Cyclodextrin glycosyltransferase (CGTase) or [1,4-α-D-glucopyranosyl]-transferase
is an extracellular enzyme, which degrades starches into cyclodextrin
(CDS) molecules via cyclization reaction. Cyclization happens when a linear
oligosaccharide (starch) chain is cleaved and the new reducing end sugar
is transferred to the non-reducing end sugar of the same chain. Therefore
cyclodextrins are cyclic oligosaccharides consisting of 6-12 units of
glucose joined by the α-1, 4-linkages. CGTases also catalyses two
intermolecular transglycosylation reactions: coupling, in which a cyclodextrin
ring is cleaved and transferred to an acceptor maltooligosaccharide substrate
and disproportionation, in which a linear maltooligosaccharide is cleaved
and the new reducing end sugar is transferred to an acceptor maltooligosaccharide
substrate. Besides these reactions, the enzyme has a weak hydrolyzing
activity (Penninga et al., 1995; Bart et al., 2000). Cyclodextrins
with 6, 7 and 8 glucose units are most common and also known as α-,
β-and γ-cyclodextrin, respectively.
CGTases with varying properties are produced by bacteria
mainly belonging to the Bacillus species, by submerged culture
in a complex medium (Adriana et al., 2002). Some of the known sources
of CGTase producers are Bacillus macerans, Bacillus subtilis,
Bacillus stereothermophillus, Bacillus megaterium, Klebsiella
pneumonia and micrococcus species. Alkalophilic microorganism is also
known to produce unusual enzyme that can be used in industrial and other
processes. All known CGTases (Bart et al., 2000) produce a mixture
of cyclodextrins (and linear malto-oligosaccharides) when incubated with
starch. The CGTase crude enzyme isolated from local Bacillus sp.
produces alpha (α) and beta (β) cyclodextrin only.
However a CGTase, which only produces a single type of
cyclodextrin, is industrially favorable. The aims of this research are
to optimise CGTase production using local isolated strain by Response
Surface Methodology approach and finally production of cyclodextrin using
the optimized condition obtained.
MATERIALS AND METHODS
Screening and isolation of microorganism: Soil
samples were collected from various places such as sago plantation, herbs
plantation and soil near marketplace in Malaysia. Criteria for soil selection
were mainly based on its soil`s pH (pH 5-6). Collected soil samples were
suspended in sterile saline and the solid particles were allowed to settle.
Plating was done at high pH (pH 7-9) to discourage the growth of fungal
species and screening was done according to Park et al. (1989).
Bacterial colonies with yellowish clearance zone were selected and streaked
onto Horikoshi-Phenolphthalein (PHP) plate for several times until uniform
colonies were formed. Suspensions of vegetative cells were grown in Horikoshi
Broth (HB) for 24 h at 37 °C, mixed with sterilized glycerol (20%
v/v) and kept in 1 mL aliquots at -80 °C until further used.
Morphological characterizations: Microscopic morphological
characteristics include the shapes of cells and the characteristic arrangements
of cells in groups. On the other hand, the macroscopic properties of pure
cultures including the colour of colonies, shape and odour can be considered
as among the morphological characteristics of microbes.
Biochemical identification of microorganism: Biochemical
test was used to characterize the isolated microorganisms. API 50 CHB/E
Medium kit was used in assistance of confirmation of the genus of bacteria
based on biochemical reaction.
Preparation of bacterial inoculums: Bacillus
sp. (Strain MK6) were grown in 20 mL Horikoshi medium (Park et
al., 1989) contained the following solutions after autoclaving. Solution
1: Starch 1.0% (w/v), peptone 0.5% (w/v), yeast extracts 0.5%(w/v), K2HPO4
0.1% (w/v), MgSO4 0.02% (w/v). Solution 2: Na2CO3
1.0% (w/v). The culture was incubated at 37 °C, agitated at 200 rpm
for 18 h. Cells were harvested by centrifugation at 5000 rpm for 5 min
and washed once with normal saline solution (0.85% w/v NaCl) and were
then suspended in normal saline solution to give an optical density reading
of 0.5 at 660 nm, using a UV-spectrophotometer.
