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Initial Screening of Fermentation Variables for the Production of Cyclomaltodextrin Glucanotransferase (CGTase) from Local Isolated Bacillus stearothermophillus HR1.

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Dev. Chem. Eng. Mineral Process. 13(5/6), pp. 541-547, 2005.
Initial Screening of Fermentation Variables
for the Production of Cyclomaltodextrin
Glucanotransferase (CGTase) from Local
Isolated Bacillus stearothermophillus HR1
T.E. Wong', R.M. Illias*',M.M. Salleh2, 0. Hassan3,
K. Kamaruddin4, R.A. Rahman' and A.F. Ismail'
Faculty of Chemical Engineering and Natural Resources Engineering,
Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia
Department of Biology, Universiti Teknologi Malaysia, 81310 Skudai,
Johor, Malaysia
School of Chemical and Food Sciences, Universiti Kebangsaan
Malaysia, Bangi, Selangor, Malaysia
Bioprocess & Chemical Technology Centre, SIRIM Berhad, Malaysia
This paper presents results on the effect of fermentation variables on CGTase
production from Bacillus stearothermophilus HRl. Initial screening of temperature,
pH and agitation speed was determined using a 2-level full factorial design. In this
study, we have improved the production of cyclodmtrin glucanotransferase (CGTase)
from Bacillus stearothermophilus HR1 in jlasks using the experimental design
approach. The regression model generated for the correlation of CGTase with the
variables was 99.97% accurate. Results of the screening process indicated that pH
and agitation speed were of greater signlficance compared to temperature. Results
suggested that only two variables, namely pH and agitation speed, should be
considered in firture optimization studies. Temperature was least significant and it
was set at 55*C where CGTase production of 10.41 Ulml was slightly higher
compared to temperatures of 3 PC, 45"C, 50°C and 60°C.
Cyclodextrins (CDs), also known as Schardinger dextrins, are oligosaccharide rings
comprising a-1,4-linked glucopyranose residues. The most common types of CDs
are a-,p- and y-cyclodextrins, which are made up of 6,7 or 8 molecules, respectively.
* Author for correspondence (
T.E. Wong,R.M.Illias, M.M. Salleh, et al.
CDs have found many uses in a variety of industries, especially in the food,
pharmaceutical, chemical, cosmetic, and agricultural industries. CDs are able to form
inclusion complexes with a wide variety of guest molecules, either organic or
inorganic, without changing the properties of the guest molecule. This is attributed to
the fact that CD molecules are torus-shaped structures with hydrophilic hydroxyl
groups as the exterior cavity, and hydrophobic C-H groups and glycosidic oxygen as
the interior cavity. CDs are also able to stabilize labile materials, mask off odours,
emulsify oils, and change viscous or oily compounds into powder.
Of the three main types of CDs produced, p-CD is more widely used and
developed in many applications. p-CD is readily separated from the reaction mixture
without any treatment with organic solvents, and its inclusion complexes can be easily
prepared, due to its low solubility in water. Efforts are focused on finding suitable
CGTase producers for efficient production of p-CD since enzymes capable of
producing predominantly a particular type of CD can reduce subsequent purification
costs, and hence are commercially desired.
In general, CDs are produced by starch degradation upon the action of the enzyme
Cyclodextrin glucanotransferase (CGTase; EC, a type of extracellular
enzyme excreted by bacteria. All CGTase produced are mixtures of a-,0- and ycyclodextrins; their proportion being characteristic of the individual enzymes. Many
types of bacteria have been identified as producers of CGTase, at different rates and
conditions. CGTase producers can be found in various places such as soil, waste,
plantation, hot springs, and even deep-sea mud [ 11. Bacillus coagulans [2], Bacillus
circulans [3], Bacillusfirmus [4], Bacillus stearothennophilus [5], Bacillus macerans
[6], Bacillus lentus [7], Klebsiella pneumonia [S], Klebsiella oxytoca [9],etc., are all
known CGTase producers.
Little information is available on the effects of fermentation conditions on
CGTase production. Therefore, in this study, a preliminary screening process was
carried out using Response Surface Methodology (RSM). Variables such as
temperature, pH and agitation were studied in shake flasks, and results were analyzed
using a two-level h l l factorial experimental design. This allowed for the
determination of the main variables influencing CGTase production.
Materials Used and Experimental Methods
(i) Preparation of bacterial inoculum
Bacillus stearothermophilus HR1 was grown in 20 ml of optimized medium with the
composition of 1.602% (w/v) sago starch, 2.0% (w/v) peptone from casein, 0.14%
(wh) K2HP04 and 0.02% (w/v) CaCl2.2Hz0 with initial pH 7.54 in a 250ml conical
flask. The culture was incubated at 50°C with shaking at 200 rpm for 18 hours. The
cells were then centrihged at 5000 rpm for 5 minutes, washed once with normal
saline solution to give an optical density (OD) reading of 0.5 at 550 nm.
(ii) Preparation of crude enqyme
Ten percent (vh) of the bacteria inoculum was inoculated into 100 ml of medium in a
500 ml conical flask. After incubation, bacterial cells were separated by centrifuging
at 5000 rpm for 2 minutes and clear supernatant was used as crude enzyme.
