymes that randomly cleave α-1,4 link-

ages between adjacent glucose units in the linear amylose

chain and ultimately generate glucose, maltose, and malto-

triose units. This class of industrial enzymes constitutes

approximately 25% of the enzyme market covering many

industrial processes such as sugar, textiles, paper, brewing,

baking, distilling industries, preparation of digestive aids,

production of cakes, fruit juices, starch syrups and

pharmaceuticals [1-3]. In all these processes, thinning and

liquefaction of starch is prerequisite and is carried out at

elevated temperatures using thermostable amylases [4].

Biosynthesis of thermostable amylolytic enzymes is rarely

observed in natural bacterial strains [5,6]. Various species

of the genus Bacillus are widely used as producers of

commercial thermostable amylases [5,7,8]. Alicyclobacillus

acidocaldarius ATCC 27009, which grows at 45 to 73oC

and pH 2~4, is a commercial source of acido-thermophilic

enzymes, including amylopullulanase, cyclase, esterase,

endoglucanase, and α-glucosidase [9]. The requirement for

thermostable enzymes with improved properties has initiat-

ed a continuous search for thermophilic microorganisms

producing novel amylases for industrial applications.

Amylases are a group of enzymes that have been found in

several microorganisms i.e. bacteria, actinomycetes, yeast

and fungi and vary not only in their types, but also in their

G. Satheesh kumar*, P. SreenivasuluDepartment of Virology, Sri Venkateswara University, Tirupati 517-502,IndiaTel: +91-97-0104-2538; Fax: +91-877-224-9611E-mail: satheesh.mcb@gmail.com

M. Subhosh Chandra, Yong-Lark Choi*Department of Biotechnology, College of Natural Resources and LifeScience, Dong-a University, Busan 604-714, KoreaTel: +82-51-200-7585; Fax: +82-51-200-6536E-mail: ylchoi@dau.ac.kr

G. Satheesh kumar*, K. V. MallaiahDepartment of Microbiology, Acharya Nagarjuna University, Nagarjunanagar 522-510, Guntur, India

RESEARCH PAPER

2 Biotechnology and Bioprocess Engineering 15: 000-000 (2010)

range of activity depending on temperature, pH, substrate

concentration, inhibitory substances, reducing agents, and

surfactants [5]. Preliminary studies conducted at laboratory

scale, showed that when cultured at 60oC, a newly isolated

thermophilic A. acidocaldarius strain from our culture

abundantly produced an extracellular amylolytic enzyme

that was stable at elevated temperatures and may be of

potential interest for industrial applications. The present

study was conducted to biochemically characterize the

enzyme.

2. Materials and Methods

2.1. Bacterial strain and growth conditions for α-

amylase production

Thermophilic bacteria were isolated from different en-

vironmental samples collected around Tirupati, Chittoor

district, Andhra Pradesh, India. The standard microbio-

logical “starch-iodine plate” assay was used for prelimin-

ary screening for extracellular α-amylase production at

60oC and promising isolates obtained from the hot water

effluent of a boiled rice production mill were used for

further studies. The selected bacterial isolate was tentative-

ly identified as Bacillus sp., based on culturing, Gram’s

staining and spore staining methods. Later, the Microbial

Type Culture Collection Centre, Chandigarh, India, identi-

fied the bacterial culture as A. acidocaldarius MTCC 8766.

The bacteria was cultured for extracellular α-amylase

production in SYT broth (10 g potato soluble starch, 3 g

yeast extract, 3 g tryptone, 1.2 g K2HPO4, 0.2 g KH2PO4,

0.02 g MgSO4, and 0.01 g CaCl2; per liter with the final pH

adjusted to 6.0) at 60oC for 24 h, in an orbital shaking

incubator set at 120 rpm. The crude culture broth was

centrifuged at 10,000 rpm for 30 min at 4oC and the clear

supernatant used for assay of α-amylase activity.

2.2. Assay of α-amylase

The α-amylase activity was routinely measured at 60°C in

a 2 mL reaction mixture that contained 1 mL of 1.0%

(w/v) solution of soluble starch (from potato, SD fine -

chemicals), 0.9 mL of 0.1 M potassium phosphate buffer

(1 mM CaCl2 at pH 6.0) and 0.1 mL of a suitably diluted

enzyme solution. The reducing sugars formed by starch

hydrolysis were measured using the dinitrosalicylic acid

procedure and measuring the absorption at 540 nm using a

spectrophotometer [10]. One unit of enzymatic activity was

defined as the amount of enzyme that produced 1 μM per

min of reducing sugar (glucose) under the assay conditions.

