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To cite this article: Noura El-Ahmady El-Naggar , Haroun S.A , Eman A. Owis & Sherief A.A (2014): Optimization of -
Glucosidase Production by Aspergillus Terreus Strain Emoo 6-4 Using Response Surface Methodology Under Solid-State
Fermentation, Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2014.940968
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Optimization of -Glucosidase Production by Aspergillus terreus Strain Emoo 6-4
Using Response Surface Methodology Under Solid-State Fermentation
The corresponding author's contact information: Dr. Noura El-Ahmady Ali El-Naggar,
Address: Bioprocess Development Department, Genetic Engineering and Biotechnology
Research Institute, City of Scientific Research and Technological Applications, New
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Borg El- Arab City, 21934, Alexandria, Egypt Tel: (002)01003738444 Fax: (002)03
4593423 E-mail: nouraelahmady@yahoo.com
Abstract
A total of Forty-two morphologically different fungal strains were isolated from different
soil samples and agricultural wastes and screened for -glucosidase activity under solid
state fermentation. Eight species were chosen as the most active -glucosidase producers
produced by Aspergillus terreus which showed the highest activity and subjected to full
Burman design (PBD) and Box Behnken design (BBD) were applied for optimization
EMOO 6-4. Fifteen variables including temperature, pH, incubation time, inoculum
experimental runs. Among the fifteen variables, NaNO3, KH2PO4 and Tween 80 were
found as the most significant factors with positive effect on -glucosidase yield. Box-
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Behnken design was used for further optimization of these selected factors for better -
INTRODUCTION
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a synergistic action on the degradation of cellulose with endoglucanase (EC 3.2.1.4) and
sugars through the concerted action of cellulolytic enzymes has great importance, since
For instance, the sugars produced can be converted to ethanol and/or lactic acid.[1,2]
randomly at multiple internal sites in the amorphous regions of cellulose fiber opening-up
exo-1,4--D-glucanase, EC 3.2.1.91) that act to liberate cellobiose from the reducing and
3.2.1.21) which liberate glucose from cellobiose. [3] Very strong activity of -glucosidase
is thus needed for the pretreatment step of lignocellulose before a further conversion. In
addition to the role in cellulose degradation, -glucosidase has also been attributed to
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several other applications. This includes the applications in pharmaceutical, cosmetic and
glucose. One problem is the shortage of beta-glucosidase which causes cellobiose, a very
production. Several approaches have been used to increase the amount of -glucosidase
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-glucosidases have been isolated from many different fungal species including
Enzyme production is one of the most important applications of solid state fermentation
(SSF). The technique of solid state involves the growth of microorganisms on moist
solids in absence or near absence of any free-flowing water. It may prove more efficient
in producing certain enzymes and metabolites.[7,8] SSF has several advantages including
low waste water output, reduced energy requirements, simple fermentation media, ease of
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The traditional approach of optimization processes based on a single variable search
technique is incapable in detecting the true optimum when a number of variables are
come together.[10] The statistical designs such as the Plackett-Burman design and
response surface method are effective methods for optimization of the operational
conditions of factors for optimization of culture conditions for desirable responses, and
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evaluating the relative significance of several affecting factors in the presence of complex
interactions.[12] RSM used for finding out the optimum conditions for -glucosidase
production.[13]
The present study aims to describe the isolation of fungal strains, which have -
solid state fermentation by using Aspergillus terreus strain EMOO 6-4. The levels of the
activities of Aspergillus terreus grown on rice straw under SSF have been studied. It
showed the highest - glucosidase activity (3097.6 Ug-1), while CMC-ase and avicelase
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Different soil samples were collected from different localities in Dakahlia governorate;
Hurghada, El-Arish and Assiut in Egypt. Rice straw was collected from Dakhahlia in
February 2011. Samples of straw were air dried and grinded then kept in a cold place
during transportation and storage. Compost sample was obtained from Egyptian
Company for Recycling Solid Wastes which located at Kalapsho area and Belqas region
in October 2010. Fungal isolates that able to degrade agriculture wastes were isolated
using dilution plate method using cellulose agar medium which consists of (g/L):
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cellulose powder 10; (NH4)2SO4 0.5; K2HPO4 1; KCl 0.5; MgSO4.7H2O 0.2; CaCl2 0.1;
yeast extract 0.5; FeSO4.7H2O 0.01 and agar 20. Rose-bengal was added as a
bacteriostatic agent at a low concentration. The plates were incubated at 30 for 7 days,
and the resulting colonies were purified on Potato-Dextrose Agar medium (PDA). The
stock cultures of the isolated fungi were maintained on PDA slants. The cultures were
transferred every month, and fresh cultures were used for experimental work.
