ENZIMAS Exoglucanasa_proypur

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Production, Purification, and Characterization

of Exoglucanase by Aspergillus fumigatus

Raja Tahir Mahmood &Muhammad Javaid Asad &

Nazia Mehboob &Maria Mushtaq &

Muhammad Gulfraz &Muhammad Asgher &

Nasir M. Minhas &Saqib Hussain Hadri

Received: 2 September 2012 /Accepted: 7 April 2013 /

Published online: 25 April 2013# Springer Science+Business Media New York 2013

Abstract Fungi are considered good producers of industrially valuable enzymes with higher

enzymatic activities. Among these cellulases are group of extracellular enzymes commonly

employed in many industries for the hydrolysis of cellulolytic material. Aspergillus fumigatus

produced exoglucanase having high enzymatic activity (83 U/gds) during the solid-state

fermentation of wheat straw under optimum physical and nutritional conditions. Maximum

production was obtained after 72 h of fermentation, at 55 C temperature, pH 5.5, 80 %

moisture level, and 2 mL fungal inoculum. Production was further increased by the addition

of fructose (0.3 %) as additional carbon source, peptone (0.4 %) as nitrogen source, Tween-80

(0.3 %) as surfactant, and ammonium sulfate (0.2 %) in media. Exoglucanase was 2.30-folds

purified by adding 40 % ammonium sulfate with volumetric activity 95.4 U/gds and specific

activity 14.74 U/mg. Further, it was 5.18-folds purified by gel filtration chromatography with

volumetric activity 115.2 U/gds and specific activity 33.10 U/mg. Purified exoglucanase has

maximum activity at 55 C and pH 4.8 using 1 % Avicel aqueous solution as substrate. TheKmandVmaxwere 4.34 mM and 7.29M/min, respectively. Calcium, magnesium, and zinc ions

have positive effect on exoglucanase activity.

Keywords Aspergillus fumigatus . Wheat straw . Avicel . Ammonium sulfate

Introduction

Lignocellulosic material is consider the most abundant and renewable biological

source of fermentable sugars on biosphere. It has the potential to be converted it into

many useful by-products by enzymatic hydrolysis like human nutrients, biofuel, etc.

[13]. Cellulose can be converted into fermentable sugars through acidic or enzymatic

Appl Biochem Biotechnol (2013) 170:895908

DOI 10.1007/s12010-013-0227-x

R. T. Mahmood (*) :M. J. Asad :N. Mehboob :M. Mushtaq :M. Gulfraz :N. M. Minhas :S. H. Hadri

PMAS, Arid Agriculture University, Islamabad, Punjab, Pakistan

e-mail: [email protected]

M. Asgher

University of Agriculture Faisalabad, Faisalabad, Punjab, Pakistan


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hydrolysis and then can be used for the production of organic acids [4], ethanol [5],

and other important chemicals [6, 7].

Estimated cellulosic and lignocellulosic material production is 15 1012 tons per

year [8]. Cellulose is linear polymer of D-glucose linked through (14) glycosidic

linkage [9, 10]. Hydrolysis of these linkages is necessary to obtain benefits fromcellulosic material [11].

Cellulases are the group of extracellular enzymes including endoglucanase (E.C.

3.2.1.4), exoglucanase (E.C. 3.2.1.91), and -glucosidase (E.C. 3.2.1.21). These act

sequentially on cellulose and convert it into glucose molecules [1214]. Exoglucanase

acts on reducing and non-reducing ends of oligosaccharides and releases cellobiose

units, which consists of two or three glucose units [13, 15]. The three-dimensional

structure of active site exoglucanase has tunnel-like loop for interaction with substrate

through hydrogen binding [16].

Production of cellulases has been studied extensively in the past few years, due to their

applications in various industries [2, 8, 17]. Cellulases production can be increased by

studying microbial strain, media composition, and other factors that control growth and

production [18]. Different lignocellulosic materials are used for economic enzymes produc-

tion like sawdust [19], corn cobs [20], bagasse [21], wheat straw [22], rice straw [23], and

wheat bran [20].

In recent years, interest towards solid-state fermentation (SSF) is increasing due to its

some additional advantages like lower capital expenditure, cheaper fermentation media,

superior productivity, reduced energy requirements, and absence of rigorous control of

fermentation parameters as well as less waste output [2426].

Aspergillus fumigatus is a thermophilic saprophytic fungus belongs to PhylumAscomycota. It is mostly found in soil and other decaying organic matter to play important

role in the recycling of carbon and nitrogen. During the process of decaying dead organic

matter, it produces many extracellular enzymes like exoglucanase.