Production of crude CGTase: Ten percent (v/v)
of bacterial inoculums was inoculated into 100 mL of Horikoshi Medium
in a 500 mL conical shake flask. The bacteria culture was incubated at
37 °C in an incubator shaker for 24 h. At the end of incubation period,
1.0 mL of the culture was removed and separated by centrifugation at 8000
rpm for 10 min at 4 °C. The supernatant was assayed for CGTase activity
and used as crude enzyme solution.
Optimization using Response Surface Methodology (RSM)
approach: Response Surface Methodology or RSM is a collection of statistical
techniques for designing experiments, building models, evaluating the
effects of factors and searching optimum conditions of factors for desirable
responses (Montgomery, 1976). Factorial designs of a limited set of variables
is advantages in relation to the conventional method of the manipulation
of a single parameter trial, because such an approach frequently fails
to locate optimal conditions for the process due to its failure to consider
the effect of possible interaction between factors. In direct fermentation
of starch to production of CGTase, the preliminary test had indicated
that the CGTase production is significantly affected by the concentration
of starch, pH values and agitation speed. However, the true relationship
between the response, Y and the independent variables is unknown.
Therefore the Y can be written as a function of the levels of the
variables χ1 with a significant influence on CGTase production
The nature of this function is unknown but usually these
kind of responses can be approximated by a second-order polynomial, as
shown in Eq. 2:
Where, Y is the predicted response, βo,
βi, βii and βij are constant
and regression coefficients of the model and χi is the
independent variable in coded values. In this study, k = 3 because there
were three independent variables involved. Thus the mathematical relationship
connecting to the three variables and the response from Eq.
||= The predicted response,
||= Offset term,
|B1, B2, B3
||= Linear coefficients,
|B11, B22 and B33
||= The quadratic coefficients,
|B12, B13 and B23
||= The cross product coefficients,
|X1, X2 and X3
||= The independent variables.
|| Actual factor levels corresponding to the coded factor
A randomization step was conducted using completely randomized
design. Each treatment consist of 3 replicates were treated and given
a number started from 00 until 26 with 27 total treatment. Using a random
digit table, the treatment was assigned according to the number appeared
in order. The range and the levels of the variables investigated in this
study are given in Table 1. Response Surface Design
using MINITAB software (version 13.1) was used to conduct the RSM analysis.
A second-order polynomial expression of three variables as Eq.
2 was fitted. In order to visualize the relationship between the response
(Y) and the experimental levels of each factor, surface plots were generated
from the fitted second order polynomial equation. After collecting the
experimental data and determining the optimum pH, sago starch concentration
and agitation speed, optimum response was verified by real experiments
under optimum condition. Response was monitored and results were compared
with model prediction.
CGTase assay: CGTase activity was determined using
phenolphthalein assay (Kaneko et al., 1987).
Determination of cyclodextrin and sugars: The
cyclodextrin and sugars concentration was determined using HPLC. Samples
were first centrifuge to remove suspended solids and biomass. Centrifugation
was done at 12,000 rpm for 10 min. The supernatant was then filtered through
a nylon filter paper with 0.45 μm pore size. The liquid chromatograph
comprised of a Jasco-PU 980 pump (Jasco, Japan) and a differential refractive
index detector (Perkin Elmer LC-25, USA) with a sensitivity of 5x10-5
RIU. Data integration was done using Borwin Software package V1.21 for
liquid chromatography integration. The column used was Merck NH2
column (Purospher Star; 5 μm, 250x4.6 mm). Injections were carried
out using a 20 μL sample loop at room temperature (25-28 °C)
with 75% acetonitrile as the mobile phase with the flow rate of 1.0 mL
min-1. Samples (optimized crude enzyme) and cyclodextrin standard
was prepared using deionized water.