The isolated fungi were subjected to identification using an Imaging Analysis System
using Soft-Imaging GbH software at the Regional Center for Mycology and
the cultures were dried and coated with gold. The gold-coated specimen was examined at
species was made with the help of universally recommended keys for identification of
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Aspergillus species.[14] For molecular identification, it is important to use a pure
cultivated fungus. Fungus are picked up with a sterilized toothpick, and suspended in 0.5
10,000 rpm for 10 min. After removal of supernatant, the pellet is suspended in 0.5 mL of
InstaGene Matrix (Bio-Rad, USA). Incubated at 56oC for 30 min and then heated at
100oC for 10 min. After heating, supernatant can be use for PCR. Fragments of the ITS1-
5.8S-ITS2 were amplified by the use of the primers ITS1 and ITS4.[15] The final volume
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of each reaction was 25 l, containing 2.5 l of buffer (200 mM Tris- HCl, pH 8,4 - 500
mM KCl, 1x concentrated), 2.0 l of dNTPs (2.5 mM), 1.5 l of each of the primers ITS1
and ITS4 (Invitrogen 10 pmol ml-1), 1.0 l MgCl2 (50 mM), 0.2 l Taq DNA
polymerase (5 U ml-1), 2 l DNA (5 ng ml-1) and 14.3 l of distilled water. The reaction
conduct 35 cycles after an initial denaturation of 4 min at 92C. Each amplification cycle
consisted of three steps: denaturation (92C, 40 s), annealing (55C, 1 min and 30 s) and
elongation (72C, 2 min). Final elongation at 72C for 5 min was used. The amplified
(Amersham Biosciences). The conditions for injection and electrophoresis were 2 Kv/60
The basal medium used in solid state fermentation consists of (g/L): (NH4)2SO4 0.5;
K2HPO4 1; KCl 0.5; MgSO4.7H2O 0.2; CaCl2 0.1; yeast extract 0.5; FeSO4.7H2O 0.01, all
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of these components were dissolved in 1L of 0.1M sodium acetate buffer (pH 5.5)[17],
Fungal isolates were grown on cellulose slants at 28 for 5 days. 10 mL of sterile basal
medium were added to prepare fungal spore suspension. From this suspension, 3.0 mL
were used to inoculate 1.0 gm of the sterilized straw sample in 250 mL Erlenmeyer flask
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Fugal isolates were cultured on rice straw according to the modified method of
Purkarthofer et al.[18] as follows: 1.0 g from rice straw was placed in 250 mL Erlenmeyer
flask as a carbon source. 3.0 mL of nutrient basal buffered medium (pH 5.5) were added
to each flask. All flasks were autoclaved at 121 for 30 min, and then cooled. The
sterilized flasks were inoculated by 1.0 mL of spore suspension of each fungal strain
under controlled conditions. Control flasks remained without inoculation. All flasks were
The isolated fungal strains were cultured on rice straw for selecting the most active
Purkarthofer et al.[18].
