ORIGINAL PAPER

Purification and characterization of cyclodextrinb-glucanotransferase from novel alkalophilic bacilli

Ashraf F. Elbaz • Ahmed Sobhi • Ahmed ElMekawy

Received: 21 June 2014 / Accepted: 24 October 2014 / Published online: 4 November 2014

� Springer-Verlag Berlin Heidelberg 2014

Abstract The discovery of novel bacterial cyclodextrin

glucanotransferase (CGTase) enzyme could provide

advantages in terms of its production and relative activity.

In this study, eight bacterial strains isolated from soils of a

biodiversity-rich vegetation in Egypt based on their

hydrolyzing activity of starch, were screened for CGTase

activity, where the most active strain was identified as

Bacillus lehensis. Optimization process revealed that the

using of rice starch (25 %) and a mixture of peptone/yeast

extract (1 %) at pH 10.5 and 37 �C for 24 h improved the

bacterial growth and enzyme activity. The bacterial

CGTase was successively purified by acetone precipitation,

gel filtration chromatography in a Sephadex G-100 column

and ion exchange chromatography in a DEAE-cellulose

column. The specific activity of the CGTase was increased

approximately 274-fold, from 0.21 U/mg protein in crude

broth to 57.7 U/mg protein after applying the DEAE-cel-

lulose column chromatography. SDS-PAGE showed that

the purified CGTase was homogeneous with a molecular

weight of 74.1 kDa. Characterization of the enzyme

exhibited optimum pH and temperature of 7 and 60 �C,

respectively. CGTase relative activity was strongly inhib-

ited by Mg2?, Zn2?, Al3? and K?, while it was slightly

enhanced by 5 and 9 % with Cu2? and Fe2? metal ions,

respectively.

Keywords Bacillus � Cyclodextrin b-glucanotransferase �Purification � Starch

Introduction

The life sciences are quickly improving toward developing

powerful enzymes which permit the synchronized appli-

cation of enzymes with other technologies [1]. Cyclodex-

trin glycosyltransferases (CGTases) (EC 2.4.1.19)

exemplify one of the most vital microbial groups of amy-

lolytic enzymes that have the ability to catalyze cycliza-

tion, disproportionation, hydrolysis and coupling reactions

[2, 3]. CGTases are mainly employed in the industrial

production of cyclodextrins (CDs) through the degradation

of starch and related sugars by intramolecular transglyco-

sylation (cyclization) reaction catalyzed by the CGTase

enzyme [4]. CDs have three main structural types based on

the number of glucose units; a-, b-, and c-CD that have six,

seven, and eight a-1, 4 linked glucose units, respectively

[5]. The assembly of glucose units in a CD molecule leads

to the formation of a void trimmed cone with a hydro-

phobic internal hollow and a hydrophilic external surface,

which allows CDs to form different complexes with several

inorganic and organic compounds, leading to positive

modifications in physical and chemical characteristics of

the introduced molecules [6, 7]. As a result, CDs are

generally employed as complexing agents in pharmaceu-

tical, food, and cosmetic industries [4]. Therefore, there is

growing interest in the development of effective biological

production of CD, which is capable of addressing different

industrial applications.

All authors equally contributed to this paper.

A. F. Elbaz � A. Sobhi � A. ElMekawy (&)

Genetic Engineering and Biotechnology Research Institute,

University of Sadat City (USC), Sadat City, Egypt

e-mail: ahmed.elmekawy@gebri.usc.edu.eg;

amelmeka@gmail.com

A. F. Elbaz

Chemical and Biomedical Engineering Department, Ohio State

University (OSU), Columbus, OH, USA

123

Bioprocess Biosyst Eng (2015) 38:767–776

DOI 10.1007/s00449-014-1318-y

A mixture of different ratios of a-, b- and c-CDs is

usually produced by most CGTases, according to the

source of the CGTase rather than the reaction conditions.

