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Use of Agro Industrial Residues for the Production of Amylase by Penicillium sp. for Applications in Food Industry

Department of Biotechnology, Lovely Professional University, Phagwara, Punjab, India

*Corresponding Author:

Arora N
Department of Biotechnology
Lovely Professional University
Phagwara, Punjab, India
Tel: +9666665287
E-mail: nidhi.arora2492@gmail.com

Received date: April 13, 2017; Accepted date: April 28, 2017; Published date: May 05, 2017

Citation: Arora N, Kaur S, Kaur S (2017) Production of Amylase by Penicillium sp. for Applications in Food Industry. J Biotechnol Biomater 7:256. doi:10.4172/2155-952X.1000256

Copyright: © 2017 Arora N, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

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Abstract

Glucoamylase production has been investigated by solid state fermentation using agro-industrial residues, rice bran, wheat bran and banana peel by Penicillium sp. Out of the different agricultural residues tested for enzyme production, rice bran yielded the maximum glucoamylase activity (10.29 ± 0.07 Units/ml), protein content (1.22 ± 0.0014 mg/ml) and specific activity (8.4 Units/mg protein) with Penicillium sp. after 7th day of fermentation as compared to other agro residues. Media supplementation with carbon and nitrogen sources enhances the enzyme activity. For maximum production of enzyme, sucrose (13.70 ± 0.77 Units/ml) (1% mass level) and yeast extract (14.41 ± 0.07Units/ml) (1% mass level) as carbon and nitrogen source respectively were found optimum on rice bran with Penicillium sp. Optimum enzyme activity was observed at 90°C, pH 9. The partial purification of enzyme from rice bran by Penicillium sp. was done with 60% ammonium sulphate precipitation showed maximum enzyme activity. Enzyme hydrolysis showed maximum activity (3.69 Units/ml) with potato starch from rice bran by Penicillium sp.

Keywords

Amylase; Glucoamylase; Asperigillus; Rhizopus; Bradford method; Penicillium sp.; Enzyme

Introduction

Recent discoveries on the use of microorganisms as sources of industrially relevant have led to an increased in the application of microbial enzymes in various industrial processes. Nowadays, the use of microorganism as biotechnological source of industrially relevant enzymes has interest in exploration of extracellular enzymatic activities in several microorganisms. Amylases are used in dextrose production, in the baking industry, in the brewing of low-calorie beer and in whole gain hydrolysis for the alcohol industry [1].

The amylase family has two major classes, namely amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3). Alpha-amylase are extracellular enzymes which hydrolyze starch into maltose, glucose and maltotriose by cleaving the 1,4-D- glucosidic linkages between adjacent glucose units in the linear amylose chain [2].

Glucoamylase (GA)

Hydrolyses single glucose units from the non-reducing ends of amylase and amylopectin in a stepwise manner and produce glucose as the sole end-product from and related polymers. Unlike alpha amylase, most glucoamylases are also able to hydrolyse the 1,6-linkages at the branching points of amylopectin, at a lower rate than 1,4-linkages [3]. Alpha amylase is secreted as a primary metabolite and its secretion is growth associated [4]. It is an important group of enzymes in starch processing. They are second to the proteases in worldwide distribution and sales among industrial enzymes.

Production of glucoamylase (GA) is performed mostly by two methods, Solid State or Solid-Substrate Fermentation (SSF) and Submerged Fermentation (SMF) [5]. SSF has many advantages over SMF, such as simple technique, superior productivity, low capital investment, low energy requirement and less water output, better product recovery and lack of foam build up and reported to be the most appropriate process for developing countries [6]. Selection of a suitable microorganism is one of the most important in SSF. Several groups of microorganism such as filamentous fungi group can grow on solid substrates.

Agro-industrial residues are generally considered the best substrates for the SSF processes and enzyme production by SSF is not an exception to that [7]. Use of suitable low cost fermentation medium for production of α-amylase by using agricultural by products has been reported [8].