Materials and Methods

Substrate

Wheat straw was selected as a lignocellulosic substrate for A. fumigatus due to its highcellulose percentage (3540 %), availability in Pakistan, and good for the growth of

cellulolytic microorganisms [8]. Substrate was dried in sunlight for 10 days and then

oven-dried for 24 h. It was then ground to powder in the Department of Soil Science,

PMAS-Arid Agricultural University Rawalpindi and packed in air tight plastic jars.

Fermentative Organism

Thermophilic A. fumigatus isolated from the soil in the temperate region of Pakistan was

used for the current study. It was identified in The Department of Plant Pathology, PMAS-Arid Agriculture University Rawalpindi, Pakistan.

Maintenance of Organism

A. fumigatus was maintained on potato dextrose agar media in Industrial Environmental

Biotechnology Lab of Biochemistry Department, PMAS-Arid Agricultural University

896 Appl Biochem Biotechnol (2013) 170:895908


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Rawalpindi [26]. For its use in the fermentation process, it was preserved on inoculum

media; flasks containing inoculum media were adjusted at pH 5 and autoclaved at standard

conditions. Flasks were then inoculated aseptically with loopful of fungal spores from

preserved slants and placed in shaking incubator at 180 rpm and 55 C for 72 h. The

conidial (spores) suspension was adjusted between 107

and 109

conidia/mL with the help ofa hemocytometer and biomass monitor (ABER 220).

Fermentation Process

SSF is the growth of microorganisms on moist solid supports, including inert carriers and

insoluble substrates, and has more production [27]. Duplicate flasks (250 mL) containing 5 g

of grounded wheat straw, moisten with mineral salt solution (70 % of total dry material) and

having pH 5.0 (1 N HCl/NaOH), were used. Flasks were autoclaved, inoculated aseptically

with 2 mL of inoculum, and incubated at 55 C for 72 h.

Optimization of Cultural Conditions

Fungal culturing required specific conditions which must be maintained throughout the

media to obtained better growth and production. The following cultural conditions were

optimized to increased exoglucanase production:

Fermentation period:A. fumigatuswas cultured from 24 to 120 h for the optimization of

the most suitable fermentation period.

Incubation temperature: A. fumigatus was incubated for 72 h (optimum) at different

temperatures ranging from 45 to 65 C having gap of 5 C.Optimization of pH: For the optimization of pH, A.fumigatuswas cultured at different

pH levels ranging from 4 to 6. It was maintained with the help 1 M HCl/NaOH.

Moisture level: Moisture level was optimized with mineral salt solution from 50 to

90 % of total dry contents.

Optimization of Nutritional Conditions

Fungi need certain nutrients for growth, which it obtained from the substrate and media.

Addition of these nutrients in the media increased its growth and production of extracellularenzymes. Different nutrients were added in the media and their effect on exoglucanase

production was checked. Different concentrations (w/w) from 0.1 to 0.5 % of each of the

nutrient were added and the most suitable were selected.

Effect of Carbon Source Glucose and fructose were used as additional carbon sources in the

media.

Effect of Nitrogen Source Peptone and urea were added in the media as additional nitrogen

sources.

Effect of Surfactants Three surfactantsSDS, Tween-80, and Tween-20were employed

to check their effect on exoglucanase production.

Effect of Mediator Cane molasses, yeast extract, and ammonium sulfate were used as

mediators to enhance the production of exoglucanase.

Appl Biochem Biotechnol (2013) 170:895908 897


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Enzyme Extraction

After 72 h, the flasks were harvested for the extraction of exoglucanase by contact method.

In each flask, 50 mL of distilled water having pH 5.5 (optimum) was added and shaken at

120 rpm for 1 h in a shaking incubator at room temperature. Mixture was then filtered byusing Whatman no. 1 filter paper and filtrate was centrifuged at 10,000 rpm for 15 min at 4 C

to remove all the spores and other impurities. Crude enzyme obtained after centrifugation was

stored at 4 C before performing assay [2].

Exoglucanase Assay

Exoglucanase activity was checked by mixing 1 mL of crude enzyme and 1 mL of

Avicel solution (1 %) in a test tube against blank lacking enzyme solution. The pH of

the mixture was maintained with phosphate buffer (1 mL) having pH 5 [ 2]. The test

tubes were incubated at 55 C for 30 min and then 3 mL of dinitrosalicylic acid(DNS) was added and tubes were placed in boiling water for 15 min. DNS react with

enzymatically digested products (cellulobiose) and produced complexes; concentration

of these complexes were checked by taking OD at 540 nm in a spectrophotometer

[22].