Accordingly, CGTase is categorized into three distinct

types, a-CGTase, b-CGTase and c-CGTase based on the

main CD produced [8]. Fewer bacterial strains can produce

b-CGTase, compared to those that can produce a-CGTases

[9]. Bacteria are still considered as the main CGTases

producers, since Bacillus macerans was discovered as the

first CGTase producer [10]. A wide range of bacterial

groups have been observed to produce CGTase, which are

anaerobic thermophilic, aerobic thermophilic, aerobic

mesophilic and aerobic alkalophilic bacteria. Different

bacterial genera, that are known to produce CGTase,

include Bacillus [2, 9, 11–14], Amphibacillus [15], Ther-

moanaerobacterium [16, 17] and Pyrococcus [18]. Despite

the important role of CDs for several industrial fields, their

expansive applications are still considerably restricted due

to their high prices and low yields [19]. As a result, the

tracking down of new sources for CGTases has a wide

technical and practical impact on the enzymatic production

of CDs [20].

The arable lands of Shebin El Qanater are nutrient-rich

soils that support a wide vegetation diversity, due to the

continuous development of microbial enzymatic systems

contained within the soil for the efficient metabolism of

organic matter. Additionally, enzymatic diversity of

CGTase among alkalophils of Egyptian soil source has

barely been profiled. Accordingly, a systematic study on

the potential diversity of alkalophils from soil habitats of

Egypt would be of great significance. Therefore, the main

objective of this study was to isolate new bacterial CGTase

producer strains from Egyptian soil, in which CGTase was

purified from the soil bacterial isolates and the enzyme

properties were characterized. Also, more investigations

were accomplished to find out the main factors that influ-

ence the bacterial enzyme production.

Materials and methods

Collection of soil samples and isolation of bacteria

Soil samples were collected from rice, corn, potato, wheat

and sweet potato cultivated fields in Shebin El Qanater (El-

Kalubia governorate 30�18045.100N, 31�19022.200E, Egypt),

in clean plastic bags and kept at 4 �C. The general steps to

obtain the purified CGTase enzyme from these samples are

schematized in Fig. 1 and detailed in the next few sections.

The starch-hydrolyzing bacteria were isolated based on

starch hydrolysis activity [21]. Each soil sample (1 g) was

suspended in 10 mL of sterilized water and one drop of the

soil suspension was inoculated onto alkaline starch-agar

plates (1 % soluble starch, 0.5 % peptone, 0.5 % yeast

extract, 0.1 % K2HPO4, 0.02 % MgSO4�7H2O, 1 %

Na2CO3 and 1.5 % agar; pH 10.5). After 24 h of incubation

at 37 �C, the plates were stained with 0.02 % iodine in 0.2

KI solution. Colonies that showed clear zones were con-

sidered as a starch hydrolyzing enzyme producer.

Screening of isolates for CGTase activity

The isolated bacteria were screened on Horikoshi II agar

plate (1 % soluble starch, 0.5 % peptone, 0.5 % yeast

Fig. 1 Overview of the isolation, identification and characterization process of the CGTase isolated from Egyptian soil originated bacteria

768 Bioprocess Biosyst Eng (2015) 38:767–776

123

extract, 0.1 % K2HPO4, 0.02 % MgSO4�7H2O, 1 %

Na2CO3, 0.03 % phenolphthalein (PHP), 0.01 % methyl

orange and 1.5 % agar) according to the method described

by Park et al. [22]. After 24–48 h of incubation at 37 �C,

yellowish colored zones were detected around the CGTase

producing isolates. These bacterial isolates were trans-

ferred to slants of the same growth culture medium without

PHP and methyl orange and maintained at 4 �C. The

selected isolates were identified by GEN III MicroLog

system (BIOLOG, U.S.A) in the Egyptian microbial cul-

ture collection (Cairo Mircen), faculty of Agriculture, Ain-

Shams University, Egypt.