Fungal species such as Asperigillus, Rhizopus and Penicillium are considered to be the chief producers of amylase [9,10]. Among large number of agro-industrial wastes (wheat bran, wheat straw, rice bran, rice husk, rice straw, sago hampas, grapevine trimmings dust, saw dust, corncobs, coconut coir pith, banana waste, tea waste, cassava waste, palm oil mill waste, aspen pulp, sugar beet pulp, sweet sorghum pulp, apple pomace, peanut meal, rapeseed cake, coconut oil cake, mustard oil cake, cassava flour, wheat flour, corn flour, steamed rice, steam pretreated willow, starch and bagasse ) [11] are available as substrate for the production of enzyme.

In the present study glucoamylase production has been studied with a natural isolate of the fungus Penicillium sp. using the rice bran waste obtained from local market of Haryana along with wheat bran and banana peel as the growth substrates.

Methods

Microorganism and its maintenance

Penicillium sp. was propagated on Potato Dextrose Agar (PDA) medium. Slants were grown at 30°C for 5 days and stored at 4°C. Subculturing was done after every 15 days.

Preparation of basal media

Solid state fermentation with the agro residues wheat bran (WB), Rice Bran (RB) and Banana Peel (BP) was done by moistening with mineral salt solution having composition (mg/gds) (NH₄)2SO₄ 4, MgSO₄.7H₂O 1, FeSO₄.7H₂O 0.02, K₂HPO₄ 1.4 and KH₂PO₄ 0.6.The pH of the media was adjusted to 7.0 [5].

Enzyme production and extraction

The production media contained 10 g of solid substrate and 20 ml mineral solution in 250 ml Erlenmeyer flasks initially maintained at pH 7. The media were inoculated with 2 ml of inoculum having 106 spores/ ml collected from 72 h grown culture of Penicillium sp. at 28 ± 2°C. Inoculated production media were incubated under static conditions at 28 ± 2°C and enzyme production was checked after every 24 h for 5 days. Enzyme was extracted in 100 ml, 0.1 M sodium acetate buffer on a rotary shaker at 100 rpm for 60 min. The content was filtered through muslin cloth and the filtrate was used as the enzyme source.

Measurement of protein content and enzyme activity of crude enzyme extract

Protein estimation: Protein content was determined by using Bradford method. Bradford reagent was made by dissolving 100 mg Coomassie Blue G-250 in 50 ml of 85% ethanol and adding 100 ml of 85% (w/v) phosphoric acid and make volume to 1 L. 1 ml of Bradford reagent was added to protein sample and incubated at room for 10 min. Standard curve was prepared by using BSA, stock solution having concentration of 100 mg/ml. The absorbance of each sample was measured at 595 nm using a UV-visible spectrophotometer [12].

Enzyme activity: The enzyme activity was determined by incubating a reaction mixture containing 0.95 ml of 0.1 M sodium acetate buffer (pH 5), 1.0 ml starch solution (1%, w/v) and 0.05 ml of crude enzyme at 55°C for 15 min [13]. The reducing sugar released after 10 min were measured with 3,5-dinitrosalicyclic acid (DNSA) reagent [14] using glucose as a standard. Glucoamylase activity unit (U) was expressed as the amount of enzyme releasing 1 μmol of glucose equivalent per min under assay condition and enzyme activity is expressed in terms of units per gram dry fermented substrate (U/gdfs).

Growth kinetics

Wheat bran, rice bran and banana peel (10 g) were kept separately in a 250 ml flask and then moistened with 20 ml of mineral solution containing (mg/gds) (NH₄)2SO₄ 4, MgSO₄.7H₂O 1, FeSO₄.7H₂O 0.02, K₂HPO₄ 1.4 and KH₂PO₄ 0.6, initially maintained at pH 7 [5] and sterilized at 121°C for 30 min. The fermentation process was started by adding 2 ml of spore suspension as prepared above. The whole content was mixed thoroughly and then incubated at 30°C for 3 days, 5 days, 7 days and 10 days in a stationary condition.