2.3. Purification of α-amylase

Bacterial growth and enzyme production was carried out in

SYT broth medium at 60oC for 24 h. Crude culture broth

(100 mL) was centrifuged at 10,000 rpm for 30 min at 4oC

and the clear supernatant mixed with ammonium sulphate

to a final concentration of 70% saturation. The concent-

rated enzyme from ammonium sulphate precipitation was

collected by centrifugation at 15,000 rpm for 30 min 4oC

and the precipitate was dissolved 3 mL of phosphate buffer

0.1 M, pH 6.0, and the mixture was loaded on Sephadex

gel exclusion column (Sigma, Sephadex G 80-120, 1.9 X

30 cm) which was equilibrated prior with the same buffer.

The enzyme was eluted at a flow rate of 0.5 mL/min using

the same buffer. The active fractions were combined and

loaded on anion exchanger (Biorad, anion exchanger, 5 mL

matrix in 10 mL surgical syringe) which was prior equi-

librated with 50 mM Tris-HCl pH 8.3. The bound protein

was eluted using 50, 100, 150, 200, 250, and 300 mM

NaCl concentrations. The active fractions were combined

and used in further experiments as the purified enzyme.

The enzyme activity in different stages of purification was

determined as described above and the protein concent-

ration was measured by using bovine serum albumin (BSA)

as the standard [11].

2.4. Denaturing (SDS-PAGE) and non denaturing

(Native) PAGE analysis of α-amylase

SDS-PAGE was performed for the purified enzyme as

described by Laemmli [12] using a 12% resolving slab gel.

After electrophoresis, the enzyme protein band was visuali-

zed by staining with Coomassie brilliant blue R-250. The

enzyme separation and starch hydrolytic activity in native

polyacrylamide gel (activity staining) analysis was per-

formed by excluding the 2-ME and SDS in PAGE

preparations and adding 0.01% solution of soluble potato

starch to produce an 8.0% PAGE resolving gel mixture.

The experiment was performed at 4oC to avoid tracking

starch hydrolytic effects of the enzyme in resolving gel.

After separation, the protein associated with amylase activity

was visualized by incubating gel at 60oC for 10 min in

20 mL of potassium phosphate buffer (0.1 M, pH 6.0

containing 1 mM Ca2+) and flooding the gel with Iodine

solution (I2 0.33% and KI 0.96%), to detect the enzyme

activity.

2.5. Chromatography of hydrolysis products

The purified enzyme (50 μL), plus substrate solution 1.0

mL (1% soluble potato starch in phosphate buffer, 1 mM

Ca2+), was incubated at 60oC for 20 min for determination

of enzyme activity. A 50 μL aliquot was collected from the

enzyme reaction mixture and the enzyme activity was

stopped by adding 5 μL of 0.1 N NaOH. The hydrolysis

products were separated by thin-layer chromatography on

silica gel using a mixture of butanol/ ethanol/water (3:5:2,

Purification and Characterization of Highly Thermostable α-amylase from Thermophilic Alicyclobacillus acidocaldarius 3

v/v) as the mobile phase [13]. A 1 mM solution of glucose

and maltose was used as standard. Carbohydrates were

visualized by spraying the plate with detection reagent (3 g

phenol, 5 mL 95% H2S04, and 95 mL ethanol) followed by

incubating the plate for 10 min at 120°C [14].

2.6. Effect of temperature, pH, and enzyme stability

The effect of assay temperature ranging from 30 to 90oC

and pH of 4, 5 (0.1 M acetate buffer), 6, 7, 8 (0.1 M

potassium phosphate buffer), and 9 (0.1 M glycine-HCl

buffer) on α-amylase activity was determined. Thermal

stability of the enzyme was examined by incubating the

enzyme at 30~80oC for 2 hours in 0.1 M potassium phos-

phate buffer, pH 6.0 without substrate and then measuring

the enzyme activity. The pH stability was determined by

incubating the enzyme at pHs of 4~9 in the same above

buffers for 12 h without substrate and then measuring

remaining enzyme activity.

2.7. Effect of denaturing agents and heavy metal ions

The effect of denaturing agents and metal chelating agents

such as SDS, TritonX-100, Tween-20, and Tween-80 at

0.02, 0.04, and 0.06%, urea at 0.5 and 1.0%, EDTA at 20,

40, and 60 mM and various heavy metal ion salts at 1 mM

concentrations on the α-amylase activity was determined

by incorporating the agents into the assay mixture.

2.8. Enzyme kinetics

One of the most fundamental factors affecting enzyme

activity is substrate concentration. The effect of substrate

concentration on activity is usually expressed in Km and

Vmax values using a double reciprocal Lineweaver-Burke

plot. In the present study these values were determined by

varying the substrate concentration from 0.2 to 1.0%. The

data was analyzed and plots were drawn using the

GraphPad Prism 5.0 software package.