Cultivation and optimization of enzyme production

Each conical flask (250 mL) containing 50 mL of sterile

Horikoshi II broth (without dyes) was inoculated by 1 % of

18 h old Bacillus lehensis strain MLB2 broth culture, and

incubated for 24 h at 37 �C with shaking at 200 rpm unless

otherwise mentioned. At the end of cultivation, the cells

were centrifuged under cooling at 5,000 rpm for 5 min and

the supernatant was used for assaying protein content and

enzyme activity. The enzyme production was optimized

using several growth conditions. These included the dif-

ferent carbon sources substituting soluble starch (potato

starch, corn starch, rice starch, dextrin, sweet potato starch

and glucose), main carbon source concentrations

[0.5–3.5 % (w/v), with increment of 0.5 %], nitrogen

sources [combined 0.5 % (w/v) of yeast extract and 0.5 %

(w/v) of peptone, 1 % (w/v) of corn steep liquor, barley

flour, whey protein or tryptone], main nitrogen source

concentrations [0.5, 1, 1.5 and 2 % (w/v)], inoculum vol-

umes (2, 4, 6, 8 and 10 %), harvesting times (48, 72, 96 h),

incubation temperatures (25, 30, 33, 37 and 40 �C) and

initial pH values (7, 8, 9, 9.5 and 10.5).

The CGTase activity was measured as b-CD forming

activity by the PHP method [23]. A reaction mixture, of

1 mL of 2 % soluble starch in 50 mM Tris–HCl buffer (pH

7) and 50 lL of crude enzyme, was incubated at 55 �C for

10 min. A mixture of 4 mM PHP (4 mL) in ethanol and

125 mM Na2CO3 (pH 11) was added and the color inten-

sity was measured by spectrophotometer at 550 nm. One

unit of the CGTase activity was defined as ‘‘the amount of

enzyme that catalyzes the production of 1 lmol of b-CD

per minute under the reaction conditions’’ [23]. A standard

curve was prepared using various concentrations

(0.04–0.22 lmol) of b-CD in 50 mM Tris HCl buffer (pH

7). The protein content was assayed by Lowry method [24].

Also, the starch in the culture was determined according

to Kitahata et al. [25] method which is based on the col-

orimetric reaction of starch with iodine that would result in

a change of the color from dark blue to purplish blue.

Glucose determination was carried out using glucose

diagnostic kit (Linear ChemicalsTM, USA) that is based on

the colorimetric Trinder reaction [26, 27]. The filtered cells

were washed twice with distilled water and dried in an

oven at 95 �C for 24 h. The samples were placed in a

desiccator to absorb excess moisture before weighing.

b-CD forming activity was measured according to the

method of Goel and Nene [23]. A mixture of culture filtrate

(1 mL) and 4 mM PHP (4 mL) was added to Na2CO3

(125 mM, pH 11), and the color intensity was measured at

550 nm. The b-CD concentration in the culture filtrate was

quantified according to a standard curve of b-CD concen-

trations ranged from 0 to 5 mg/mL.

Bioreactor fermentation

The production of CGTase enzyme from the isolated strain

was executed in a 5 L total volume batch bioreactor (New

Brunswick ScientificTM, U.S.A-BIOFLO 310) containing

2 L working volume of the production medium under the

pre-optimized growth conditions. The fermentation culture

medium was aerated by agitation at 200 rpm and airflow

rate of 1.5 vvm, under uncontrolled pH conditions [12].

Samples were withdrawn on 2 hours basis to examine the

biomass amount, starch/glucose concentrations, b-CD, pH

and enzymatic activity.

Purification of CGTase enzyme

Solvent precipitation was performed by the slow addition

of absolute acetone to 100 mL of the crude enzyme under

cooling and stirring conditions until acetone concentration

reached 50 % [28]. The precipitated enzyme was left under

cooling for 2 h, after which it was centrifuged under

cooling and the precipitate was re-dissolved in 10 mM

Tris–HCl buffer (pH 8), then dialyzed against the same

buffer. The enzyme solution was assayed for its protein

content and CGTase activity, then applied into Sephadex

G-100 column equilibrated using 10 mM Tris–HCl buffer

(pH 8.0), then washed with the same buffer. The retained

proteins were subsequently eluted with the same buffer

containing 0.1 M NaCl at a flow rate of 55 mL/h. Fractions

(10 mL) were collected and assayed for CGTase activity

and protein content. The active fractions were pooled and

concentrated using a cellophane membrane with a 40-kDa

molecular weight cut-off [29].

