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Preparative Biochemistry and Biotechnology


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Optimization of -Glucosidase Production by


Aspergillus Terreus Strain Emoo 6-4 Using Response
Surface Methodology Under Solid-State Fermentation
a b b b
Noura El-Ahmady El-Naggar , Haroun S.A , Eman A. Owis & Sherief A.A
a
Department of Bioprocess Development , Genetic Engineering and Biotechnology Research
Institute, City of Scientific Research and Technological Applications , Alexandria , Egypt
b
Department of Botany, Faculty of Science , Mansoura University , Egypt
Accepted author version posted online: 18 Jul 2014.

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

To link to this article: http://dx.doi.org/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

Noura El-Ahmady El-Naggar1, Haroun S.A2, Eman A. Owis2, Sherief A.A.2


1
Department of Bioprocess Development, Genetic Engineering and Biotechnology
Research Institute, City of Scientific Research and Technological Applications,
Alexandria, Egypt, 2Department of Botany, Faculty of Science, Mansoura University,
Egypt

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

and were subjected to primary morphological identification. -glucosidase was highly

produced by Aspergillus terreus which showed the highest activity and subjected to full

identification using scanning electron microscopy and molecular identification. Plackett

Burman design (PBD) and Box Behnken design (BBD) were applied for optimization

of different factors affecting -glucosidase production by Aspergillus terreus strain

EMOO 6-4. Fifteen variables including temperature, pH, incubation time, inoculum

size, moisture content, substrate concentration, NaNO3, KH2PO4, MgSO4.7H2O, KCl,

CaCl2, yeast extract, FeSO4.7H2O, Tween 80 and (NH4)2SO4 were screened in 20

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 -

Glucosidase production. The maximum -glucosidase production was 4457.162 (Ug-1).

KEYWORDS: -glucosidase, Aspergillus terreus, optimization, Plackett-Burman and

Box-Behnken design, solid state fermentation, identification

INTRODUCTION
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-glucosidase (EC 3.2.1.21) is well-known as a component of cellulase complex and has

a synergistic action on the degradation of cellulose with endoglucanase (EC 3.2.1.4) and

exoglucanase (EC 3.2.1.91). Degradation of lignocellulosic materials to monomeric

sugars through the concerted action of cellulolytic enzymes has great importance, since

sugars can serve as raw materials in a number of biotechnological production processes.

For instance, the sugars produced can be converted to ethanol and/or lactic acid.[1,2]

Microbial cellulases are multienzyme complex that work synergistically to attack

crystalline cellulose producing glucose as a final product. It consist of three major

components; endoglucanase (endo-1,4--D-glucanase, EC 3.2.1.4) which attack

randomly at multiple internal sites in the amorphous regions of cellulose fiber opening-up

sites for subsequent attack by the cellobiohydrolases; exoglucanases (cellobiohydrolases;

exo-1,4--D-glucanase, EC 3.2.1.91) that act to liberate cellobiose from the reducing and

non-reducing ends of cellulose chains and -glucosidases (1,4--D-glucosidase, EC

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

detergent industries.[4] -glucosidase hydrolyzes the glucose dimmers, cellobiose, to

glucose. One problem is the shortage of beta-glucosidase which causes cellobiose, a very

potent inhibitor of endoglucanases and cellobiohydrolases, to be accumulated. The low

amount of -glucosidase results in a shortage of capacity to hydrolyze the cellobiose to

glucose resulting in accumulation of cellobiose and a feedback inhibition of enzyme

production. Several approaches have been used to increase the amount of -glucosidase
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in cellulose conversion to glucose. One approach is to produce -glucosidase using

microorganisms and add the beta-glucosidase exogenously to endoglucanase and

cellobiohydrolase to enhance the cellulose hydrolysis.