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Preparation Of Enzyme Solution
The fermentation cultures were soaked with 50 mL of (0.1M) sodium acetate buffer (pH
5.5), then they were shaken using incubating shaker for 60 min at room temperature and
centrifuged at 5000 rpm for 15-20 min to remove all fungal cells and residue of
substrate.[19, 20] The clear extract represented the enzyme solution and was used for
al.[21], 0.2 ml of crude enzyme was diluted with 0.4 mL of 0.1 M acetate buffer (pH 5.5)
added. The mixture was incubated at 35oC for 1.0 hr. Two mL of 1.0 M sodium carbonate
solution was added. The liberated -nitrophenol was then measured at 420 nm using
Inc.U.S.A). One unit of -glucosidase is defined as the amount of enzyme which releases
The amount of reducing sugars was determined as reported by Nelson[22] and Somogyi[23]
method.
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The soluble protein concentration was spectrophotometrically determined by the method
Design.
Screening process was carried out by conducting the experiments to determine which
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including temperature, pH, incubation time, inoculum size, moisture content, substrate
20 runs. The low level (1) and high level (+1) of each factor are listed in Table 1. All
experiments were carried out in duplicates and average of -glucosidase activities was
taken as the response. Based on the enzyme activity, the factorial experiment was
analyzed using ANOVA or regression analysis. From the regression analysis the
variables, which were significant (P < 0.1), were considered to have greater impact on the
Y = 0 + i X i (1)
Where, Y is the response variable (-glucosidase activity), 0 is the model intercept and
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Optimization Of -Glucosidase Production By Box-Behnken Design.
BoxBehnken design is one of response surface designs, especially made to require only
three levels, coded as 1, 0, and +1. The three coded variables (X1, X2 and X3) were
-glucosidase activity. All experiments were carried out in duplicates and average of -
glucosidase activities was taken as the response. The experimental results of RSM were
fitted via the response surface regression procedure, using the following second order
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polynomial equation:
Y = 0 + i X i + ii X i 2 + ij X i X j (2)
i ii ij
coded levels of independent variables. However, in this study, the independent variables
were coded as X1, X2, and X3. Thus, the second order polynomial equation can be
presented as follows:
Statistical Analysis
The experimental data obtained was subjected to multiple linear regressions using
Microsoft Excel 2007 to evaluate the analysis of variance (ANOVA) and to estimate the
main effect, t-value, p-value and confidence level. The quality of fit of regression model
was expressed via the correlation coefficient (R), the coefficient of determination (R2)
and the adjusted R2, and its statistical significance was determined by an F-test. Optimal
value of activity was estimated using the solver function of Microsoft Excel tools. The
statistical software package, STATISTICA software (Version 8.0, StatSoft Inc., Tulsa,
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USA) was used to plot the three-dimensional surface plots, in order to illustrate the
relationship between the responses and the experimental levels of each of the variables
A total of Forty-two morphologically different fungal strains were isolated from different
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soil samples and agricultural wastes and screened for -glucosidase activity under SSF
and the results were recorded (Figure 1). Upon initial screening, it appeared that most of
the isolates (90.48%) were able to produce -glucosidase activity while only 9.52% of the
isolates were unable to produce this enzyme. -glucosidase producing isolates were
categorized into 4 groups according to the -glucosidase activity; very strong (2001-3034
Ug-1), strong ((1001-2000 Ug-1)), moderate (501-1000 Ug-1), weak (1-500 Ug-1), the four
groups were represented by 19.05, 19.05, 21.43 and 30.95%, respectively (Figure 1).