The gel filtered concentrated enzyme was then loaded to

DEAE-cellulose 52 column (1.5 9 40 cm) equilibrated

with 10 mM Tris–HCl buffer (pH 8.0) [30]. After washing

the column with the same buffer, the enzyme was eluted

with a linear gradient of sodium chloride (0–0.5 M) in the

same buffer at a flow rate of 120 mL/h. Fractions (10 mL)

were collected for the quantifying of enzyme activity and

protein content. Concentrations of active fractions were

Bioprocess Biosyst Eng (2015) 38:767–776 769

123

achieved using a cellophane dialysis membrane (40-kDa

cut-off).

Polyacrylamide gel electrophoresis

Molecular weight of the purified enzyme was determined

by sodium dodecyl sulfate–polyacrylamide gel electro-

phoresis (SDS-PAGE) according to Laemmli [31] on a

vertical slab gel using 12 % polyacrylamide gel. Protein

bands were visualized by staining with Coomassie brilliant

blue R.

Kinetic characterization

The Woolf plot was applied to determine the rate of

CGTase enzymatic reaction in terms of Km and Vmax. The

enzyme activity was tested at different pH values (4–10) at

55 �C for 10 min by using 0.1 M sodium acetate buffer

(pH 4–6), 50 mM Tris–HCl buffer (pH 7–8) and 0.1 M

glycine-NaOH buffer (9–10) [32]. Stability of the purified

enzyme was examined at different pHs by incubating

0.1 mL of the purified CGTase with 0.2 mL of 0.1 M

sodium acetate buffer (pH 4–6), 50 mM Tris–HCl buffer

(pH 7–8) or 0.1 M glycine-NaOH buffer (9–10) at 55 �C

for 1 h [33].

Also, optimum temperature of the purified CGTase was

determined according to Sian et al. [34] by reacting the

enzyme with soluble starch in 50 mM Tris–HCl buffer pH

7 at different temperatures, ranging from 40 to 90 �C for

10 min. Temperature stability of the purified enzyme was

measured using the method described by Jemli et al. [32].

The pure enzyme was incubated in 50 mM Tris–HCl buffer

pH 7 at temperatures ranged from 40 to 90 �C for 1 h.

Moreover, the effect of some metal ions on the CGTase

activity was investigated by its incubation with 1 mM of

each of Ca2?, Mg2?, Ba2?, Zn2?, Mn2?, Cd2?, Al3?, K?,

Cu2? and Fe2? in 50 mM Tris–HCl buffer pH 7, for

30 min at 30 �C [30]. In all cases, the profile of the relative

activity versus each kinetic parameter was plotted by

considering the enzyme activity of the optimum parameter

as 100 %.

Results and discussion

Screening of isolates for CGTase activity

CGTase producing bacteria were isolated by primary

screening for starch-hydrolyzing activity on a starch-agar

plate [35, 36]. Eight bacterial colonies having the ability to

hydrolyse the starch were isolated (Fig. 2a), as CGTase is

considered as one of the amylolytic glycosylase family

[37]. Out of the eight isolates, only two isolates (A and B)

were able to form yellowish zones with different diameters

around their colonies upon the screening of the isolates for

CGTase production on the Horikoshi medium plate con-

taining PHP (Fig. 2b, c). Although the two isolates have

the ability to produce CGTase enzyme, which convert the

starch into CDs, but based on good reproducibility of

CGTase activity, isolate (A) was superior and therefore it

was chosen for further study.