Separation and identification of sugars by thin layer chromatography

Sugars get separated on the basis of differential adsorption onto silica gel. The reaction mixture contains 1 ml of enzyme and 1 ml of standard solution which was prepared as 1% solution of standard sugars such as glucose, maltose in 10% isopropanol (v/v).Carefully the solution of individual standard sugars and the mixture of the samples applied on the separate marked spots. The chromatogram was developed with solvent system of ethyl acetate:iso-propanol:water:pyridine (26:14:7:2, v/v). Due to capillary action solvent reaches the marked line on the upper side of the silica plate. Plate was removed from chromatographic chamber and let it dry at room temperature. The spots were visualised by spraying aniline diphenylamine reagent (mix 5 volumes of 1% aniline and 5 volumes of 1% diphenylamine in acetone with 1 volume of 85% phosphoric acid) and heating at 100°C. The sugar spots appeared as dark brown spots.

Identification of the sugars was done by comparing the relative fraction (R_f) values of the samples with that of the standards.

R_f=Distance travelled by unknown amino acid/Distance travelled by solvent system

Characterization of glucoamylase

Soluble starch used in assay system for enzyme-substrate reaction was supplemented as 1%. Enzyme activity was checked individually at four different pH as 3, 5, 7 and 9 of the 0.1 M Sodium acetate buffer used and at four different temperatures 35°C, 55°C, 75°C and 90°C.

Effect of temperature: To check the optimum temperature for glucoamylase activity, crude enzyme of 7th day extract was incubated with 1% soluble starch in 0.1 M acetate buffer, pH 5 at different temperatures as 35°C, 55°C, 75°C and 90°C. Enzyme activity was checked individually.

Effect of pH: To check the optimum pH for glucoamylase activity, the pH of 0.1 M sodium acetate buffer was varied as 3, 5, 7 and 9 and the enzyme activity was checked individually.

Effect of carbon and nitrogen additives: The optimum SSF medium of wheat bran and rice bran was supplemented with different carbon (glucose, fructose, sucrose and starch) and nitrogen sources (urea, yeast extract, soybean meal and peptone) at 1% (by mass) level to study their effect on GA production by Penicillium sp. The fermentation was carried out at pH 5 and 30°C for 7 days [15].

Partial purification of crude enzyme

Ammonium sulfate precipitation: 100 ml of the crude enzyme were first brought to 40% saturation with solid ammonium sulfate (enzyme grade) and the process was carried out at 4°C [16]. The whole apparatus was placed on a magnetic stirrer and ammonium sulphate was allowed to dissolve in the enzyme solution. The precipitated proteins were regimented by centrifugation for 15 min at 1000 rpm. The resulted pellet was dissolved in 3 ml of 0.2 M phosphate buffer at pH 6.2.The supernatant was applied again with ammonium sulfate to achieve 60% saturation. Both enzyme activity and protein content were determined for each separate fraction [17].

Dialysis: Pellet was dissolved in sodium acetate buffer (0.2 M) and dialysed against the same buffer (pH 5) with 3 equal changes of buffer after every hour to ensure the complete removal of the ammonium salt, enzyme was dialysed for 3 h at 4°C. The dialysed fraction referred to as partially purified glucoamylase [18].

Application of glucoamylase

Enzyme hydrolysis of various starches: The ability of the crude amylase to hydrolyze different native starches was studied using corn and potato starches. Commercially available soluble starch was used as standard. The assay mixture consisted of 1% of various starch in 0.2 M citrate-phosphate buffer (pH 6) and 0.05 of the enzyme. Enzyme assay was carried out after incubation at 40°C for 10 min [19].

Results and Discussion

Glocoamylase production using solid substrates

Rice bran, wheat bran and banana peel was used as substrate for the production of glucoamylase by Penicillium sp. for 5 days in solid state fermentation. The results showed that maximum activity was shown by Penicillium sp. on rice bran (8.029 ± 0.014 Units/ml) followed by wheat bran and banana peel.

Growth kinetics

Wheat Bran, Rice Bran and Banana Peel (10 g) which were incubated for 3 days, 5 days, 7 days and 10 days in stationary condition showed enzyme activity. Penicillium sp. on rice bran (10.29 ± 0.07 Units/ml) at 7th day showed maximum enzyme activity followed by wheat bran and banana peel. After 7 days enzyme activity decreased (Figure 1).