-glucosidases have been isolated from many different fungal species including

Ascomycetes and Basidiomycetes. However, Aspergillus is by far the most efficient

producer of -glucosidase among the microorganisms investigated such as A. niger, A.

glaucus and A. oryzae.[ 5,6]

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

aeration and reduced bacterial contamination.[9]

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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

parameters.[11] Response surface methodology (RSM) is a collection of mathematical and

statistical techniques for designing experiments, building models, searching optimum

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 -

glucosidase activities from Egyptian soils; using PlackettBurman design as a statistical

approach for identifying significant variables influencing -glucosidase production under

solid state fermentation by using Aspergillus terreus strain EMOO 6-4. The levels of the

significant variables were further optimized using Box-Behnken design. Cellulolytic

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

activities were 12.75 Ug-1 and 6.95 Ug-1 respectively.

MATERIALS AND METHODS

Sample Collection, Isolation And Purification Of The Fungal Strains

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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.

Identification Of Fungal Isolate

The isolated fungi were subjected to identification using an Imaging Analysis System

using Soft-Imaging GbH software at the Regional Center for Mycology and

Biotechnology (RCMB), Al-Azhar University. For scanning electron microscopy (SEM),

the cultures were dried and coated with gold. The gold-coated specimen was examined at

different magnifications with Analytical Scanning Electron Microscope (Jeol JSM-6360

LA operating at 20 Kv) at the Central Laboratory, City of Scientific Research and

Technological Applications, Alexandria, Egypt; the identification of fungal genera and

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

ml of sterilized saline solution in a 1.5 mL centrifuge tube. Centrifugation takes place at

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

mixture was placed in a PTC-100 thermo-cycler (MJ Research, Inc.) programmed to

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

fragments were analyzed by gel electrophoresis in 1x TEB buffer according to Sambrook

and Russell [16]. Sequencing was performed on a Mega-BACETM 1000 sequencer

(Amersham Biosciences). The conditions for injection and electrophoresis were 2 Kv/60

s and 6 Kv/230 min, respectively.

Preparation Of Basal Medium

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],

autoclaved at 121oC and 1.5 atm for 20 min.

Preparation Of Fungal Inoculum

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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during solid state fermentation.

Solid State Fermentation (SSF)

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

incubated at 30 for 6 days.

Survey For -Glucosidase Production Of Isolated Fungi

The isolated fungal strains were cultured on rice straw for selecting the most active

cellulolytic strain for further investigations according to the modified method of

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

assaying -glucosidase activity.


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Determination Of -Glucosidase Activity

-glucosidase activity is generally determined by measuring the release of p-nitrophenol

from p-nitrophenyl--D-glucopyranoside according to the method of Christakopoulos et

al.[21], 0.2 ml of crude enzyme was diluted with 0.4 mL of 0.1 M acetate buffer (pH 5.5)

and 0.4 mL of 0.0136 M of -nitrophenyl--D-glucopyranoside (Sigma Aldrich) was

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

Spectro UV-VIS RS spectrophotometer (Serial number: UV-VIS 0478; Labomed

Inc.U.S.A). One unit of -glucosidase is defined as the amount of enzyme which releases

one mole of -nitrophenol per minute under assay conditions.

Estimation Of Reducing Sugars

The amount of reducing sugars was determined as reported by Nelson[22] and Somogyi[23]

method.

Determination Of Soluble Protein

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The soluble protein concentration was spectrophotometrically determined by the method

of Bradford[24]. The protein concentration was calculated from a standard curve

constructed using bovine serum albumin (BSA).

Screening Of Factors Affecting -Glucosidase Production By Plackett-Burman

Design.

Screening process was carried out by conducting the experiments to determine which
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variables significantly affect -glucosidase production. Fifteen independent variables

including temperature, pH, incubation time, inoculum size, moisture content, substrate

concentration, NaNO3, KH2PO4, MgSO4.7H2O, KCl, CaCl2, yeast extract, FeSO4.7H2O,

Tween 80 and (NH4)2SO4 were chosen to be screened by Plackett Burman experiment in

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

enzyme activity and were further optimized by Box-Behnken design.

PlackettBurman experimental design is based on the first order model:-

Y = 0 + i X i (1)

Where, Y is the response variable (-glucosidase activity), 0 is the model intercept and

i is the linear coefficient, and Xi is the level of the independent variable.

