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Analysis, gene cloning and expression of two α­amylases from  Bacillus cereus

Zhang Lin­lin, Wang Qi, Li Rong­xi, Wang Yong­jun and Mei Ru­hong

Chinese Journal of Agricultural Biotechnology / Volume 6 / Issue 02 / August 2009, pp 135 ­ 140DOI: 10.1017/S1479236209002538, Published online: 30 October 2009

Link to this article: http://journals.cambridge.org/abstract_S1479236209002538

How to cite this article:Zhang Lin­lin, Wang Qi, Li Rong­xi, Wang Yong­jun and Mei Ru­hong (2009). Analysis, gene cloning and expression of two α­amylases from  Bacillus cereus. Chinese Journal of Agricultural Biotechnology,6, pp 135­140 doi:10.1017/S1479236209002538

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Analysis, gene cloning and expression of twoa-amylases from Bacillus cereus

Zhang Lin-lin1,2, Wang Qi1,*, Li Rong-xi2, Wang Yong-jun1 and Mei Ru-hong2

1Department of Plant Pathology, China Agricultural University, Beijing 100094, China; 2Department

of Agronomy, Inner Mongolia Agricultural University, Huhehaote 010018, China

Received 25 January 2008; Accepted 7 May 2008

First published in Journal of Agricultural Biotechnology 2008, 16(4): 676–680

AbstractBacillus cereus can produce a-amylase, which has important industrial production value and

can endure high temperatures. The enzymatic characteristics of two a-amylases from B. cereus

(B905 and B904) were studied. The results showed that both preserved their activity at

90–100 �C. Their thermal stability and enzymatic activity did not depend on Ca2+. However,

their protein molecular weights were obviously different. The structural genes of amy905 and

amy904 were cloned successfully using the polymerase chain reaction (PCR) and expressed in

Escherichia coli. The amy904 and amy905 genes were 1362 bp and 1761 bp long and their

protein molecular weights were about 55 kDa and 68 kDa, respectively.

Keywords: Bacillus cereus; a-amylase; gene clone; gene expression

Introduction

Amylase, the enzyme that hydrolyses starch, is found

widely in animals, plants and microbes (Li and Cai, 2004).

Nowadays, amylase is the most important, industrially

produced and, so far, the most frequently used enzyme in

starch processing (Kong and Wang, 1989; MacGregor and

Janecek, 2001). Starch liquefaction is usually conducted

under high temperatures (Du and Hao, 2006), therefore

high-temperature resistance and thermal stability are the

main features of interest regarding the use of amylase

(Guo and Li, 2006). The a-amylase from B. cereus

presents these characteristics as well as a high activity

(Kang and Lee, 2004).

Bacillus cereus B905 and B904 are beneficial endo-

phytic bacteria isolated from plants. The results from

greenhouse and field experiments have shown that both

strains promote plant growth and have a prophylactic

effect on plants. Before colonizing the plant host, these

two strains use available nutrients to intensify their meta-

bolism and increase their population (Kang and Lee,

2004). Extracellular amylase activities have been found

on both strains.

In this experiment, the characteristics of extracellular

amylase of B. cereus B905 and B904 strains were analysed

and the amylase genes were cloned. Results from this

study could help in revealing the molecular mechanism

of plant colonization by Bacillus cereus, and provide new

genetic resources for the industrial production of highly

thermoresistant amylase.

Materials and methods

Bacterial strains and plasmids

The bacterial strains and plasmids used in this experiment

are shown in Table 1. Strains were cultured on plates of

liquid or agarose Luria–Bertani (LB) media. The selective

medium for a-amylase was LBS (LB medium with 1%

soluble starch), supplemented, when necessary, with

50mg/ml ampicillin (Amp) to ensure plasmid stability in

the bacteria.* Corresponding author. E-mail: wangqi@cau.edu.cn

g China Agricultural University 2009 Chinese Journal of Agricultural Biotechnology 6(2); 135–140ISSN 1479-2362 doi:10.1017/S1479236209002538

Enzymes and biochemical reagents

Tris-saturated phenol and Amp were produced by Beijing

Xinjingke Biotechnology (Beijing, China). Restriction

endonucleases XbaI, HindIII and BamHI were from

Promega (Wisconsin, USA). Agarose was from Spain,

packed by TanWei Biotechnology (Beijing, China). dNTP

came from TaKaRa (Dalian, China) and polymerase Taq

DNA and ligase T4 DNA were purchased from TanWei

Biotechnology. Other analytically pure reagents were all

of domestic origin.