Only 8 species were chosen as the most active -glucosidase producers. The -
glucosidase activity of the selected strains was confirmed using rice straw as carbon
source showed that all the various isolates were capable of producing -glucosidase in
different levels. The eight species were subjected to morphological identification, which
resulted that these fungi were belonging to only one class: Ascomycetes and they were:
strain EMOO 13-3. -glucosidase was highly produced by Aspergillus terreus which
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niveus showed the lowest activity. Aspergillus terreus was selected as the most suitable
The electron micrograph showed in Figure 2. Aspergillus terreus species grow as buff to
micrographs show that the conidiophores of Aspergillus terreus were typically long,
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columnar, 60.5 m in diameter and smooth giving rise to sub-globose vesicles with 15.4
m diameter that were biseriate, primary and secondary sterigmata are 6.4 x 2.3 and 6.0
The nucleotide sequence of Aspergillus sp. has been compared with other Aspergillus
the sequence was assembled and deposited in the NCBI Genbank with accession number
JX885883. The phylogenetic analysis of Aspergillus sp. isolate EMOO 6-4 revealed
100% similarity with Aspergillus terreus. A phylogenetic tree (Figure 3) based on 18S
neighbor-joining method of Saitou and Nei[25] with MEGA4.[26] This tree shows the close
phylogenetic association of strain EMOO 6-4 with other Aspergillus terreus isolates.
Design.
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A total of 20 runs were performed and the results of experiments are presented along
with the experimental, predicted activity of -glucosidase and residuals (Table 2). The
results showed a wide range of difference in the -glucosidase yield. The minimum and
maximum activities obtained were 2653.76 Ug-1 and 4136.27 Ug-1 respectively.
Statistical analysis of the responses were performed which is represented in Table 3. The
main effects of the examined variables on -glucosidase production were calculated and
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presented graphically in Figure 4. Main effects allowed the determination of the effect of
each variable. As shown, it was found that all variables except temperature, pH,
incubation time, substrate amount, moisture content and MgSO4.7H2O within the test
range had a positive effect on -glucosidase production. The Pareto chart illustrated the
showed the high positive significance by (16.17%). Next to NaNO3, Tween 80 showed
higher positive effect by (9.24%). Interestingly incubation time showed the highest
The value of the determination coefficient (R2 = 0.9682) indicates that 96.82% of the
variability in the response was attributed to the given independent variables and only
3.18% of the total variations are not explained by the independent variables. In
addition, the value of the adjusted determination coefficient (Adj. R2 = 0.8493) is also
very high which indicates a high significance of the model. A higher value of the
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independent variables,[27] this indicated a good correlation between the experimental
and predicted values. Thus, the analysis of the response trend using the model was
considered to be reasonable.
The model F value of 8.1431 implies that the model is significant. The values of
Significance P < 0.1 (0.0279) indicate model terms are significant. A comparison was
made between the experimental and the predicted results by the model as presented in
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and P-values, which are listed in Table 3. Variables with confidence levels above
90% (P < 0.1) were considered significant. Based on the statistical analysis of confidence
level of the fifteen variables shown in Table 3, NaNO3, KH2PO4, incubation time and
Tween 80 had high confidence levels above 90% and hence, they were considered the
probability value of 0.001, was determined to be the most significant factor, followed by
NaNO3 (0.008), Tween 80 (0.047) and KH2PO4 (0.072). NaNO3, KH2PO4 and Tween 80
production. On the basis of the calculated t-values, NaNO3, KH2PO4 and Tween 80 were
By neglecting the terms that were insignificant (P > 0.072), the first order polynomial
independent variables:
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Y( glucosidase ) = 327.891 312.226 ( X 3 ) + 193.176 ( X8 ) + 94.663 ( X9 ) + 110.386 ( X14 )
+ 193.176 ( X8 ) + 94.663 ( X9 ) + 110.386 ( X14 ) + 193.176 ( X8 ) + 94.663 ( X9 ) + 110.386 ( X14 )
(4)
Where Y is the response (-glucosidase production), and X3, X8, X9, and X14 are
amount 0.5 (g /250 mL flask), NaNO3 0.5%, KH2PO4 0.5 %, KCl 0.07%, MgSO4.7H2O
0.01%, CaCl2 0.05%, FeSO4.7H2O 0.0002%, yeast extract 0.07%, Tween 80 0.02%,
(NH4)2SO4 0.2%, pH 4.5, temperature 25C, moisture content 1 (mL /g dry substrate),
inoculum size 3 (mL /g dry substrate) and incubation period 3 days. The -glucosidase
activity produced from the optimized culture conditions was 4120 Ug-1 showed about
1.33 fold increase than that obtained from the un-optimized medium(3097.6 Ug-1).