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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

chosen to be optimized by Box-Behnken design (BBD) in order to obtain the maximum

-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

In which Y is the predicted response, 0 is the regression coefficients, i is the linear

coefficient, ii is the quadratic coefficients, ij is the interaction coefficients), and Xi is the

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:

Y = 0 + 1 x1 + 2 x2 + 3 x3 + 12 x1 x2 + 13 x1 x3 + 23 x2 x3 + 11 x12 + 22 x22 + 33 x32 (3)

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

utilized in this study.

RESULTS AND DISCUSSION

Isolation And Selection Of The Fungal Strains For -Glucosidase Production

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:

Emmonsia crescens, Aspergillus niveus, Paecilomyces variotii, Aspergillus terreus strain

EMOO 6-4, Aspergillus alliaceus, Talaromyces oxalicum and Talaromyces pinophilus

strain EMOO 13-3. -glucosidase was highly produced by Aspergillus terreus which

showed the highest activity, followed by Talaromyces pinophilus, while Aspergillus

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niveus showed the lowest activity. Aspergillus terreus was selected as the most suitable

strain for the following experiments in our study.

Identification Of The Most Active -Glucosidase Producers

The electron micrograph showed in Figure 2. Aspergillus terreus species grow as buff to

yellow brown colonies. Microscopically, conidiophore is 5.2 m in diameter. The

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

X 1.2 m in dimensions resprectively. Conidia are smooth walled, globose to slightly

elliptical and striate and 2.1 m in diameter.

The nucleotide sequence of Aspergillus sp. has been compared with other Aspergillus

spp. sequences using BLAST algorithm at the website http://www.ncbi.nlm.nih.gov, and

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

rRNA sequences of members of Aspergillus terreus was constructed according to the

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.

Screening Of Factors Affecting -Glucosidase Production By Plackett-Burman

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

order of significance for the variables affecting -glucosidase production in Plackett-

Burman Experimental Design as shown in Figure 5. Among the 15 variables, NaNO3

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

negative significance and effect by (26.135%).

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

correlation coefficient (R= 0.9840) signifies an excellent correlation between 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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Table 2. The significance of each coefficient was determined by student's t-test

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

most significant parameters influence -glucosidase production. Incubation time, with a

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

were the positive significant variables affecting -glucosidase production, while

incubation time was the negative significant parameter affecting -glucosidase

production. On the basis of the calculated t-values, NaNO3, KH2PO4 and Tween 80 were

chosen for further optimization using Box-Behnken statistical design.

By neglecting the terms that were insignificant (P > 0.072), the first order polynomial

equation was derived representing -glucosidase production as a function of the

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

incubation time, NaNO3, KH2PO4 and Tween 80; respectively.

In a confirmatory experiment, to evaluate the accuracy of Plackett-Burman, a


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medium which expected to be near optimum of the following composition: Substrate

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).

Statistical Analysis Of Box-Behnken Design For -Glucosidase Production

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

from the Placket Burmann design.

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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

experiment was slightly different from predicted by the model.

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

confirmed to be significant. The analysis of variance (ANOVA) was presented in Table

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

degree of significance that the linear and quadratic coefficients of X1 (NaNO3), X2

(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

that 92.23% of the model affected by these variables.

In order to evaluate the relationship between dependent and independent variables and to

determine the maximum -glucosidase production corresponding to the optimum levels

of NaNO3 (X1), KH2PO4 (X2) and Tween 80 (X3), a second-order polynomial model

(Equation 2) was proposed to calculate the optimum levels of these variables. By

applying the multiple regression analysis on experimental data, the second-order

polynomial equation that defines predicted response (Y) in terms of the independent

variables (X1, X2 and X3) was obtained:

Y( glucosidase ) = 3318 165.25 X1 + 588.43 X2 + 32.49 X 3 211.78 X1 X 2


(5)
+ 253.5X1 X 3 + 175.68 X 2 X 3 345.35 X12 + 387.07 X2 2 185.71 X 3 2

Where the Y is the predicted response, X1 the coded value of NaNO3, X2 the coded value

of KH2PO4 and X3 the coded value of Tween 80.