Enzyme Activity

One unit of enzyme activity is the amount of enzyme which released 1 mol of the product

per minute.

Protein Estimation

Protein contents in crude and purified samples were estimated according to biuret method

using bovine serum albumin (BSA) as a standard.

Purification of Exoglucanase

Exoglucanase produced under optimized conditions was purified for further characteriza-

tion. The following methods were used for purification of exoglucanase.

Ammonium Sulfate Precipitation

Ammonium sulfate causes the precipitation of proteins by decreasing their solubility. The

crude exoglucanase was partially purified by adding different concentrations of (NH4)2SO4,

e.g., 20, 30, 40, 50, and 60 % in 10 mL of crude enzyme. Partially purified enzyme samples

were subjected to activity assay and biuret assay to find the protein concentration.

Gel Filtration Chromatography

Gel filtration chromatography (5 % silica gel column) was used for further purifica-

tion of exoglucanase. Silica was dissolve in sodium citrate buffer having pH 5.

Different elutions were subjected to enzyme activity assay and biuret assay. Elution

having the maximum activity was further used for characterization of different kinetic

parameters.



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Characterization of Exoglucanase

Partially purified exoglucanase was subjected to characterization of optimum pH, tempera-

ture, and kinetic parameters.

Optimization of pH for Exoglucanase Activity

Purified exoglucanase was subjected to activity assay at pH values 4.5, 4.8, 5.0, 5.5, and 6.0.

Sodium citrate buffer was used to maintain pH of reaction mixture.

Optimization of Temperature for Exoglucanase Activity

Exoglucanase was subjected to activity assay at different temperature ranging from 45 to

65 C at pH 4.8 (optimum).

Effect of Substrate Concentration on Exoglucanase

The effects of different concentrations of Avicel ranging from 2 to 10 mM, on exoglucanase,

were determined to obtain the MichaelisMenten kinetic constants (Km and Vmax).

Effect of Metal Ions on Exoglucanase Activity

The effect of different metal ions like Ca2+, Mg2+, and Zn2+ on exoglucanase activity was

checked by adding different concentrations of calcium chloride, magnesium chloride, andzinc chloride ranging from 0.1 to 0.5 % in the reaction mixture.

Results and Discussion

Optimization of Exoglucanase Production

Maximum exoglucanase production (64.2 U/gds) was observed after 72 h of fermentation

(Fig. 1). After that, production was decreased possibly due to depletion of nutrients and

accumulation of waste material. A. fumigatus gave maximum production of cellulolytic

Fig. 1 Optimization of fermentation period for exoglucanase by A.fumigatus



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enzymes after 72 h of incubation [27]. Current results are in line with the results of that

reported by Shafique et al. [2] for exoglucanase by fungal source.

During the next steps, different physical and nutritional parameters were opti-

mized to increase the production of exoglucanase. Moisture levels were used

ranging from 50 to 90 %; maximum production was observed at 80 % of moisturelevel (45.2 U/gds). Further increase showed decrease in production due to decrease

in the growth of fungus because of poor aeration [12] of the media (Fig. 2). A 70

80 % moisture level of substrate is better for the production of cellulolytic enzymes

from fungus [2, 3]. Maximum exoglucanase production was observed at pH 5.5 of

growth media (Fig. 3); exoglucanase remained stable between pH 4 and 6. After

that, its activity decreased due to the acidic nature of enzyme and decreased in the

stability of enzymes due to interaction of ions with the side groups of amino acids.

Optimum pH 5.5 and acidic range were also reported in many other research studies

[12, 28].

For the optimization of incubation temperature, A. fumigatus was grown at tem-

perature ranging from 45 to 65 C. Maximum exoglucanase production was observed

at 55 C and further increase in temperature cause decrease enzyme activity (Fig. 4).

An initial increase in temperature enhanced enzyme activity, possibly due to increase

in kinetic energy of exoglucanase and increase interaction between enzyme and

substrate. Decrease in enzyme activity at higher temperature is due to denaturing of

structure of proteins. Optimum temperature around 50 C for the growth was also

reported by Gautham et al. in 2011 [14].