Identification of bacterial strain

The selected bacterial isolate was identified by the Mic-

roLog system as B. lehensis strain MLB2. It was charac-

terized as being a rod-shaped, Gram-positive bacterium

with the capacity to thrive at high pH conditions even up to

pH 11. The phylogenetic analysis (Fig. 3) revealed that the

homology between the isolated strain and the nearest

identified phylogenetic neighbor is 99 %. This analysis

confirmed the novelty of the isolated strain as a source for

Fig. 2 Isolation of starch-hydrolyzing bacteria and screening of their

CGTase activities. a Colony with clear zone on alkaline starch-agar

plate. b Variations in zones diameter of isolates B1 and B2 indicating

different CGTase activities and c Yellowish zones around the CGTase

producing colonies (color figure online)

770 Bioprocess Biosyst Eng (2015) 38:767–776

123

the CGTase, although several Bacillus species (B. aga-

radhaerens [21], B. firmus [38], Alkalophilic B. licheni-

formis [39], Bacillus. sp. NR5 UPM [40] and

B. pseudalcaliphilus [11] and B. macerans [9, 14]) were

considered as strong sources of such enzyme.

Optimization of CGTase production

Several growth conditions were screened for the maximum

activity of CGTase enzyme. The results revealed that the

maximum CGTase production (0.21 ± 0.08 U/mL)

accompanied with high specific activity (0.1 U/mg) was

obtained using rice starch as a carbon source at a concen-

tration of 25 g/L (Table 1). Alternatively, the other carbon

sources showed a lower CGTase activity ranging from 0 to

0.15 ± 0.06 U/mL for glucose and corn starch, respec-

tively. Glucose is not suitable as an inducer for the CGTase

production, but it served as a carbon source for biomass

production [41], hence CGTase production is repressed by

glucose and induced by starch [42]. Rice starch granules

have the smallest granular size and contain a high content

of amylopectin, which is crucial for CGTase production,

but high concentrations of the starch tend to reduce the

CGTase production by increasing the viscosity of the cul-

ture, which led to a poor oxygen uptake [43]. Also, deg-

radation of the high starch concentration may produce

more glucose and small oligosaccharides units, which in

turn suppresses the CGTase production.

Besides, nitrogen is considered as one of the main

building blocks in bacterial metabolism. The direct effect

of nitrogen source and its content was observed, where

peptone and yeast extract mixture was superior, in terms of

CGTase activity (0.34 ± 0.07 U/mL) and specific activity

(0.15 U/mg), compared to other sources (Table 1). The

nitrogen source is very important in regulating the key

enzymatic systems involved in nitrogen assimilation [44].

Also, organic nitrogen sources are serving as important

substrates for improving cell growth and increasing

enzyme production. Yeast extract and peptone are the most

common nitrogen sources used in CGTase production

because they contain some micronutrients that are essential

for growth and production of the enzyme [45]. The lowest

content of peptone and yeast extract mixture (total 10 g/L

with ratio of 1:1) was favorable for enzyme production

(0.36 ± 0.09 U/mL and 0.16 U/mg) (Table 1). Increase of

peptone and yeast extract concentrations in the medium

leads to reduction in the activity and specific activity of

CGTase. In this context, Singh et al. [46] kept the level of

yeast extract and peptone as low as possible to avoid the

repression of CGTase caused by nitrogen source

assimilation.

The culturing of the CGTase producer strain was ini-

tially optimized in terms of inoculum size and initial pH to

improve the enzyme production in the culture medium.

Increasing the inoculum size by fivefolds (from 2 to 10 %)

leads to a 19 % increase in the CGTase activity to reach a

maximum production of 0.43 ± 0.01 U/mL with inoculum

size 10 % (Table 1). Also, a gradual increase in the

CGTase production from 0.25 ± 0.06 to 0.45 ± 0.01 U/

mL and its specific activity was noticed with cumulative

pH values (Table 1), which verified the alkalophilic nature

of the strain under study. Similar results have been reported

for alkalophilic Bacillus species [37, 47], in which the

optimum initial pH for the CGTase production was 10.5.