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Rajoka et al. [29] reported that NaNO3 was the best nitrogen source for different

cellulases production. In addition; many investigators reported that addition of NaNO3 as

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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for cellulase production of all post-harvest fungi. CMCase productivity by Aspergillus

fumigates were highly increased by addition of NaNO3 as N-sources to the fermentation

medium.[33] Nitrogen is important for -glucosidase production as it is the main building

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

unstable for the microorganisms

KH2PO4 showed a strong positive linear effect on the cellulases activity. The cellulase

activity was recorded to increase with an increase of KH2PO4 whose concentration of

0.75 mg/L was seen to provide a maximum cellulase activity.[36] KH2PO4 showed

positive significantly effect on cellulases production. A maximum activity of -

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

contributory effect in the production of cellulases.

Chellapandi and Jani[40], indicated that high concentration of Tween 80 has been recorded

to increase the production of -glucosidase. Cellulase production by Aspergillus terreus


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was enhanced with the addition of Tween 80 in the culture. The highest cellulase yield

was obtained in medium containing 2 ml L-1 of Tween 80. At higher concentrations of

Tween 80 (> 2 ml L-1), the cellulase yield did not increase. The stimulatory effect of

surfactants may be a consequence of its action on cell membranes causing increased in

permeability by promoting the release of cell-bound enzymes.[41] Furthermore different

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

activity of cellulase by Aspergillus terreus. As the concentration of Tween 80 increased,

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

Tween 80 was improved by 1.6 times as compared to fermentation without surfactant.[42]

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

enzymes. Tween 80 at a concentration of 2 mL-1 was optimum for the production of

cellulase and -glucosidase by mixed culture of Trichoderma reesei and Aspergillus

phoenicis on dairy manure.[44] Cellulases production by Trichoderma reesei strain QM-

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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

when the production medium supplemented with Tween-80.[46] Highest -glucosidase

activity was found in the presence of the surfactant (Tween 80) at optimum

concentrations. Surfactants such as Tween 80 have been described as significantly factor

that increase the production of microbial enzymes[47] including fungal -glucosidases.[45]


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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.

KH2PO4 exhibited an important effect on the -glucosidase yield; the activity of -

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

increase with increasing KH2PO4.

Figure 6B showed the effects of NaNO3 and Tween 80 on the -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 -

glucosidase activity. The effect of KH2PO4 and Tween 80 on -glucosidase activity

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

and Tween 80.


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Verification Of The Model

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

optimal levels of the process variables for -glucosidase production by Aspergillus

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

design is used as a statistical approach for identifying significant variables affecting -

glucosidase production under solid state fermentation; the levels of the significant

variables were further optimized using Box-Behnken design. The maximum -

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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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Table 1. Experimental independent variables at two levels used for the production of -

glucosidase by Aspergillus terreus strain EMOO 6-4 using PlackettBurman design.

Code Independent variables Levels

-1 +1

A Temperature (C) 25 35

B pH 4.5 6.5

C Incubation time (d) 3.0 5.0


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D Inoculum size (ml/g dry substrate) 1.0 3.0

E Moisture content (ml/g dry substrate) 1.0 5.0

F Substrate amount (g/250ml flask) 0.5 2.0

G NaNO3 (%) 0.1 0.5

H KH2PO4 (%) 5

I MgSO4.7H2O (%) 0.01 0.05

J KCl (%) 0.02 0.07

K CaCl2 (%) 0.01 0.05

L Yeast extract (%) 0.02 0.07

M FeSO4.7H2O (%) 0.00 0.0002

N Tween 80 (%) 0.00 0.02

O (NH4)2SO4 (%) 0.07 0.2

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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 -

glucosidase activities and residuals.