Enzymological studies of a-amylase

Plaque detection and determination of enzymeactivity of amylaseB. cereus 905 and 904 were inoculated in solid medium

LBS (Zhou and Ma, 2006). After cultivation at 32 �C for

72 h, 3 mmol/l iodine and 10 mmol/l KI were added for

staining (Zhang and Zhang, 2004). Enzyme activity was

determined following QB/T1803 (Niu and Xu, 2006).

(Enzyme activity: when 1 g enzyme powder or 1 ml

enzyme liquid dissolves 1 mg soluble starch at 60 �C in

1 min, it is one enzyme activity unit expressed in U/g or

U/ml.)

Electrophoretic analysis of amylasesAfter 48 h the culture was centrifuged and the supernatant

was taken as a crude extract of amylase. An aliquot of 5ml

SDS-PAGE upper sample buffer was added to 20ml crude

extract. After electrophoresis by native-PAGE, the gel,

containing 0.5% soluble starch, was treated overnight with

Tris–HCl (pH 6.8) for protein renaturation, and also for

iodine staining activity.

Detection of heat resistance of amylaseEnzyme solutions were treated at different temperatures

(40–100 �C) for 10, 30, 60, 120 and 130 min, respectively.

Then, they were rapidly cooled on ice to room

temperature. The relative activity of the residual enzymes

was measured by comparison with the enzyme activity of

the unheated enzyme solution (100%).

Detection of the acid and alkali resistance of amylaseEnzyme solutions were diluted in buffer solutions at dif-

ferent pH (Cl–HCl pH 2.0–2.2, Gly–HCl pH 3.0, HAc-NaAc

pH 4.0–5.0, Tris–HCl pH 6.0–8.0, Gly–NaOH pH 9.0–10.0,

Na2HPO4–NaOH pH 11.0, KCl–NaOH pH 12.0–13.0)

(Deutch, 2002). After incubating at 40 �C for 1 h, the resi-

dual enzyme activity was measured (the highest enzyme

activity was considered as 100%).

Detection of the impact of metal ions and proteaseinhibitor on enzyme activityDifferent metal ions and the protease inhibitor sodium

dodecyl sulphate (SDS) and ethylenediaminetetraacetic

acid (EDTA) were added into the reaction systems at a

final concentration of 1.0 mmol/l. The enzyme solution

without metal ions was used as a control.

Cloning and expression of genes encoding a-amylase

Total DNA extraction from B. cereusOne Bacillus cereus strain was selected and put in 5 ml LB

liquid at 37 �C, before shaking at 200 rpm until OD600 =0.8. The bacteria were collected from 1 ml culture by

centrifugation and were extracted in 600ml Tris–EDTA

(TE) buffer pH 8.0, containing 2 mg/ml lysozyme, 10% SDS

and 20 mg/ml protease K. After 1 h in a water bath at 37 �C,

solutions of 5 mol/l NaCl and cationic surfactant hexa-

decyltrimethylammonium bromide (CTAB)/NaCl (10%/

0.7 mol/l) were added and well mixed. After incubation in

a water bath at 65 �C for 10 min, the same volume of

phenol/chloroform (1 : 1,v/v) was added and the super-

natant was precipitated with 0.6 volumes of isopropanol.

The precipitate was washed in 70% ethanol, air-dried and

dissolved in 20ml TE buffer (pH 8.0) and preserved

according to Xia (2002).

Table 1. Bacterial strains and plasmids used

Bacterial strains orplasmids Relevant characteristics Source

Escherichia coliDH5a

f80 d lacZDM15 recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 deoRD(lacZYA-argF )U169 F -

This lab

E. coli BL21 hsdS gel(lcIts857 ind1 Sam7 nin5 lac UV-5-T7) This labBacillus cereus 905 Wild type This labB. cereus 904 Wild type This labpET-22b (+) Expression vector; T7 promoter; KanR This labpET-amy904 pET-22b(+) harbouring the entire open reading frame of amy904 from

B. cereus B904Thiswork

pET-amy905 pET-22b(+) harbouring the entire open reading frame of amy905 fromB. cereus B905

This work

136 Zhang Lin-lin et al.

Cloning of amylase genesBased on B. cereus a-amylase DNA sequences in

GenBank, the following primers were designed:

AmyS-5: (50-ACAGGATCCATGCGTGTGGGGAAAATAC-30);

AmyS-3: (50-ACACTCGAGTTTCCGTCTCTTTTTAACC-30).