RESULTS AND DISCUSSION
Isolation of microorganism: A total of 250 isolates
have been successfully isolated in this study. CGTase activity was detected
using plate assay containing soluble starch as substrate and phenolphthalein
as indicator. However strain MK6 gave the highest CGTase activity through
the formation of large diameter (5 cm) of clearance zone as a qualitative
Morphological characteristics: Gram staining was
done to determine the basic morphological characteristics of the isolated
microorganism. Gram staining showed strain MK6 was gram positive, rod
in shape with rounded ends
Biochemical identification of strain MK6 using API
50 CHB/E medium: Biochemical identification was done after selection
of the best strain for production of CGTase enzyme. The choices of medium
used in this experiment depend much on the knowledge of morphological
characteristic of the strain MK6 done earlier. API 50 CHB/E medium was
used based on these characteristics as shown in Table 2.
Characteristics above are indicative of a Bacillus
sp. Therefore further confirmation using biochemical testing were done
using the API 50 CHB/E medium, analyzed using APILAB PLUS (V 3.2.2). During
incubation, carbohydrates were fermented to acids which resulted in a
decrease in pH, detected by the colour change of the indicator. Result
obtained showed good identification to the genus of the isolated bacteria,
which is of Bacillus sp. with 85% similarity.
Preliminary study for optimization of CGTase production
The effect of pH on CGTase activity and stability:
Enzyme activity was measured using the standard assay method by varying
the pH values ranging from pH 3 to 10 at 70 °C. It was observed that
optimum pH of the crude CGTase was at pH 6.0 (Fig. 1).
Nonetheless, the enzyme showed only slight activity at pH 4.0 and 9.0.
|| Summary of morphological characteristics of strain
||Effect of pH on the enzyme activity of Bacillus
MK6 after 10 min incubation
suggests that the CGTase from Bacillus MK6 requires
a near neutral pH range to perform its cyclization reaction. Extreme pH
values were unsuitable for the enzyme to carry out cyclization activity.
Most of the reported CGTase exhibited optimum pH ranging from 5.0 to 8.0
(Ho et al., 2004). Some purified CGTase enzyme however may exhibit
more than 1 optimum peak. This phenomenon suggested the existence of more
than one peak were due to presence of different CGTases (acid-, neutral-and
alkaline) in the culture filtrate (Turnes and Bahar, 1996). It was also
reported that at 60 °C, the CGTase from alkalophilic Bacilllus
sp. showed stability over a wide range of pH of 6-10. However, the enzyme
activity decreased drastically beyond that range and almost lost its activity
below pH 4.0 and above pH 11.0 (Cao et al., 2005).
The effect of temperature on CGTase activity and stability:
The effect of different temperatures on CGTase activity was measured at
pH 6.0 following standard assay. The optimum temperature for CGTase from
Bacillus MK6 was 70 °C using sago starch as the substrate (Fig.
2). It is interesting to note the presence of another peak at 90 °C,
suspected to be an isozyme of crude CGTase used, having different physical
characteristics such as temperatures or pH but catalyzes the same reaction.
Most reported alkalophillic bacteria have optimum temperatures ranging
from 45 to 60 °C, except for Bacillus stearothermophillis which
have optimum temperature of 80 °C (Rita and Rajni, 2002). There are
no other known reports of CGTase with optimum temperature exceeding 60
°C for alkalophillic bacteria, except the one reported by Tien (2001),
where its optimum temperature was found to be at 70 °C. Presence of
other protein in crude CGTase enzyme increases its stability hence giving
another optimum peak at 90 °C.
The effect of temperature on stability of crude CGTase
enzyme was also investigated and the enzyme was observed to be stable
ranging from temperatures 30-80 °C (for 1 h incubation). The isolated
crude CGTase enzyme was found active even at 90 °C in the first 40
min incubation period before being totally inactivated (Fig.
3). It may be due to influence of other proteins presence in crude
enzyme that enhanced its stability over high temperature.