The CGTase enzyme production depends not only on

nutritional factors, but also is influenced by other growth

conditions such as harvesting time and incubation tem-

perature. Temperature affects the biosorption and bioac-

cumulation process in bacterial cells by influencing the

enzymatic system [48]. The optimum temperature for

CGTase production was 37 �C, with the enzyme reaching

its maximum production after 24 h (Table 1). It is well

known that the cell enters the stationary phase and stop

doubling by extending the incubation period due to

depletion of critical nutrients, which explains the decrease

Fig. 3 Phylogenetic dendogram showing the position of the bacterial

soil isolate in relation to the most correlated similar strains

Bioprocess Biosyst Eng (2015) 38:767–776 771

123

in CGTase production, protein content and CGTase spe-

cific activity. Additionally, the decrease in enzyme pro-

duction was caused by the accumulation of certain by-

products, i.e. glucose and maltose, as a result of cellular

metabolic activity [35].

Batch fermentation

The batch production of CGTase by B. lehensis in a bench-

top bioreactor was carried out under the pre-optimized

growth conditions, including the rice starch as carbon

Table 1 Effect of different

growth conditions on the

CGTase activity

a One unit of the CGTase

activity was defined as the

amount of enzyme that

catalyzes the production of

1 lmol of b-CD per minute

under the reaction conditions

Tested factor Factor’s levels CGTase activity

(U/mL)aProtein

(mg/mL)

Specific activity

(U/mg)

Carbon source (1 %) Soluble starch 0.08 ± 0.01 2.12 ± 0.07 0.04

Potato starch 0.11 ± 0.03 2.26 ± 0.05 0.05

Corn starch 0.15 ± 0.06 2.43 ± 0.08 0.06

Rice starch 0.21 ± 0.08 2.09 ± 0.04 0.1

Sweet potato starch 0.09 ± 0.01 1.97 ± 0.03 0.05

Dextrin 0.13 ± 0.02 2.1 ± 0.08 0.06

Glucose 0 2.21 ± 0.06 0

Rice starch (g/L) 5 0.09 ± 0.01 2.26 ± 0.09 0.04

10 0.17 ± 0.07 2.1 ± 0.04 0.08

15 0.22 ± 0.04 2.12 ± 0.11 0.1

20 0.25 ± 0.08 2.12 ± 0.26 0.12

25 0.3 ± 0.02 2.07 ± 0.09 0.15

30 0.28 ± 0.03 2.2 ± 0.16 0.13

Nitrogen source (1 %) Peptone ? yeast

extract

0.34 ± 0.07 2.31 ± 0.08 0.15

Tryptone 0.3 ± 0.05 2.38 ± 0.14 0.13

Corn steep liquor 0.05 ± 0.01 1.45 ± 0.34 0.04

Barley flour 0.07 ± 0.01 1.3 ± 0.09 0.05

Whey protein dil. 0.07 ± 0.01 0.71 ± 0.04 0.1

Peptone ? yeast extract (g/L,

1:1)

10 0.36 ± 0.09 2.19 ± 0.17 0.16

20 0.32 ± 0.06 2.89 ± 0.16 0.11

30 0.29 ± 0.07 3.18 ± 0.27 0.09

40 0.3 ± 0.05 3.34 ± 0.31 0.08

Inoculums size (%) 2 0.38 ± 0.08 2.13 ± 0.11 0.17

4 0.38 ± 0.04 2.1 ± 0.13 0.18

6 0.4 ± 0.08 2.15 ± 0.08 0.19

8 0.41 ± 0.06 2.19 ± 0.24 0.19

10 0.43 ± 0.01 2.2 ± 0.17 0.2

Harvesting time (h) 24 0.45 ± 0.18 2.1 ± 0.23 0.21

48 0.21 ± 0.08 1.76 ± 0.09 0.12

72 0.14 ± 0.02 1.73 ± 0.07 0.08

Initial pH 7 0.25 ± 0.06 2.3 ± 0.37 0.11

8 0.27 ± 0.04 2.2 ± 0.29 0.12

9 0.29 ± 0.07 2.13 ± 0.18 0.14

9.5 0.37 ± 0.09 2.17 ± 0.21 0.17

10.5 0.45 ± 0.01 2.1 ± 0.15 0.21

Incubation temperature (�C) 25 0.31 ± 0.03 2.12 ± 0.11 0.15

30 0.35 ± 0.02 2.17 ± 0.09 0.16

33 0.42 ± 0.06 2.07 ± 0.17 0.2

37 0.45 ± 0.81 2.12 ± 0.23 0.21

40 0.37 ± 0.08 2.22 ± 0.16 0.17

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source, pH 10.5, 37 �C and 10 % (v/v) inoculum. The

biomass concentration was increased from 1.2 ± 0.08 g/L,

at the start of incubation, to 7.1 ± 0.43 g/L after 22 h and

then decreased to 6.7 ± 0.39 g/L after 24 h of incubation.