AmyL-5: (50-ACAGGATCCGATGCTTAAAGAAGC-30);

AmyL-3: (50-ACACTCGAGCCATTTTAATATGGAG-30).

The underlined regions indicate restriction sites, for

BamHI in AmyS-5 and AmyL-5 and for XhoI in AmyS-3

and AmyL-3.

PCR amplifications with genome DNA encoding

B. cereus 905 and B. cereus 904 were carried out, using

the following parameters: denaturation at 94 �C for 5 min;

35 cycles of denaturation at 94 �C for 30 s, annealing at

50 �C for 30 s, and elongation at 72 �C for 40 s; extension

at 72 �C for 10 min. The amplified fragment was recovered

by electrophoresis after double digestion. The recovery

product was inserted into pET-22b(+) after a double

digestion with BamHI and XhoI. Escherichia coli BL21

(DE3) was transformed with the resulting plasmid for

protein expression and sequence analysis (Wang and

Peng, 2007).

Expression of amylase genes in E. coliRecombinant E. coli was cultured overnight at 37 �C in

5 ml LB containing 100mg/ml AMP. It was then inoculated

in 50 ml LB [1% (v/v)] and cultured at 37 �C until OD600 =0.6–0.8. The inducer isopropyl b-D-thiogalactopyranoside

(IPTG) was added at a final concentration of 1 mmol/l.

The culture was incubated at 37 �C for 5 h to induce the

expression of target genes.

Induced bacteria (1 ml) were centrifuged at 12 000 rpm

for 30 s. The pellet was washed with 50 mmol/l

phosphate buffer solution (pH 6.0) and received the

same volume of SDS upper sample buffer (40 mmol/l

Tris–HCl, 10% glycerol, 2% SDS, 5% mercaptoethanol and

0.1% bromophenol blue). The tube was bathed in boiling

water for 5 min, rapidly cooled on ice for 2 min and cen-

trifuged at 5000 rpm for 10 min. A 12% SDS-PAGE of 5ml

supernatant was carried out (uninduced bacteria were

used as a control).

Results

Amylase test of the two strains

After B905 and B904 were cultured in LBS medium for

48 h, they were stained with iodine. The results showed a

bright circle of starch degradation around the growing

colonies (Fig. 1).

The native PAGE, protein renaturation staining and

iodine activity showed differences in the form of the active

protein between these two bacteria (Fig. 2), suggesting

that they (AMY905 and AMY904) are different amylases.

Effect of pH on enzyme activity and stability

The crude amylase enzymes from the two B. cereus

strains were extracted and tested for acid and alkali

resistance at 40 �C. The results showed that their levels of

sensitivity to acidity and alkalinity were similar. The

optimum pH ranged between 6.0 and 10.0. In this range,

both amylases retained more than 50% enzyme activity

in 48 h (Fig. 3), which meant that the two enzymes could

have relatively high activity over a wide pH range.

1 2

Fig. 1. Activity of Bacillus cereus on the 1% starch of LBS substrate. 1, B. cereus 905; 2, B. cereus 904. (See online for acolour version of this figure.)

Analysis, gene cloning and expression of a-amylases from B. cereus 137

Impact of temperature on enzyme activityand stability

The heat resistance test revealed that the a-amylase

generated from both strains maintained high activity in

the temperature range of 40–100 �C (Fig. 4). Their

activities were more than 70 U/ml for both strains,

showing that they were both high-temperature resistant

a-amylases. Below 100 �C, the enzyme activity of strain

B904 was lower than that of strain B905. Their a-amylases

exhibited high heat stability without Ca2+ dependence

(see below). After treatment at 100 �C for 2 h, the enzyme

activity of the amylases were 80% and 60% for B904 and

B905, respectively.

Impact of metal ions and protease inhibitor onenzyme activity

At room temperature and pH 6.0, the crude enzyme was

treated with 1.0 mmol/l different metal ions and protease

inhibitor for 30 min. The enzyme activity was measured

and compared. The results showed that Ca2+, Na+, Li+,

NH4+, Fe2+ and Mg2+ had a slight promoting effect on

the amylase secreted from B905. Whereas Ca2+, Na+, Li+,

NH4+ and Fe2+ had almost no effect on the amylase

from B904, some inhibitory effect was noticed with Mg2+.