Cao et al. (2005) found that the CGTase isolated
from alkalophilic Bacillus sp. in China shows wide thermal stability
(40-70 °C) when kept in buffer at pH 8.5. However, above 70 °C,
rapid loss in activity occurred and only 14% of activity remained at 80
°C. It was also reported that CGTase are more resistant to thermal
denaturation in the
||Effect of temperature on the activity of CGTase isolated
from Bacillus MK6 after 10 min incubation
||Temperature stability of crude enzyme at 60 min incubation
presence of its substrate, product and calcium ions,
respectively. Although without the addition of any additives, the isolated
enzyme has shown high stability over wide range of pH and temperature
(Rita and Rajni, 2002).
It was reported that only CGTase from Bacillus steareothermophillus,
or thermal tolerant organism, will show high stability over high temperature.
These phenomenon may be due to different enzyme characteristic as compared
to those enzymes isolated from mesophilic bacteria. In thermophilic bacteria,
presence of abundant ionic bonding and hydrophobic bonding and also increase
in hydrophobic interaction between protein subunit (enzyme) brings to
the stability of thermal enzyme (Suelter, 1985).
Growth profile for CGTase enzyme production: Production
of CGTase enzyme followed a similar enzyme production pattern (Fig.
4). The enzyme synthesis begins from the early exponential phase.
However the maximum CGTase activity as measured by dextrinizing activity
was obtained at 22nd-26th h of cultivation. From literature CGTase production
by Bacillus cereus was at its peak during the 16-20 h of incubation
||Production profile of CGTase by Bacillus MK6
(Jamuna et al., 1993). While for B. circulans
alkalophillic sp. exhibits cyclizing activity after 40 h of growth with
a long lag period (Mäkelä et al., 1988). Production of
CGTase was observed only at the end of the stationary phase for alkalophillic
Bacillus sp. G1 (Tien, 2001) and Bacillus agaradhaerens
reaches stationary phase after 11 h of incubation with maximum enzyme
production of 0.31 U mL-1 after 25 h cultivation (Rita and
From the above comparisons, CGTase production from Bacillus
MK6 was suggested as non-growth associated as the enzyme production was
maximum at the late stationary phase. During the stationary phase, sporulation
begins which appear to trigger enzyme synthesis for higher activity (Kabaivanova
et al., 1999). CGTase production produced in shake flasks
was studied by Thatai et al. (1999) and found to be at 7.5 U mL-1
after 24 h growth for alkalophillic Bacillus sp.
Nogrady et al. (1995) and Pocsi et al.
(1998) suggested that extracellular CGTase are not involved in the degradation
of starch in the exponential phase growth of the bacteria, because they
are usually released into the culture medium when all the starches has
been consumed. The enzyme was probably attached to the cell membrane during
the exponential phase; it was retained between the cell membrane and the
cell wall during the early stationary phase and was only released into
the culture medium in the late stationary phase and during the cell lysis.
Optimization of CGTase production by RSM approach:
Using completely randomized design, the experiments with different combination
of sago starch concentrations, pH and agitation speed was assayed and
calculated after Bacillus MK6 inoculum that was cultivated at 37
°C for 22 h. The results obtained were analyzed using analysis of
variance (ANOVA) as appropriate to the experimental
||Analysis of variance for the regression model of response,
Y obtained from the response surface experiment
design used. The regression (Eq. 4)
obtained after analysis of variance gives the production of CGTase from
Bacillus MK6 as a function of the different initial pH (X1),
variables of sago starch concentration (X2, g L-1)
and agitation (X3, RPM). All terms regardless of their significance
was included in the following second-order polynomial equation:
The regression model consisted of 1 offset, 3 linear,
3 quadratic and 3 interaction terms were generated when using the MINITAB
version 13.0 software. The model was tested for adequacy and the quality
of fit by the analysis of variance (Table 3). The variance
ratio of the regression mean square gives a value of 10.64 [Tabulated
F-value, F0.05 (9, 17) = 3.01]. As a practical rule, a model
has a statistical significance when the calculated F-value is at least
3-5 times greater than the tabular value (Silva et al., 1999).