Based on the results of Placket-Burman design, NaNO3 (X1), KH2PO4 (X2) and Tween 80
(X3) were chosen to be optimized by Box-Behnken design (BBD) in order to obtain the
maximum -glucosidase activity. Each factor in the design was studied at three different
levels (1, 0, 1) as shown in Table 4. A total of 15 runs were used to optimize the range
and levels of chosen variables, 3 runs (run 13-15) have been performed with the tested
variable parameters at middle level (center point runs). The parameters that were not
tested in this Box-Behnken design were applied at high and low level based on the results
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The results presented in Table 4 showed that the minimum -glucosidase activity
was (2499.733 Ug1) with specific activity 18.9 U/mg protein that observed in run 7
under the conditions of (0.3%) of NaNO3, (0.5%) of KH2PO4 and (0.03%) of Tween 80.
On the other hand, the maximum activity was (4457.162 Ug1) with specific activity 20.5
U/mg protein that were achieved in run 12 under the conditions of (0.5 %) of NaNO3,
(0.7%) of KH2PO4 and (0.03%) of Tween 80. A comparison was made between the
experimental results and results predicted by the model. The predicted and observed
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responses were presented in Table 4. It can be seen that the enzyme activity from the
The value of R was 0.9735 and this value indicates a high degree of correlation
between the experimental and the predicted values by 97.35%. The value of
determination coefficient R2 (0.94785), and the measure of fit of the model was about
94.8%, which indicates that only about 5.2% of the variation was unable to be explained.
This implies a satisfactory representation of the process by the model and the model was
5. It showed that P-value is less than 0.1 (0.0101) at confidence level of 95% so this was
a significant model, the degrees of freedom are 9 equal to the number of independent
observations, or are the number of values in the final calculation of a statistic that we can
vary. Also it refers to a positive whole number that indicates the lack of restrictions in our
calculations. The regression coefficients and the corresponding P-values were presented
in Table 6. The P-values were used as a tool to check the significance of each coefficient,
which also indicated the interaction strength of each parameter. The smaller the P-values
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were, the bigger the significance of the corresponding coefficient. [28] The significance of
each coefficient was determined by t-values and P-values which are listed in Table 6. The
P-values denotes the significance of the coefficients and also important in understanding
the pattern of the mutual interactions between the variables. It can be seen from the
(KH2PO4) are significant, meaning that they can act as limiting factor and little variation
in their values will alter the product production rate. Furthermore, the probability values
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of the coefficient suggest that among the three variables studied, X1 (NaNO3) and X3
(Tween 80) showed maximum interaction between the two variables (0.0777), indicating
In order to evaluate the relationship between dependent and independent variables and to
of NaNO3 (X1), KH2PO4 (X2) and Tween 80 (X3), a second-order polynomial model
polynomial equation that defines predicted response (Y) in terms of the independent
Where the Y is the predicted response, X1 the coded value of NaNO3, X2 the coded value
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Rajoka et al. [29] reported that NaNO3 was the best nitrogen source for different
a nitrogen source stimulates cellulase production during SSF.[30] Riswan et al.[31] also
reported that the cellulase activity increases with increase in NaNO3 and thereafter
cellulase activity decreases with further increase. Furthermore Rajmane and Korekar [32]
indicated that nitrate source like sodium nitrate stimulated the cellulase activity while,
ammonium sources in the form of nitrate, phosphate and sulphate were proved inhibitory
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block of proteins and is one of the main constituents of protoplasm. It was found that all
the nitrogen sources supported cellulase production.[34] Also Mangat and Mandahr[35],
showed that nitrogen sources greatly influence cellulases biosynthesis hence they should
be used with proper concentration. High concentrations of nitrogen sources are usually