Tr A B C D E F G H I J K L M N O Experimental - Spe Pre Resi

ial glucosidase cifi dict dual

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 - 1 - 1 - - - 3741.577 9.8 372 12.1

1 1 1 1 1 1 1 9.38 94

2 1 - - 1 1 1 1 - 1 - 1 - - - - 3086.962 9.4 319 -

1 1 1 1 1 1 1 1 0.93 103.

0 968

3 - - 1 1 1 1 - 1 - 1 - - - - 1 2794.952 9.6 279 -

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

5 1 1 1 1 - 1 - 1 - - - - 1 1 - 2653.76 7.6 263 19.8

1 1 1 1 1 1 1 3.86 95

6 1 1 1 - 1 - 1 - - - - 1 1 - 1 3154.349 8.2 316 -


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1 1 1 1 1 1 1 6.54 12.1

3 94

7 1 1 - 1 - 1 - - - - 1 1 - 1 1 3167.184 9.1 318 -

1 1 1 1 1 1 1 7.07 19.8

9 95

8 1 - 1 - 1 - - - - 1 1 - 1 1 - 2849.503 10. 296 -

1 1 1 1 1 1 1 1 9 1.17 111.

3 670

9 - 1 - 1 - - - - 1 1 - 1 1 - - 3154.349 21. 324 -

1 1 1 1 1 1 1 1 1 1 6.76 92.4

5 16

10 1 - 1 - - - - 1 1 - 1 1 - - 1 2810.996 13. 277 31.4

1 1 1 1 1 1 1 1 7 9.54 47

11 - 1 - - - - 1 1 - 1 1 - - 1 1 4120.228 20. 422 -

1 1 1 1 1 1 1 1 1 4.19 103.

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6 968

12 1 - - - - 1 1 - 1 1 - - 1 1 1 4020.752 12. 389 123.

1 1 1 1 1 1 1 5 7.53 222

13 - - - - 1 1 - 1 1 - - 1 1 1 1 3642.102 15. 376 -

1 1 1 1 1 1 1 7 5.32 123.

4 222
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14 - - - 1 1 - 1 1 - - 1 1 1 1 - 4136.272 19. 401 123.

1 1 1 1 1 1 1 9 3.05 222

15 - - 1 1 - 1 1 - - 1 1 1 1 - 1 3099.797 8.6 309 0.64

1 1 1 1 1 1 9.15 2

16 - 1 1 - 1 1 - - 1 1 1 1 - 1 - 2942.561 8.1 283 111.

1 1 1 1 1 1 0.89 670

17 1 1 - 1 1 - - 1 1 1 1 - 1 - 1 3635.684 13. 354 92.4

1 1 1 1 1 6 3.26 16

18 1 - 1 1 - - 1 1 1 1 - 1 - 1 - 3189.647 10. 322 -

1 1 1 1 1 1 7 1.09 31.4

4 47

19 - 1 1 - - 1 1 1 1 - 1 - 1 - - 3000.322 8.3 302 -

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1 1 1 1 1 1 1 0.21 19.8

7 95

20 - - - - - - - - - - - - - - - 3196.064 16. 310 92.4

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,

and P-values for each variable affecting -glucosidase activity.

Variables Coefficients Main effect t- Stat P-value Confidence

level (%)

Intercept 3277.891 6555.782 84.165 0.000 100.000

Temperature -46.850 -93.700 -1.203 0.295 70.467

pH -4.813 -9.627 -0.124 0.908 9.240


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Incubation time -312.226 -624.452 -8.017 0.001 99.869