Thus the regression is adequate in explaining the functional relationship
between the response and the independent variables. The values of determination
coefficient (R2 = 0.849, Adjusted R2 = 0.769) indicates
that 15.1 - 23.1% of the total variations are not explained by the model.
The larger the magnitude of the t-value and the smaller
the p-value, the more significant is the corresponding coefficient. For
the first order effects, judging from the regression coefficient and t-values,
it could be concluded that the sago starch percentage had the most significant
effect on CGTase production, followed by agitation speed and pH value
For the first order effects, judging from the regression
coefficient and t-values, it could be concluded that the sago starch (β2)
concentration had the most significant effect on CGTase production, followed
by agitation (β3) and pH (β1). The quadratic
main effect of agitation (p<0.001) is the only significant factor.
The pH and sago starch concentration were not significant at quadratic
level. Therefore, agitation can act as a limiting factor and a little
variation in their agitation speed may alter either growth or product
formation rate or both to a considerable extent.
||Regression coefficients, t-value and p-value of second-order
response surface equation for yield of CGTase enzyme, Y
|*: Significant at 5% level; **: Significant at 1% level;
***: Significant at 0.1% level, Linear: βo: Constant,
β1: pH, β2: Sago Starch, β3:
Agitation, Quadratic: β11: pH*pH, β22:
Sago Starch*Sago Starch, β33: Agitation*Agitation,
Interaction: β12: pH*Sago Starch, β13:
pH*Agitation, β23: Sago Starch*Agitation
||Influence of pH and percent (%) sago starch on production
of CGTase, Y when agitation was fixed at center point
The non-additive effects of pH and percent (%) sago starch
in Fig. 5 were due to the significant interaction between
the two variables. The coefficient estimated for this interaction term
has a negative sign (β12 = -0.090). Considering this interaction
only, a negative sign of β12 may include that for an increase
of the response, the coded levels of pH and % sago starch must not have
the same sign, which means when there is increase in pH then there must
be decrease in % sago starch. This may also indicate that the interaction
term is not dominated by the other terms. Figure 6 and
7, shows no interaction between pH and agitation and
% sago starch and agitation because effect of these pairs were addictive.
At optimum point, the coded value of (pH) χ1 and (% sago
starch) χ2 are 10.151 and 3.343, respectively.
The nature and concentration of the carbon-source are
highly important in enzyme production from many organisms, especially
when the carbon source also plays an important role in the enzyme induction
||Influence of percent (%) sago starch and agitation on
production of CGTase, Y when agitation was fixed at center point
||Influence of pH and agitation on production of CGTase,
Y when agitation was fixed at center point
Patkar, 2001). Comparisons with results shown by Khairizal
et al. (2004), increase in sago starch concentration will bring
to increase in CGTase production. In their research, it was found that
the concentration of 1.48% of sago starch, was able to produce 84.32 U
mL-1 of enzyme which is very high. From Fig.
6, although by increasing the concentration of sago starch will produce
higher CGTase, this is however not economically feasible as pre heat treatment
at a longer time needed to be done. Gawande et al. also commented
that above a certain concentration of carbon substrate, when other nutrients
are kept constant, catabolite repression may occur. This repression may
occur due to limitation of other media components in the culture medium.
Results reported for alkalophillic Bacillus sp. (ATCC 31007) reported
that soluble starch concentration higher than 20-30 g L-1,
resulted in low enzyme production.
For the effect of pH, optimum pH for CGTase production
was at 10.151. This is due to the alkalophillic
||Correlation between observed response and predicted
nature of the isolated organism. However, when interact
with higher concentration of sago starch, as shown in Fig.
7; surprisingly lower pH value is needed. This effect may be due to
the high starch concentration resulting in high acid production, which
decreases cell growth rate and enzyme production. This result is similar
to those reported by Gawande and Patkar (2003).