KH2PO4 showed a strong positive linear effect on the cellulases activity. The cellulase
0.75 mg/L was seen to provide a maximum cellulase activity.[36] KH2PO4 showed
glucosidase was obtained at 0.3% of KH2PO4 and that was obtained by Ghori et al. [37]. In
addition, Riswan et al.[31], indicated that production of cellulases and xylanase was
dependent on KH2PO4 and cellulase and xylanase activities increase with increasing in
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KH2PO4 up to 3.3 g/L and thereafter their activities decrease with further increase in
KH2PO4. Zhang et al. [38] also pointed that KH2PO4 had no significant effect on cellulases
activities. Furthermore, Rashid et al.[39] showed that KH2PO4 didnt show any
Chellapandi and Jani[40], indicated that high concentration of Tween 80 has been recorded
was enhanced with the addition of Tween 80 in the culture. The highest cellulase yield
Tween 80 (> 2 ml L-1), the cellulase yield did not increase. The stimulatory effect of
concentrations of Tween 80 did not affect the growth of Aspergillus terreus. However,
the addition of Tween 80 to the culture medium led to a substantial increase in the
the production of cellulase was also increased until it reached the maximum activity at 2
ml L-1 of Tween 80. Cellulase production in fermentation with the addition of 2 mL-1 of
Pardo [43] reported that the effect of surfactant on growth of fungi and cellulase
production is well known. The use of Tween 80 is beneficial as it does not denature the
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9414 and Streptomyces flavogriseus was also enhanced with the addition of 1 and 0.2%
(v/v) of Tween 80, respectively.[45] The maximum cellulase production was achieved
activity was found in the presence of the surfactant (Tween 80) at optimum
Three dimensional (3-D) surface and contour plots for the obtained responses were
drawn based on the model polynomial functions to assess the change of the response
surface. These types of plots showed effects of two factors on the response at a time. In
all the presented figures, the third factor was kept at level zero. As shown in Figure 6 A,
it can be concluded that the three-dimensional plots of the response surface for the -
glucosidase yield as related to NaNO3 and KH2PO4 with Tween 80 at a constant level.
glucosidase increases with increasing NaNO3 until certain point, further increase of
NaNO3 level resulted in gradual decrease in -glucosidase activity and the optimum
concentration is showed towards the center point, while the activity of -glucosidase
activity in presence of KH2PO4 at a constant level. The response surface plot indicated a
clear peak, which means that the optimum point was inside the design boundary well.
The plot showed that -glucosidase activity increases with the increase of NaNO3 to
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optimum level, and then declined with the increases of this factor. The highest value of -
glucosidase production was obtained with middle level of both NaNO3 and Tween 80.
This result indicates that two variables had mutually dependent influence on the -
shown (Figure 6C); there is gradual increasing in -glucosidase activity with increasing
of both KH2PO4 and Tween 80. The highest activity was shown at high levels of KH2PO4
Optimal concentrations of the factors, obtained from the optimization experiment were
verified experimentally and compared with the predicted data. The measured -
glucosidase activity was 4460 Ug-1, where the predicted value from the polynomial
model was 4405 Ug-1. The verification revealed a high degree of accuracy of the model
of more than 98.8%, indicating the model validation under the tested conditions. The
terreus strain EMOO 6-4 were NaNO3 (0.5%), KH2PO4 (0.7%) and Tween 80 (0.03%).
CONCLUSION
Fungal isolates were screened for their ability to produce -glucosidase. The most active
isolate was identified as Aspergillus terreus strain EMOO 6-4. The PlackettBurman
glucosidase production under solid state fermentation; the levels of the significant
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glucosidase production was 4457.162 (Ug-1) which attained by the medium of the
following composition: Substrate amount 0.5 (g /250 mL flask), NaNO3 0.5%, KH2PO4
0.7 %, KCl 0.07%, MgSO4.7H2O 0.01%, CaCl2 0.05%, FeSO4.7H2O 0.0002%, yeast
extract 0.07%, Tween 80 0.03%, (NH4)2SO4 0.2%, pH 4.5, temperature 25C, moisture
content 1 (mL /g dry substrate), inoculum size 3 (mL /g dry substrate) and incubation
period 3 days.