3. Results and Discussion

3.1. Production of α-amylase

We have screened and isolated many starch hydrolytic

bacteria by incubating at 60oC and the isolate obtained

from the hot water effluent of a boiled rice production mill

was found promising because it formed a significant

hydrolytic clear zone using the standard “starch iodine-

plate” assay method. In the present study, the selected

thermophilic species was identified as A. acidocaldarius

MTCC 8766 and was used for enzyme production. The

isolate produced extracellular thermostable α-amylase (776

U/mL) in SYT broth medium at 60oC incubation.

3.2. Purification and Poly Acrylamide Separation of α-

amylase Denaturing (SDS-PAGE) and Non-Denaturing

(Native) PAGE Analysis of α-amylase

The active fractions eluted following sephadex and ion

exchange column chromatography were analyzed by SDS-

PAGE and appeared as a single band with an apparent

molecular mass of 94.5 kDa. The native polyacrylamide

gel stained with iodine solution showed a single amylase

activity band, which coincided with that detected in SDS-

PAGE (Fig. 1). The purification of fermented broth

medium by ammonium sulphate precipitation followed by

Sephadex G and Biorad macroprep Q-strong anion

exchanger yielded a protein fraction with specific activity

of 3,239 U/mg, and 8.138 fold purification (Table 1).

Thermophilic microorganisms and their enzymes have

potential application in starch processing industries for

liquefaction and saccharification. Traditionally α-amylases

Fig. 1. SDS-PAGE analysis of purified amylase: Lane 1 - activefractions of sephadex purified sample, Lane 2 - marker protein,Lane 3 - Ion exchange purified enzyme, Lane 4 - marker proteins,and Lanes 5 & 6 - Native PAGE analysis of purified amylase.

Table 1. Purification of extracellular α-amylase from Alicyclobacillus acidocaldarius

Purification stepTotal protein

(mg)Total activity

(U)Specific activity (U/mg protein)

Purification fold

Percentage of recovery

1. Culture supernatant2. Sephadex gel exclusion chromatography3. Ion exchange chromatography

195.64314.2

780005400046000

39812553239

13.158.138

1006958

4 Biotechnology and Bioprocess Engineering 15: 000-000 (2010)

produced by various bacterial species have been purified

by conventional methods employing a combination of

ammonium sulphate precipitation, ion exchange chromato-

graphy, and gel filtration chromatography. Damodara Rao

et al. [15] studied a rapid method for the affinity puri-

fication of thermostable α-amylase from Bacillus licheni-

formis. There are several reports in the literature on the

purification and SDS-PAGE analysis of α-amylase produced

by various Bacillus species. The reported isoenzymes of

α-amylase are 1-3 and their Mr ranged from 42 to 150 kDa

[5]. The Mr 94.5 kDa of α-amylase of the present study is

comparable to Mr 97 kDa α-amylase produced by Bacillus

sp. [16]. Raoudha Ellouz Ghorbel et al. [17] reported a

Bacillus cohnii US147 amylase with molecular mass of

30 kDa and a single alkaline amylase band when stained

for activity in native polyacrylamide gel. The reported

activities for purified α-amylases in the genus Bacillus sp.

include B. licheniformis with 147.5 U/mg, thermoactive α-

amylase from Bacillus subtilis with 2205 U/mg, Bacillus

sp. ANT-6 with 195 U/mg, Bacillus sp. NRRL B 3881 18.5

U/mg, B. subtilis with 5,000 U/mg and Bacillus sp. L1711

with 1,483 U/mg protein [5,18-22].

3.3. Chromatography of enzyme hydrolysis products

Thin layer chromatography of hydrolysis products demon-

strated the production of maltose as an end product of α-

amylase activity (Fig. 2). Amylases are generally classified

based on starch hydrolysis products. Maltose was the

major end product and identified the enzyme as sacchari-

fying maltogenic type (α-amylase). Similar results were

reported for α-amylase of B. subtilis [23] and C. acetobut-

ylicum [24].

3.4.Temperature and pH dependence

The α-amylase activity increased with an increase in assay

temperature between 30 and 60oC and declined thereafter.

The α-amylase activity was maximum at assay temperature

of 60oC and was low at temperatures of 40 and 80oC. The

effect of temperature on enzyme stability showed no

significant loss of activity up to 60oC and more than 50%

activity remained following 2 h incubation at 70oC (Fig. 3).

The influence of pH within a range of 4~9 was studied.

The enzyme activity was stable in assays using pH of 5 to

7, and the activity increased with pH up to 6.0, but did not

show increased activity at pH > 6.0. The pH stability of the

enzyme showed more than 50% maximum activity from 4

to 7 and thereafter declined significantly (Fig. 4).

