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OPTIMIZATION OF CONDITIONS FOR THE PRODUCTION OF GLUCOAMYLASE FROM ASPERGILLUS FUMIGATUS: PURIFICATION AND KINETIC STUDIES OF GLUCOAMYLASE.

Byline: M. Tayyab, H. Ali, B. Muneer, S. Firyal, A. R. Awan, M. Wasim, F. Masood and S. Saeed

Keywords: Glucoamylase, Aspergillus fumigatus, wheat bran, optimization.

INTRODUCTION

Glucoamylases (GAs) hydrolyze starch by catalyzing [alpha] 1-4 and [alpha] 1-6 linkage from non-reducing ends (Sauer et al., 2000). Several GAs have been characterized from a variety of bacteria (Zheng et al., 2010) and fungi (Puri et al., 2013). Fungi have major contribution for the production of GAs (Chiquetto et al., 2004). Microbial strains of genera Aspergillus and Rhizopus have been predominantly used for commercial production of GAs (Li et al., 2014; Pandey, 1995). Scientists used various substrates including rice bran, wheat bran and paddy husk for the economic bulk production of GAs (Puri et al., 2013; Singh and Soni, 2001) whereas, others utilize various sources for carbon, nitrogen and phosphorous for the optimal production of GAs (Bertolin et al., 2001). A. fumigatus is a fungus found in decaying organic matter and in soil i.e. compost heaps and has contributory role in nitrogen and carbon recycling in the environment (Klich and Pitt, 1988).

Production of various enzymes from A. fumigatus has been reported previously which includes [beta]-Glucosidase (Rudick and Elbein, 1975), xylolytic enzymes (Flannigan and Sellars, 1978), cellulolytic and proteolytic enzymes (Krikstaponis et al., 2001) and [beta]-1,3-glucanases (Mouyna et al., 2013). In the present study, we reported the production of glucoamylase from A. fumigatus (GAAF) and conditions were optimized for the production of GAAF.

MATERIALS AND METHODS

Chemicals: All the chemicals utilized in the current study were purchased from Sigma Aldrich, Germany and were of purified grade.

Methods: A. fumigatus was isolated from organic waste soil samples from river Ravi located in Lahore, Punjab, Pakistan and was initially grown on Sabouraud Dextrose Agar (Dartora et al., 2002). Hemocytometer was used for counting spores and 1x106 spores per mL was utilized for inoculation (Strober et al., 2001).

Enzymes production: Sterilized fermentation medium supplemented with 2% wheat bran and nutrients [ZnSO4, (NH4)2SO4, KH2PO4, MgSO4, FeSO4 7H2O, C12H22O11 and MnSO4] (Arun et al., 2008) was inoculated followed by incubation at 37oC in an orbital shaker at 130 rpm. The harvesting was done after 4 days and the biomass thus produced was centrifuge at 6500 rpm for 15 minutes (Gupta et al., 2003). The supernatant was evaluated for enzyme activity.

Optimization of Conditions for the production of Glucoamylase: Production of GAAF was analyzed by examining the A. fumigatus growth in wheat bran medium at various temperatures ranging from 30 to 50oC and pH from 3 to 9 using 50 mM of each of sodium acetate buffer (3-5), sodium phosphate buffer (5-7) and Tris-HCl buffer (7-9) separately (Tayyab et al., 2011). In order to examine the maximal glucoamylase production, the wheat bran medium was supplemented with additional carbon (sucrose and starch), nitrogen (urea and ammonium sulphate) and phosphate (potassium mono and dihydrogen phosphate) sources at various concentrations ranging from 0.5 to 1.5%. Fungal growth and GAAF production was recorded in the above said medium after sterilization. Effect of incubation time for the maximal GAAF production was recorded by examining the fungal growth up to 5 days. GAAF production was also analyzed in the presence of Tween-80 a non-ionic detergent at a final concentration of 0.1 to 1% detergent.