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46. El-Naggar, N.E.; Abdelwahed, N.A.M. Optimization of Process Parameters For
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Table 1. Experimental independent variables at two levels used for the production of -
-1 +1
A Temperature (C) 25 35
B pH 4.5 6.5
H KH2PO4 (%) 5
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Table 2. Twenty trials of PlackettBurman design for Aspergillus terreus strain EMOO
6-4, for evaluation of fifteen variables along with the experimental, predicted -
s activity(Ug-1) c ed s
acti acti
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vit vity
(U/
mg
pro
tein
1 1 1 1 1 1 1 9.38 94
1 1 1 1 1 1 1 1 0.93 103.
0 968
1 1 1 1 1 1 1 1 5.59 0.64
4 2
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4 - 1 1 1 1 - 1 - 1 - - - - 1 1 3160.767 8.8 314 12.1
1 1 1 1 1 1 1 8.57 94
1 1 1 1 1 1 1 3.86 95
1 1 1 1 1 1 1 6.54 12.1
3 94
1 1 1 1 1 1 1 7.07 19.8
9 95
1 1 1 1 1 1 1 1 9 1.17 111.
3 670
1 1 1 1 1 1 1 1 1 1 6.76 92.4
5 16
1 1 1 1 1 1 1 1 7 9.54 47
1 1 1 1 1 1 1 1 1 4.19 103.
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6 968
1 1 1 1 1 1 1 5 7.53 222
1 1 1 1 1 1 1 7 5.32 123.
4 222
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1 1 1 1 1 1 1 9 3.05 222
1 1 1 1 1 1 9.15 2
1 1 1 1 1 1 0.89 670
1 1 1 1 1 6 3.26 16
1 1 1 1 1 1 7 1.09 31.4
4 47
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1 1 1 1 1 1 1 0.21 19.8
7 95
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 3.64 16
8
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Table 3. Statistical analysis of Plackett-Burman design showing coefficient values, t-test,
level (%)
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Table 4. BoxBehnken experimental design, the coded and actual levels of variables, the
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Table 5. Analysis of variance for optimization of -glucosidase production using Box
Behnken design.
Total 14 5027908.73
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Table 6. Estimated regression coefficients for optimization of -glucosidase activity
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Figure 1. Screening of fungal isolates for the -glucosidase production (Percentage inside
the parenthesis indicates the frequency of fungal isolates within specific category in
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Figure 2. Electron micrographs of Aspergillus terreus strain EMOO 6-4, showing (A):
micrograph at 500 x and (B): micrographs at 750x, (C): micrographs at 1000x, and (D):
micrograph at 5000x.
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Figure 3. Phylogenetic tree obtained by neighbor-joining analysis[25] of 18S ribosomal
RNA gene (partial), internal transcribed spacer 1, 5.8S rRNA gene, internal transcribed
spacer 4 and 28S ribosomal RNA gene (partial), showing the position of Aspergillus sp.
strain EMOO 6-4 within the genus Aspergillus. Only bootstrap values above 50 %,
expressed as percentages of 1000 replications, are shown at the branch points. GenBank
sequence accession numbers are indicated in parentheses after the strain names.
Phylogenetic analysis was conducted in MEGA4.[26] Bar, 0.2 substitution per nucleotide
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position.
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Figure 4. The main effects of the factors affecting -glucosidase production according to
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Figure 5. Pareto chart illustrates the order of significance of the variables affecting the -
glucosidase production by Aspergillus terreus strain EMOO 6-4 (the red color represent
negative effects and the blue color represent positive effects; Ranks (%) values ranging
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Figure 6. Response surface 3D and contour plots of -glucosidase production showing
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