Optimization of Nutritional Conditions

Effect of Carbon Source Glucose and fructose were used as additional carbon source forA.

fumigatus. Both have positive impact on fungal growth as well as exoglucanase production;

maximum activity was observed at 0.3 % of fructose (Fig. 5). Fructose is a better carbon

source than glucose because it is readily available to fungus than glucose. External carbon

sources increase the growth of fungi and production of cellulases because of their readily

availability to fungus than substrate [29]. Addition of fructose in the media enhances the

production of extracellular enzymes more than glucose [30].

Effect of Nitrogen Source Urea and peptone were used as additional nitrogen sources in themedia ofA.fumigatus. Different concentrations ranging from 0.1 to 0.5 % were added in the

Fig. 2 Optimization of moisture level for exoglucanase by A. fumigatus



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growth media and the effect was checked by measuring exoglucanase activity. Peptone

(0.4 %) was found a better nitrogen source as compared to urea (Fig. 6). Sherief et al. also

reported that peptone enhances the production of cellulases more than urea. This might be

due to the fact that peptone contains amino acids which are readily available nitrogen source

for the growth ofA.fumigatus[26]. Higher concentration of nitrogen makes substrate non-

favorable for fungi by changing its texture. Nitrogen source enhances the production of

extracellular enzymes, like cellulases [31].

Effect of Surfactants Tween-20, Tween-80, and SDS were used as surfactants to enhance

production of exoglucanase. Tween-80 at 0.3 % concentration of substrate was found as abetter surfactant than Tween-20 and SDS (Fig. 7). It increased fungal growth and

exoglucanase production by increasing permeability of wheat straw for fungus. Tween-80

enhances the production of cellulases partially by increasing the permeability of substrate

and partially by increasing interaction between substrate and enzymes [2, 32]. SDS de-

creases the production of exoglucanase because it decreases the stability of the

exoglucanase. Inhibition of production of extracellular cellulases by SDS was also reported

by Iqbal et al. [1,33].

Effect of Mediators Yeast extract, ammonium sulfate, and cane molasses were used as

mediator to study their effect on exoglucanase production by A. fumigatus. Various

Fig. 3 Optimization of pH for exoglucanase byA.fumigatus

Fig. 4 Optimization of fermentation temperature for exoglucanase byA.fumigatus



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concentrations of each ranging from 0.1 to 0.5 % were added and the effect was noted from

the exoglucanase activity. Each of the concentration has beneficial effect on exoglucanase

production but 0.2 % of ammonium sulfate was found to be most suitable for the production

of exoglucanase byA.fumigatus(Fig.8). Increase in the production of exoglucanase by the

addition of yeast extract was due to the fact that it act as nitrogen source as well as vitamin B

complex, necessary for amino acids synthesis [12,34]. Ammonium sulfate act as inorganic

nitrogen source and increases the production of enzymes [35].

Purification of Exoglucanase

Exoglucanase produce at optimized conditions has activity of 83 U/gds with specific activity

of 6.39 U/mg of protein (Table1). It was further purified by ammonium sulfate precipitation

by adding different concentrations of ammonium sulfate in 10 mL of crude enzyme. It was

2.30-folds purified with 40 % ammonium sulfate with specific activity of 14.74 U/mg of

protein (Fig. 9). Ammonium sulfate precipitates out exoglucanase by decreasing its

solubility.

Fig. 5 Optimization of carbon sources for exoglucanase production byA.fumigatus

Fig. 6 Optimization of nitrogen source for exoglucanase byA. fumigatus



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Exoglucanase was further purified by gel filtration chromatography using 5 % silica gel

column. This results in 5.18-folds increased in exoglucanase concentration with activity of

115.2 U/gds and specific activity of 33.10 U/mg of protein. Protein contents during each

purification steps were estimated by standard biuret protein assay using BSA as a standard.

The concentration of protein decreases after every step of purification, due to exclusion of

unwanted proteins. Increased in concentration of exoglucanase was indicated by the increasein specific activity of the protein. Purification of cellulases after ammonium sulfate precip-

itation and gel filtration chromatography was also reported by Asad et al. [2,12]. Increase in

the activity of exoglucanase indicates the purification of exoglucanase [2]. There was 1.80-

and 3.33-folds increase in protein concentration after ammonium sulfate precipitation and

gel filtration chromatography, respectively [36,37]. A 2.53-folds increase in concentration

of cellulase after purification by ammonium sulfate and gel filtration was also reported by

Iqbal et al. [1]. The enzyme obtained after gel filtration chromatography was used for further

exoglucanase characterization [8].