Cu2+ strongly inhibited amylase activity for the two

strains (Fig. 5). The protease inhibitors SDS and EDTA

both had a promoting effect on the amylase from the two

strains.

Cloning of genes encoding a-amylase

The gene sequences of a-amylase from B. cereus strains

in GenBank were compared. There were two completely

different a-amylase genes of B. cereus. According to gene

sequences, two primers AmyL-5/AmyL-3 and AmyS-5/

AmyS-3 were designed. The results of PCR amplification

showed that only a 1700 bp band was amplified by AmyL-

5/AmyL-3 from B905 and a 1000 band by AmyS-5/AmyS-3

from B904 (Fig. 6). The sequence analysis revealed that

B905 and B904 contained 1761 and 1362 bp DNA frag-

ments, respectively. From BLAST sequence analysis we

know that both fragments belonged to a-amylase genes,

namely amy905 and amy904, respectively.

Induced expression of a-amylase genes inEscherichia coli

We inserted amy905 and amy904 into plasmids, produ-

cing pET-amy905 and pET-amy904. E. coli BL21 was

transformed with these. After inducing expression with

ITPG, fragmented cells contained a-amylase activity. The

SDS-PAGE results showed protein bands of 55 kDa

(amy904) and 68 kDa (amy905) (Fig. 7).

1 2

Fig. 2. Identification of B. cereus amylases by nativePAGE. 1, B. cereus 905; 2, B. cereus 904.

%

Fig. 3. Effect of pH value on activity of enzymes producedby B. cereus.

Enz

yme

activ

ity (

U/m

l)

Temperature (°C)

Fig. 4. Effect of temperature on enzyme activity producedby B. cereus.

138 Zhang Lin-lin et al.

Discussion

The enzymological analysis showed that a-amylases from

B. cereus 905 and 904 (B905 and B904) conserved their

activity in a relatively wide pH range. The stability tem-

perature of the enzyme activity reached 90–100 �C, and

the thermal stability as well as the enzymatic activity did

not depend on Ca2+. Therefore, these new proteins,

which are high-temperature resistant and extracellular

Ca2+ independent, could solve ongoing problems re-

lated to amylase production. This finding will help to

improve the heat resistance of the enzyme and reduce the

200

150

100

50

0

Rel

ativ

e ac

tivi

ty

CaCl2 Na2SO4 MgSO4 FeSO4 NH4HPO4 CuSO4 EDTA SDSLiCl

905904

Fig. 5. Effects of various salts, SDS and EDTA on enzyme activity produced by B. cereus.

M 1 2bp

514842693530

2027190415871375

948831

M 3 4bp

514842693530

2027190415871375

948831

Fig. 6. PCR analysis of the a-amylase from B. cereus. Lanes: M, marker; 1 and 4, B. cereus 905 PCR product; 2 and 3,B. cereus 904 PCR product.

1 2 3 4 5 kDa

Fig. 7. SDS-PAGE of the purified Amy905/Amy904 from E. coli BL21. Lanes: 1, total protein of E. coli BL21 (DE3) withpET-amy905 induced by IPTG; 2 and 3, total protein of E. coli BL21 (DE3) with pET-amy905/904 before inductionwith IPTG; 4, total protein of E. coli BL21 (DE3) with pET-amy904 induced by IPTG; 5, standard protein marker.

Analysis, gene cloning and expression of a-amylases from B. cereus 139

dependence of its thermal stability on Ca2+. Application

of these gene resources in industrial processes involving

amylase could, to some extent, improve the efficiency

and quality of the production of high-temperature

resistant amylase and reduce production costs.

The amylase genes have been cloned in many bacteria.

Two completely different amylases exist in B. cereus. This

experiment has validated this phenomenon at the DNA

level. The differences of gene length and sequence

showed that their proteins have evolved in two entirely

different ways.

Bacterial strains B905 and B904 were both separated

from plant hosts. The amylase produced extracellularly

could very well help bacteria degrade sugar and ease its

absorption, so that bacterial nutrition could be ensured

when the bacteria colonize plant hosts. The two different

amylase genes provide potential for bacterial genetic

modification and improvement of colonization ability in

plant hosts.

Acknowledgements

This work was supported by National High Technology

Research and Development Plan ‘863 plan’ (No.

2006AA10A211) and Special Agricultural Commonwealth

Research Programs: Physaclospora piricola resistance

resources information database establishment (No.

nyhyzx07055).

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