Fig. 2. Thin layer chromatography of sugars released by hydro-lysis of soluble potato starch by partially purified α-amylase: Lane1 - glucose, 2 - maltose, 3 - soluble potato starch with enzyme,and 4 - starch without enzyme.*M.O - maltooligosaccharides.

Fig. 3. Effect of enzyme assay temperature and stability onpurified α-amylase activity.

Fig. 4. Effect of enzyme assay buffer pH and stability on purifiedα-amylase activity.

Purification and Characterization of Highly Thermostable α-amylase from Thermophilic Alicyclobacillus acidocaldarius 5

In the present study, an assay temperature of 60oC and

pH of 6.0 were found to be optimal for enzyme activity.

The effect of temperature on activity and stability of puri-

fied α-amylase showed high stability to a wide range from

40 to 70oC. This feature makes it possible to use the

enzyme at elevated temperature for starch hydrolysis. The

enzyme exhibited a wide range of acidic to neutral pH

stability. Goyal et al. [25] have reported an optimum

temperature of 70oC for Bacillus sp. I-3 α-amylase activity.

The assay pH range of 5~11 has been reported for am-

ylases produced by different bacterial species [5].

3.5. Effect of denaturing and metal ions

The extracellular thermostable α-amylase produced by A.

acidocaldarius was stable in various surfactants such as

SDS, TritonX-100, Tween-20, and Tween-80 and was

inhibited by urea and the metal chelating agent EDTA

(Fig. 5). Among the tested metal ions, calcium ions increased

the enzyme activity, while Fe2+ and Ni2+ appeared to have

no significant inhibitory effect. Mg2+, Hg2+, Zn2+, and Co2+

have inhibitory effects on α-amylase activity (Fig. 6).

The effect of surfactants on amylase activity was found

to be very low; hence the enzyme may be confidently used

in detergent industries. The denaturation with urea is due to

the presence of hydrophobic amino acid residues in its

composition [26]. The fact that EDTA produced significant

inhibition provides evidence that the enzyme’s activity is

metal dependent. From the various metal ions which were

studied for effects on the α-amylase, Ca2+ ions increased

enzyme activity where as other tested ions had either no

effect or an inhibitory effect. Ca2+ ions probably stabilized

the enzyme at high assay temperatures and thus increased

activity. Pandey et al. [5] have stated that most α-amylases

are known to be metal ion-dependent enzymes, affected by

divalent ions like Ca2+, Mg2+, Mn2+, Zn2+, or Fe2+. The

inhibition by Zn2+ was found to be an important parameter

determining the thermostability of amylase [27]. Burhan et

al. [19] and Goyal et al. [25] have reported that Ca2+

increased α-amylase activity. Similar results were obtained

with the thermostable amylase of Bacillus sp. TS-23 [28].

This could be due to the salting out of hydrophobic

residues in the protein by Ca2+, thus causing the adoption

of a compact structure. Ca-independent enzymes have also

been reported from B. thermooleovorans NP54 [29]. The

majority of α-amylases were inhibited by metal ions, and

alkaline amylases varied in their response to the chelator

EDTA [19,30]. Strong inhibitory effects of EDTA were

reported for alkaliphilic amylases from Bacillus isolates

TS-23, GM8901 and KSM-1378 [30].

3.6. Enzyme kinetics

The influence of substrate (soluble potato starch) concent-

rations from 0.1 to 1.0% was studied. The enzyme activity

increased with an increase in starch concentration from

0.1 to 0.4%, but further increase of starch concentration

Fig. 5. Effect of surfactants, EDTA, and urea on purified α-amylase activity.

Fig. 6. Effect of heavy metal ions at 1 mM concentration onpurified α-amylase activity.

Fig. 7. The effect of substrate concentration on amylase activity.The Km and Vmax values of double reciprocal Lineweaver-Burkeplot.

6 Biotechnology and Bioprocess Engineering 15: 000-000 (2010)

produced no significant increase of enzyme activity. The

Km and Vmax values were found to be 2.9 mg/mL and 7,936

U/mL respectively (Fig. 7). Similar studies were performed

using B. cohnii US147 and reported Km and V

max values for

amylase of 0.7 mg/mL and 2.2 U/mL, respectively [17].

The reported Km values for various α-amylases in the

genus Bacillus sp. include those from Bacillus sp. ANT-6

(3.85 mg/mL) and Bacillus sp. NRRL B 3881 (1.9 mg/mL)

[19,20]. In the present investigation we have purified and

characterized a highly thermostable α-amylase from newly

isolated thermophilic A. acidocaldariu, this enzyme is

attractive for applications in starch saccharification.

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