Figure 1: Effect of days on enzyme activity (U/ml) by Penicillium sp. on different substrates.

Figure 2: Thin layer chromatography of the reaction product hydrolysed by glucoamyalse from Penicillium sp. glucose and maltose was used as standard.

Thin layer chromatography of hydrolysis products

Glucoamylase is an exo-acting enzyme that catalyses the production of β-glucose from the non-reducing ends of amylase, amylopectin and glycogen. It consecutively hydrolyses α-1,4; α-1,6 and rare α-1,3 linkages [20]. TLC revealed only glucose as the hydrolysis product of starch which indicates that the crude enzyme was a glucoamylase [21]. R_f values of WB(A),WB(P), RB(A) and RB(P) are 0.85, 0.78, 0.75 and 0.89, respectively and Rf values of glucose and maltose of WB(A), WB(P), RB(A) and RB(P) are 0.85,0.89; 0.78,0.74; 0.75,0.7; 0.89,0.85, respectively (Figure 2).

Figure 3: Effect of temperature(°C) on enzyme activity (Units/ml) [WB (P)].

Figure 4: Effect of temperature(°C) on enzyme activity (Units/ml) [RB (P)].

Figure 5: Effect of pH on enzyme activity (Units/ml) [WB (P)].

Figure 6: Effect of pH on enzyme activity (Units/ml) [RB (P)].

Figure 7: Effect of different carbon and nitrogen additives on enzyme activity (Units/ml) of wheat bran (WB) and rice bran (RB) by Penicillium sp.

Table 1: Enzyme activity (Units/ml) of partial purified enzyme.

Figure 8: Effect of different starch on enzyme activity of wheat bran (WB) and rice bran (RB) by Penicillium sp.

Characterization of enzyme

Effect of temperature: Glucoamylase activity was tested at different temperatures. Penicillium sp. showed the maximum activity in case of WB (9.23 ± 0.10 Units/ml) at 55°C and for RB (10.66 ± 0.0007 Units/ml) at 90°C (Figures 3 and 4).

Effect of pH: Enzyme activity was maximum at pH 9, for Penicillium sp. on RB and WB using 1% soluble starch for enzyme assay (Figures 5 and 6).

Effect of carbon sources and nitrogen additives on enzyme activity: Addition of different sugars as additives to wheat bran and rice bran medium enhanced glucoamylase production by Penicillium sp. The maximum activity was shown by Penicillium sp. on RB with sucrose (13.70 Units/ml) as carbon source and yeast extract (14.41 Units/ml) as nitrogen source (Figure 7).

Partial purification of glucoamylase

Glucoamylase was purified by ammonium sulfate precipitation and dialysis process. Penicillium sp. in rice bran and wheat bran showed an increase in enzyme activity as comparative to crude enzyme (Table 1).

Application of glucoamylase

Enzyme hydrolysis of various starches: The rate of the amylase hydrolysis of different native starches was evaluated. Figure 8 showed that the amylase from the Penicillium sp. and A. terreus was able to hydrolyse different starches to varying degrees. Highest rate of hydrolysis was observed for potato starch, followed by corn starch (Figure 8).

Conclusion

On the basis of the finding of the present study it can be concluded that out of the all different agricultural residues which is used to test for enzyme production, rice bran has the maximum glucoamylase activity (10.29 ± 0.07 Units/ml), protein content (1.22 ± 0.0014 mg/ml) and specific activity (8.4 Units/mg protein) with Penicillium sp. at 7th day as compared to other agro residues. By usage of Media supplements with carbon and nitrogen sources increases the enzyme activity. Sucrose (13.70 ± 0.77 Units/ml) (1% mass level) and yeast extract (14.41 ± 0.07 Units/ml) (1% mass level) as carbon and nitrogen source respectively were found optimum on rice bran with Penicillium sp. Optimum enzyme activity was observed at 90°C, pH 9.The partial purification of enzyme from rice bran by Penicillium sp. was done using ammonium precipitation and showed maximum enzyme activity at 60%. Enzyme hydrolysis showed maximum activity (3.69 Units/ml) with potato starch from rice bran by Penicillium sp.

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