Enzyme Assay: Enzyme activity was determined using starch as substrate at pH 5 in 50 mM sodium acetate buffer. Regarding the enzyme activity, 0.1 mL of enzyme was mixed with 1.9 mL substrate prepared in 50 mM sodium acetate buffer, followed by incubation at 40oC for 40 mins. The production of glucose was measured by DNS method by recording the absorbance at 540 nm. The units for enzyme activity were calculated using standard curve for glucose. One unit of enzyme activity was the amount of enzyme required for the production of one micromole of reducing sugar in one minute at 40oC (Miller, 1959).

Purification of enzyme: The soluble portion after centrifugation of fungal growth was utilized for 20, 40, 60 and 80% ammonium sulphate precipitation. The precipitation was done at 4oC. Each level of ammonium sulphate precipitated sample (20, 40, 60 and 80%) was centrifuged at 6500 rpm for 15 min at 4oC. The protein pellet obtained after each step was dissolved in 20 mL of 50 mM sodium acetate buffer (pH 5) and was utilized to examine the enzyme activity. The fraction with GAAF activity was introduced into the dialysis tube and was dialyzed against 50 mM sodium acetate buffer (pH 5) for 48h by replacing the dialysis buffer after every 4h with fresh buffer. The dialyzed fraction with enzyme activity was utilized for further purification by column chromatography. The dialyzed sample (200 mg protein) was applied to pre-equilibrated DEAE Sephadex A-50 ion exchange column (12cm X 3cm).

Unbound proteins were removed by washing the column with same buffer and elution of bound protein was done using NaCl gradient (0 to 1 M) in 50 mM sodium acetate buffer (pH 5) at a flow rate of 0.5 mL/min. The fractions were collected and utilized for enzyme activity analysis. The fractions with maximal enzyme activity were pooled and applied to pre-equilibrated Sephadex G-200 gel filtration column (12cm X 3cm) followed by elution with 50 mM sodium acetate buffer (pH 5) at a flow rate of 1mL/min. The purified protein was utilized for kinetic studies of GAAF.

Kinetic Studies of Glucoamylase: Enzyme activity was examined by varying the concentration of starch from 1 to 10% under optimized conditions and the obtained data was utilized for developing the Line-weaver Burk Plot and for the estimation of kinetic parameters (Mansoor et al., 2018).

Table 1. Summary of Purification of Glucoamylase from Aspergillus fumigatus.

Purification Step###Total Protein (mg)###Total Enzyme###Specific Activity###Yield###Fold

###Activity (U)###(U/mg)###(%)###Purification

Crude Sample###790###6248###7.91###100###-

Ammonium Sulphate Precipitated###200###5500###27.5###88###3.48

Purified sample###30###2790###93###45###12

RESULTS AND DISCUSSION

We have isolated and identified four strains of A. fumigatus. The strain with maximal production of glucoamylase (11 U/mL at 35oC) was selected for the further studies. It was found that the initial increase in temperature from 30 to 40oC resulted in the increased production of GAAF from 9.33 U/mL to 17.4 U/mL. Further increase in temperature resulted in the decreased production of enzyme (Fig 1). The optimal production of GAAF was observed at 40oC that is much higher as compared to 30oC for Aspergillus oryzae (Puri et al., 2013) and 35oC for GAs from Aspergillus awamori NRRL 3112 and Aspergillus niger (Aguero et al., 1990). A linear increase in the enzyme activity was recorded with the increase in pH. Maximum activity was recorded at pH 5 when 50 mM sodium acetate buffer was used for GAAF activity (Fig 2). Our findings are comparable to previously reported results by Pandey and Radhakrishnan (1993).