Fig. 7 Optimization of surfactants for exoglucanase byA.fumigatus

Fig. 8 Optimization of mediator for exoglucanase by A.fumigatus



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Characterization of Exoglucanase

Optimum pH of Exoglucanase

To find out the optimum pH for exoglucanase, purified exoglucanase was subjected toactivity assay under different pH values (4.5, 4.8, 5.0, 5.5, and 6.0). The maximum activity

was observed at pH 4.8 and then there was a decreased in activity possibly due to change in

ionic strength of the reaction mixture that causes unstability of the protein (Fig. 10).

Exoglucanase showed resistance between pH 4.5 and pH 6.0.

Optimum Temperature of Exoglucanase

For optimum temperature, exoglucanase activity assay was performed with purified

exoglucanase at different temperature ranging from 45 to 65 C, pH was maintained at 4.8

in each case. Exoglucanase gave maximum activity at 55 C and remained active up to 60 C

(Fig. 11). After 60 C, exoglucanase activity decreased quickly due to denaturation of

enzyme structure at higher temperature.

Effect of Substrate on Exoglucanase: Determination of Km

and Vmax

To find out MichaelisMenten constants (KmandVmax) for exoglucanase, activity assay was

performed at different concentration of Avicel (2, 4, 6, 8, and 10 mM). The results of the

Table 1 Purification summary of exoglucanase byA.fumigatus

Sample Volume

(mL)

Activity

(U/gds)

Protein

(mg/mL)

Total

activity

(U/gds)

Total

protein

(mg)

Specific

activity

(U/mg)

Purification

fold

Crude enzyme 50 83 12.97 4,150 611.5 6.39 1

Ammonium sulfate purified 10 95.4 6.47 952 79.3 14.74 2.30

Gel filtration chromatography 5 115.2 3.48 576 25.35 33.10 5.18

Fig. 9 Purification of exoglucanase by ammonium sulfate precipitation



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assay were used to construct LineweaverBurk reciprocal plot between 1/[V] on Y-axis

against 1/[S] on X-axis (Fig.12). Linear equation was used to obtain the values ofKm and

Vmaxfrom the plot and these were 4.34 mM and7.29 M/mL, respectively. Values ofKmand

Vmaxindicated higher affinity of exoglucanase for its substrate (Avicel). Kmvalue of 3.8 mM

of exoglucanase fromTrichoderma reeseiwas reported by Dashtban et al. [38]. TheVmaxof

1.80 U/mL for exoglucanase using Avicel as a substrate was reported by Ayman et al. [39].

Effect of Metal Ions on Exoglucanase Activity

To find the effect of metal ions like Zn2+, Mg2+, and Ca2+, different concentrations, varying

from 0.1 to 0.5 % of zinc chloride, magnesium chloride and calcium chloride, were added in

reaction mixture. Results showed that each metal ion has positive effects on exoglucanase

activity (Fig.13), most effective one was Ca2+ ion. Increase in the activity of exoglucanase

by the addition of CaCl2 as a source of Ca2+ metal ion was also reported by Hussain et al.

[21]. Addition of Mg2+ ion increased the activity of exoglucanase produced by fungi [18].

Fig. 10 Effect of pH on exoglucanase activity by A.fumigatus

Fig. 11 Effect of temperature on exoglucanase activity by A.fumigatus



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These ions increase the production of exoglucanase by activating various processes in the

fungus like synthesis of proteins and act as a cofactor of enzymes.

Conclusion

Exoglucanase takes part in the hydrolysis of cellulose along with endoglucanase and beta-glucosidase. It hydrolyzed oligosaccharides produce by endoglucanase into tri- and disac-

charides, called cellobiose. Solid-state fermentation of wheat straw produces large amount of

exoglucanase byA.fumigatusunder optimized conditions. Addition of fructose as a carbon

source, peptone as a nitrogen source, Tween-80 as a surfactants, and ammonium sulfate as a

mediator further enhanced the production of exoglucanase. Purified exoglucanase has higher

enzymatic activity and specific activity as compared to crude form. It was 2.30-folds purified

0

0.09

0.18

0.27

0.36

0.45

0.54

-0.4 -0.2 0 0.2 0.4 0.6

1/[S] mM

1/V0uM/ml/m

in

Fig. 12 LineweaverBurk plot between 1/[S] and 1/[V0] to find out the Kmand Vmaxfor exoglucanase

Fig. 13 Effect of metal ions on exoglucanase by A. fumigatus



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