Figure 8 shows the correlation between
the predicted response and the observed response. It can be concluded
that the experimental value is close to (R2 of 0.8476) the
predicted experimental value given by the statistical software system.
Therefore nature optimal values of the test variables are as follows:
pH (χ1) = 10.151, % sago starch (χ2) =
3.343 and agitation (χ1) = 187 rpm. Using the optimal
condition, the predicted response was 2.07 U mL-1. However
the verification of results using the optimised medium was accomplished
by carrying out shake flask experiments which gave final CGTase concentration
of 2.56 U mL-1. Not only that these experimental finding are
in close agreement with the model predictions, the experimental response
value was 24% more than the predicted value. Furthermore, the experiments
are controlled where numbers of experiments needed and the combination
of parameters were clearly defined.
Production of cyclodextrin: From the optimal condition
for cyclodextrin production obtained, the enzyme reactions were done in
2% slurried soluble starch, 0.1 M acetate buffer (pH 6.0) at 60 °C
for 24 h. During incubation of CGTase enzyme in the presence of substrate,
in the beginning of the reaction, the action of CGTase on starch will
begin with the chaotic splitting of starch, followed by simultaneous cyclization.
However, this chaotic splitting will also result in the formation of malto-oligosaccharides
with various degrees of polymerization (Abelyan, 2001). CGTase from alkalophilic
organisms have been known to be mainly β-CD producers while thermophilic
and mesophillic CGTases produce a mixture of α-, β-and γ-CD,
the α form being the dominant product (Abelyan et al., 1992).
|| Production of cyclodextrin at different incubation
|Soluble potato starch of 2% were used as substrate for
cyclodextrin production and was detected using HPLC
||Time course of cyclodextrins production by Bacillus
Table 5 listed the production of CDS,
in function of time using Bacillus MK6`s crude CGTase on 2% (w/v)
slurried soluble potato starch. A maximum conversion of about 27.30% to
5.46 mg mL-1 of β-cyclodextrin was obtained after 6 h
of incubation period and for α-cyclodextrin, the highest conversion
of 19.25% corresponding to 3.85 mg mL-1 of was obtained after
24 h of incubation. However the maximum total conversion into both α-and
β-cyclodextrin were obtained at 42% only after 20 h of incubation
The production profile (Fig. 9) shows
that incubation of crude CGTase enzyme (2.5 U mL-1) per gram
substrate yields mainly β-cyclodextrin (61.6% of the total cyclodextrins
yield) with a α:β production ratio of 0.62:1. The concentration
of β-cyclodextrin slowly decrease after 20 h incubation probably
due to product degradation/decyclization in the reaction system (Kamarulzaman
et al., 2004). The above results show similarity with those reported
where the production of cyclodextrins particularly are β-cyclodextrin
accelerated in the early hours of reaction (Ho et al., 2004; Rita
and Rajni, 2002).
The effect of different carbon sources on CGTase production
showed that enzyme production was highest when sago starch was used as
carbon source. The use of hydrolyzed starch and simple sugars gave low
yield of CGTase. Completely randomized design and response surface analysis
were useful to determine the optimum levels of medium concentration and
factors that significantly influence the production of CGTase from Bacillus
MK6. The final composition of the defined medium to produce CGTase after
the optimization step was as follows: 3.343% of sago starch; initial pH
of 10.151 and agitation speed of 187 rpm. In theory, this optimized media
produces 2.069 U mL-1 of CGTase while in practical, production
of CGTase was at 2.56 U mL-1. Therefore this model was shown
to adequately predict the optimization of CGTase production from Bacillus
MK6. Results from the experiment done shown that CGTase from Bacillus
MK6 were able to produce mainly β-cyclodextrin with 27.30% for 5.46
mg mL-1 β-cyclodextrin at 6th h, whilst α-cyclodextrin
with only 19.25% for 3.85 mg mL-1 at 24th h; with a α:β
production ratio of 0.62:1. No γ-cyclodextrin was detected.