They examined 4.7 as an optimal pH for the production of glucoamylase by A. niger NCIM-1245. The results demonstrated that 3% wheat bran was the optimal substrate concentration for the production of GAAF (18 U/mL). Bhatti et al., (2007) examined the maximum production of glucoamylase from Fusarium solani when 2% wheat bran was used under optimum growth condition. When the production of glucoamylase was examined, while growing the fungal strain on the medium supplemented with additional carbon source (0.5 to 1.5%) sucrose or starch; starch put a clear enhancing effect on GAAF production. The presence of sucrose could produce a little effect on the production of enzyme. The presence of 2% wheat bran alone could produce the 11 U/mL of GAAF. This was increased to 13.15 U/mL and 15.25 U/mL in the presence of 1.25% of sucrose and starch respectively (Fig 3).

Our results are in agreement with studies on A. awamori which shows the same pattern (Pavezzi et al., 2008) where as these results are contradictory to the studies on F. solani (Bhatti et al., 2007). The supplementation of urea or ammonium sulphate (0.5 to 1.5%) as nitrogen source in the growth medium containing 2% wheat bran demonstrated that Urea enhanced GAAF production from 11 U/mL to 16 U/mL when used at a final concentration of 1.25% (Fig 3). These results are in agreement with the studies of Ellaiah et al., (2002), they examined the optimal glucoamylase production from Aspergillus sp. A3 when 1% urea was utilized as additional nitrogen source. The presence of ammonium sulphate put an inhibitory effect on the enzyme production. The presence of 1.5% ammonium sulphate decreased the production from 11 U/mL to 6.7 U/mL (Fig 3). This decrease in the activity is might be due to low fungal growth in the presence of ammonium sulphate.

The presence of mono or di-potassium hydrogen phosphates didn't show a significant effect on the production of glucoamylase (data not shown). The incubation time is very important parameter for the optimal production of enzyme. The incubation of A. fumigatus at 40 oC for a period of 2 days didn't put a significant effect on the production of GAAF (11.51 U/mL). Whereas, incubation up to 3rd day resulted, increased production of GAAF (21.02 U/mL), which clearly indicates, the fungal growth. Further incubation put an adverse effect on microbial growth. These results were contradictory to previously reported data, as maximum production of glucoamylase was recorded on 4th day from F. solani (Bhatti et al., 2007) or 5th day from A. oryzae (Puri et al., 2013) when, fungal strains were incubated at 30 oC. The presence of Tween-80 showed enhancing effect on the glucoamylase production.

The enzyme activity was found to be increased from 10.5 to 19.6 U/mL when the concentration of Tween-80 was increased from 0.1 to 0.7% respectively. Further increase in Tween-80 beyond 0.7% resulted in decreased activity. GAAF purification studies demonstrated 12 fold purification with 45% yield (Table 1) after ion exchange and gel filtration chromatography (Fig 4A). The purity analysis of the purified enzyme showed a clear band on SDS-PAGE having an approximate size of 60 kDa (Fig. 4B). Previous reports demonstrated the production of 74 kDa glucoamylase from A. niger (Deshmukha et al., 2011) while A. awamori has three glucoamylases having size 59.1, 87.1 and 109.6 kDa (Negi et al., 2011). The glucoamylase yield in present study was quite high as compared to 8% for glucosidase from the same strain (Rudick and Elbein, 1975). The Line-weaver Burk plot (Fig 5) demonstrated the Km and Vmax values of 9.1 mg/mL and 40 uM min-1 mg-1 respectively, when starch was used as a substrate.

Conclusion: This study demonstrated that Aspergillus fumigatus has strong potential for the glucoamylase production using agricultural wastes as carbon source. Optimization studies revealed the highest glucoamylase activity in shake flasks containing 3% wheat bran supplemented with 0.7% Tween-80 (23 U/mL) followed by 1.5% of urea (28 U/mL) at a temperature of 40oC in the presence of 50 mM sodium acetate buffer (pH 5). The ability of the fungal strain to utilize the agricultural waste as carbon source for the production of glucoamylase makes it unique machine for the production of enzymes. To our knowledge this is the highest glucoamylase activity produced by Aspergillus fumigatus sp. reported so far.

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Publication:Journal of Animal and Plant Sciences
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