During the early stage of the fermentation process, the

CGTase production started after 4 h of incubation with an

observed activity of 0.07 ± 0.01 U/mL. Synthesis of the

CGTase was gradually increased after 10 h when the

growth entered the stationary phase (Fig. 4). The maxi-

mum CGTase production (0.45 ± 0.07 U/mL) was

obtained after 24 h of the fermentation process with a

corresponding specific activity of 0.21 U/mg and protein

content of 2.1 ± 0.18 mg/mL. It was observed that the

maximum CGTase production was achieved after 96 % of

the rice starch was consumed.

The pH of the culture was allowed to drop naturally

without control. The CGTase production was maximum

when the pH of the medium dropped to 9.6 at the end of

fermentation process (Fig. 4). The pH reduction could be

due to the substantial amount of acetic acid released during

the bacterial growth. The high glucose concentration in the

culture medium could lead to a considerable drop in pH, as

the glucose is converted to some organic acids, causing a

decrease in pH [49].

Purification of CGTase

The crude CGTase was purified to homogeneity through

three consecutive steps, starting with 50 % acetone pre-

cipitation, followed by gel filtration chromatography

(Sephadex G-100 column) and finally ion exchange chro-

matography (DEAE-cellulose column). The fraction pre-

cipitated with acetone showed a total enzyme activity of

315 ± 2.73 U, representing 46.6 % of the recovered

activity with a total protein content of 278 ± 3.77 mg

(Table 2). Six fractions (3–8) eluted from the Sephadex

G-100 column (Fig. 5a), possessed the highest recovered

activities, were pooled resulting in a total CGTase activity

of 162 ± 2.62 U and specific activity of 8 U/mg (Table 2),

while the enzyme eluted from the DEAE-cellulose column

(collected from fraction 27 to 34) resulted in a 274-fold

purification with a recovered activity of 7.7 % (Fig. 5b).

The overall purification process resulted in an increase in

specific activity from an average of 0.21 U/mg protein in

crude broth to 57.7 U/mg protein after the DEAE-cellulose

column chromatography step (Table 2). The results were

comparable to formerly reported ones, i.e. the CGTase

enzyme produced by Paenibacillus pabuli has a final spe-

cific activity of 4,000 U/mg and 23-fold purification [32],

while purified CGTase enzyme from alkalophilic B. firmus

was obtained by 80.6 % with 23.1-fold purification [50].

SDS-PAGE revealed that the molecular weight of the

purified CGTase was 74.1 kDa, with a single protein band

shown on the stained gel, to point out the purified enzyme

homogeneity (Fig. 6). Most of the previously purified

CGTases produced from different Bacillus sp. had a

molecular weight ranging from 68 to 88 kDa [34].

Enzyme characterization

The activity of the purified CGTase was measured at dif-

ferent pHs and temperatures by the standard assay method.

The optimum pH was estimated to be 7, after testing a pH

range between 4 and 10 (Fig. 7a), which is within the range

(5–9) of most CGTases from bacterial sources [51, 52].