Substrate amount -69.954 -139.908 -1.796 0.147 85.311

Inoculum size 36.582 73.163 0.939 0.401 59.924

Moisture content -62.895 -125.789 -1.615 0.182 81.837

NaNO3 193.176 386.352 4.960 0.008 99.229

KH2PO4 94.663 189.325 2.431 0.072 92.807

MgSO4.7H2O -13.477 -26.954 -0.346 0.747 25.326

KCl 77.014 154.027 1.977 0.119 88.085

CaCl2 7.060 14.119 0.181 0.865 13.502

Yeast extract 25.992 51.984 0.667 0.541 45.894

FeSO4.7H2O 56.798 113.595 1.458 0.218 78.151

Tween 80 110.386 220.772 2.834 0.047 95.286

(NH4)2SO4 82.790 165.579 2.126 0.101 89.931

t student's test; p corresponding level of significance

Multiple R 0.9840; R Square 0.9682; Adjusted R Square 0.8493

F value 8.1431; Significance F (p value) 0.0279

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Table 4. BoxBehnken experimental design, the coded and actual levels of variables, the

experimental, predicted -glucosidase activities and residuals as influenced by NaNO3

(X1), KH2PO4 (X2) and Tween 80(X3).

Trials Variables (%) -glucosidase Predicted Residuals Specific activity

X1 X2 X3 activity (Ug-1) activity (U/mg protein)

1 -1 (0.3) -1(0.3) 0(0.02) 2762.863 2724.7573 38.1058 11.3

2 1(0.7) -1(0.3) 0(0.02) 2727.565 2817.8153 -90.2502 20.4


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3 -1(0.3) 1(0.7) 0(0.02) 4415.446 4325.1958 90.2502 31.1

4 1(0.7) 1(0.7) 0(0.02) 3532.999 3571.1048 -38.1057 24.8

5 -1(0.3) 0(0.5) -1(0.01) 3276.287 3173.2013 103.0858 19.1

6 1(0.7) 0(0.5) -1(0.01) 2567.12 2335.6783 231.4418 10.7

7 -1(0.3) 0(0.5) 1(0.03) 2499.733 2731.1748 -231.4418 18.9

8 1(0.7) 0(0.5) 1(0.03) 2804.579 2907.6648 -103.0857 10.5

9 0(0.5) -1(0.3) -1(0.01) 2932.935 3074.1265 -141.1915 10.1

10 0(0.5) 1(0.7) -1(0.01) 2981.068 2787.7320 193.3360 11.6

11 0(0.5) -1(0.3) 1(0.03) 3706.28 3899.6160 -193.3360 14.1

12 0(0.5) 1(0.7) 1(0.03) 4457.162 4315.9705 141.1915 20.5

13 0(0.5) 0(0.5) 0(0.02) 3318.003 3318.003 0.0000 12.6

14 0(0.5) 0(0.5) 0(0.02) 3318.003 3318.003 0.0000 12.6

15 0(0.5) 0(0.5) 0(0.02) 3318.003 3318.003 0.0000 12.6

Multiple R 0.9735; R Square 0.94785; Adjusted R Square 0.85398

F value 10.09; Significance F (p value) 0.0101

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Table 5. Analysis of variance for optimization of -glucosidase production using Box

Behnken design.

df Sum of squares Mean square F Sig.(P-value)

Regression 9 4765702.81 529522.53 10.09 0.0101

Residual 5 262205.91 52441.18

Total 14 5027908.73

df : Degree of freedom, F: Fishers's function, Sig.: Corresponding level of significance


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Table 6. Estimated regression coefficients for optimization of -glucosidase activity

using BoxBehnken design.

Variables Coefficients Main effect t -stat P-value

Intercept 3318 6636 25.09 0.0000

X1 -165.25 -330.51 -2.04 0.0967

X2 588.43 1176.86 7.26 0.0008

X3 32.49 64.98 0.4 0.7048


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X1X2 -211.78 -423.57 -1.84 0.1236

X1X3 253.5 507 2.21 0.0777

X2X3 175.68 351.37 1.53 0.1855

X12 -345.35 -690.71 -2.89 0.0339

X22 387.07 774.14 3.24 0.0227

X32 -185.71 -371.43 -1.55 0.1799

t student's test; p corresponding level of significance

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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

relation to the total isolates).


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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

the PlackettBurman experimental results.


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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

from 0.403 to 26.135).


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Figure 6. Response surface 3D and contour plots of -glucosidase production showing

the interaction between KH2PO4, Tween 80 and NaNO3.


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