Production of CGTase@om Local Isolated Bacillus stearothermophillus HRl
(iii) CGTase assay
Activity of the CGTase was measured by the method of Kaneko [lo], with slight
modification. Reaction mixture containing 40 mg of soluble starch in 1.O ml of 0.1M
sodium phosphate buffer (pH 6.0) and 0.5 ml of CGTase solution was incubated at
90°C for 10 minutes. The reaction was stopped by adding 30 ml of 30 mM NaOH and
0.5 ml of 0.02% (w/v) phenolphthalein in 5 mM Na2C03 solution. The colour
intensity was then measured at 550 nm after standing at room temperature for
15 minutes. Blanks without the enzyme were run with each batch of assays. Standard
curves were plotted using 0.5 mg of p-CD (Fluka) and 0.5 ml water to replace soluble
starch and crude enzyme, respectively. One unit of enzyme activity was defined as
the amount of enzyme that forms 1 pmol of p-CD per minute.
(iv) Experimental design
Experimental design was carried out based on the established method [ 11, 121. Initial
screening was performed using a Z3 full factorial experimental design for 3
independent variables (temperature, pH and agitation speed), leading to a total of 8
sets of experiments. Every set of experiments requires 6 conical flasks, each
containing 100 ml of medium. Enzyme activity was assayed at 24 hours. The coded
values for different variables for screening are shown in Table 1, and the design
matrix is shown in Table 2.
Agitation speed
Actual Value
Coded Value
Table 2. 2'fulI factorial experimental design for screening.
T.E. Wong, R.M.Alias, M.M.Salleh, et al.
Results and Discussion
The screening experiment was conducted in a randomized order to avoid systematic
bias. Interpretation of results was analyzed using the analysis of variance (ANOVA)
as appropriate to the experimental design used. The results are included in Table 3.
The regression model for CGTase activity correlated with the factors can be
written as:
Y = 4.55 + 0.049A + 4.55B- 0.33C + 0.049 AB + 0.079AC - 0.33BC
where Y = CGTase activity; A = temperature; B= pH; and C = rpm.
The probability value (p-value) for each term and interaction is listed in Table 3.
A p-value of less than 0.05 defines the factors as significant. The model is significant
with a probability of 0.03 (see Table 3). This means that the regression model
generated to describe the correlation of CGTase with the factors analyzed was
The predicted coefficient of determination, R2 for the model was 0.9997. This R2
value confirmed that the variability in the CGTase activity could be associated to the
experimental factors to the extent of 99.97%, with only 0.03% of the model not
explained by the equation. The variable with the largest effect was pH (factor B).
This factor has the largest coefficient estimate as written in Equation 1.
The half normal plot can be used to determine the significant factors affecting the
response. This plot is constructed so that if there were no important effects, all the
points would fall on a straight line [12]. In Figure 1, the effect for pH (factor B)
obviously falls far away from the line, so we believe that it represents a strong signal.
Next we see that the effect of agitation speed (factor C) and the effect of interaction
between agitation speed and pH (factor BC) are slightly away from the line and is
probably a signal.
Production of CGTasefrom Local Isolated Bacillus stearothermophillus HR I
A: temperature
B: pH
C: rpm
% probability
Half Normal plot
Figure 1. Half normal plot of screening process.
In contrast, temperature (factor A) did not impose a significant effect on CGTase
production. However, temperature does influence the lag phase and the exponential
phase of cell growth. This is a logical observation since with increase in temperature,
the enzymatic and chemical reaction w i h cells will also increase, thus showing
rapid growth. At a certain maximum temperature, the protein, nucleic acid and other
cellular components in the cells may be irreversibly damaged, thus causing death.
It is observed that temperature has the least effect on CGTase production and is
therefore advisable to select a suitable temperature. Cultures were incubated at 37"C,
45"C, 5OoC, 55°C and 60°C. At 55"C, CGTase activity was slightly higher at 10.41
U/ml compared to 37°C (9.97 U/ml), 45°C (10.08 U/ml), 50°C (10.37 U/ml) and 60°C
(9.88 U/ml).Th~shigher value does not show a marked increase from the CGTase
activity recorded at 50°C, thus proving that temperature did not have a major effect on
CGTase production. However, other researchers reported a range of different
temperatures for different strains of B. stearothermophilus, such as B.
stearothermophilus 2/2 was cultured at 60°C [ 131, B. stearothermophilus No. 239 at
55°C [14], and B. stearothermophilus also at 55°C [15].
Our results have indicated the direction for future optimization studies of pH and
agitation speed with the temperature set at 55°C.
T.E.Wong, R.M.Illias, M.M. Salleh, et al.
Temperature ('C)
+- 24 hours - B--
48 hours
Figure 2. CGTase activities after 24 and 48 hours incubation at various
temperatures; the highest CGTase activity was detected at SS'C for assays carried
out after 24 and 48 hours.
An initial screening process was carried out to determine the significant fermentation
variables influencing CGTase production from Bacillus stearothermophilus HR1.
Based on the results presented fiom the screening process, it was found that pH and
agitation speed are the only variables that influence CGTase production. The
regression model has shown that the predicted coefficient of determination is accurate
to 99.97%. The optimal temperature for CGTase production was found to be at 55°C.
Thus future optimization will only take the two factors, i.e. pH and rprn, into
A Temperature ("C)
C Agitation speed (rpm)
Production of CGTasefiom Local Isolated Bacillus stearothermophillus HRI
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production, initial, isolated, stearothermophilus, bacillus, local, screening, variables, fermentation, hr1, cyclomaltodextrin, cgtase, glucanotransferase
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