The enzyme lost its activity considerably when incubated

at a pH less than 5 or more than 9. More than 50 % of the

Fig. 4 Time course of CGTase producer strain of Bacillus lehensis in

batch mode fermentation

Table 2 Summary of the

purification steps of the CGTase

enzyme from Bacillus lehensis

Purification step Total

activity (U)

Total protein

(mg)

Specific activity

(U/mg)

Purification

fold

Recovered

activity (%)

Crude enzyme 675 ± 3.07 3,150 ± 6.31 0.21 1 100

Acetone (50 %)

precipitation

315 ± 2.73 278 ± 3.77 1.1 5.2 46.6

Sephadex G-100 162 ± 2.62 20.3 ± 0.56 8 38 24

DEAE-cellulose 52 ± 1.02 0.9 ± 0.08 57.7 274 7.7

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total activity was reserved after treatment of the enzyme in

the pH range of 6–8. The CGTase enzyme showed a pH

stability in the range of 7–8, with a maximum stability at

pH 7, retaining almost more than 80 % of its initial activity

within this range, while the enzyme was less stable outside

this pH range (Fig. 7c). The stability of purified CGTase

fell within a limited pH range if compared to the one

purified from B. megaterium (pH 6–10.5) [53] or Klebsiella

pneumonia (pH 6–9) [54].

On the other hand, a temperature range (40–90 �C) was

examined for the enzyme activity. The optimum tempera-

ture was found to be 60 �C, and the enzyme activity

sharply declined at temperatures above 80 �C (Fig. 7b).

This value was quite similar to the optimum temperature

range of CGTase previously reported [5, 53, 55]. The

enzyme showed thermal stability up to 50 �C for 1 h

incubation at pH 7. Nevertheless, about 17 and 55 % of its

activity was lost at 70 and 90 �C, respectively (Fig. 7d).

Different extents for the CGTase thermal stability were

previously reported, i.e. several enzymes from Bacillus sp.

(30–80 �C) [51], (30–50 �C) [47] and (40–70 �C) [30].

The CGTase enzyme was inhibited by an array of metal

ions to different extents, where the enzyme was slightly

inhibited by Ca2?, Ba2?, Mn2? and Cd2? and strongly

inhibited by Mg2?, Zn2?, Al3? and K?, while the enzyme

relative activity was improved by 105 and 109 % with Cu2?

and Fe2? metal ions, respectively (Fig. 8). In comparable

studies, the enzyme activity produced from B. megaterium

was inhibited by Zn2?and Ag?, but enhanced by Sr2?, Mg2?,

Co2?, Mn2?, and Cu2? ions [53], whereas, the CGTase

activity produced from alkalophilic Bacillus sp. was inhibited

by Zn2? ions, yet stimulated by Ca2? and Mg2? ions [52].

Incubation of the enzyme with various concentrations of

soluble starch showed that the Km and Vmax values obtained

were 2.2 ± 0.17 mg/mL and 7.8 ± 0.38 mg b-CD/mL/

min, respectively. These values indicated that the produced

enzyme by B. lehensis had a relatively high affinity for the

soluble starch substrate, compared to previously reported

Km values for various CGTases, namely CGTase from al-

kalophilic Bacillus sp. (0.15 mg/mL) [34] and K. pneu-

moniae (1.35 mg/mL) [54], with the same substrate.

Conclusion

CGTase from the soil alkalophilic B. lehensis has been

successfully purified and its production was optimized. The

optimum carbon and nitrogen sources were observed to be

rice starch and 1:1 mixture of peptone and yeast extract,

respectively. Under optimized conditions, 0.45 ± 0.07 U/

mL of the enzyme was produced by the isolated strain

through batch fermentation process. The enzyme exhibits

good thermostability with high affinity for substrate

as indicated by its Km and Vmax values. CGTase from

B. lehensis is a good candidate especially for b-CD

Fig. 5 Elution profile of the bacterial CGTase from chromatography

column. a Sephadex G-100 column and b DEAE-cellulose DE-52

column

Fig. 6 Molecular weight of the purified CGTase enzyme

774 Bioprocess Biosyst Eng (2015) 38:767–776

123

production from starch. The overall reaction and product

specificity of this enzyme could be upgraded by protein

engineering, whereas the extracellular production of the

bacterial CGTase could be enhanced by increasing the

number of genes’ copies that code for it. Moreover, new

CGTase structure could be designed and produced to

improve existing enzyme activity or create new one [1].

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