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Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger; its purification and characterization
JRRAS206_proof ■ 18 November 2015 ■ 1/7 J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s x x x ( 2 0 1 5 ) 1 e7
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Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger; its purification and characterization Q4
Ishtiaq Ahmed a,*, Muhammad Anjum Zia a, Muhammad Azhar Hussain a, Zain Akram b, Muhammad Tahir Naveed c, Azin Nowrouzi d a
Department of Biochemistry, University of Agriculture Faisalabad, Pakistan Department of Chemistry, University of Sargodha, Pakistan c Center of Agriculture Biochemistry and Biotechnology (CABB), University of Agriculture Faisalabad, Pakistan d Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, Iran b
article info
abstract
Article history:
Agro-industrial residues are primarily composed of complex polysaccharides that
Received 30 August 2015
strengthen microbial growth for the production of industrially important enzymes. Pecti-
Received in revised form
nases are one of the most widely disseminated enzymes in bacteria, fungi and plants.
29 October 2015
Czapeck media supplemented with orange waste peel as carbon source under submerged
Accepted 4 November 2015
fermentation process Aspergillus niger presenting the preeminent enzymatic production.
Available online xxx
On partial optimization culture showed the maximum enzyme yield (117.1 ± 3.4 mM/mL/ min) at 30 C in an orange waste peel medium having pH 5.5 and substrate concentration
Keywords:
(4%) after 5th day of fermentation. The produced enzyme was purified by ammonium
Aspergillus niger
sulfate precipitation and ion exchange chromatography. A purification fold of 5.59 with
Orange waste peel
specific activity and % recovery of 97.2 U/mg and 12.96% was achieved respectively after gel
Pectinases
filtration chromatographic technique. The molecular weight of purified pectinase from A.
Purification
niger was 30 kDa evidenced by SDS-PAGE. Pectinase activity profile showed purified enzyme
SDS-PAGE
was optimally active at pH ¼ 7 and 55 C. The maximum production of pectinase in the presence of cheaper substrate at low concentration makes the enzyme useful in industrial sectors especially for textile and juice industry. Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1.
Introduction
Pectin comprises of D-galacturonic acid occurred in a (1e4)chain, naturally esterifies with methoxy groups and natural
sugars occupy the side chains. Occasionally, rhamnose group present in chain unsettles the chain helix formation (Shembekar & Dhotre, 2009). In the last two decades of twentieth century, an industrial revolution happened in
* Corresponding author. E-mail address: [email protected] (I. Ahmed). Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications. http://dx.doi.org/10.1016/j.jrras.2015.11.003 1687-8507/Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Ahmed, I., et al., Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger; its purification and characterization, Journal of Radiation Research and Applied Sciences (2015), http://dx.doi.org/10.1016/ j.jrras.2015.11.003
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developing countries in citrus producing and processing area (Juices, Oils and other by-products). Pakistan being an agricultural country produces millions of tons of fruits and vegetables annually. Mango, citrus, apple, banana and dates are the highest yielding fruits in Pakistan. The cell wall and middle lamella are enriched of pectin ingredients (Alkorta, Garbisu, Llama, & Serra, 1998). Carbohydrates and proteins are the major but the fat contents are in low amount in dried citrus peel. Significant quantity of pectin in citrus peel urges us to use them as a substrate for microorganism to produce pectinolytic enzymes. Microbial systems enhance pectinolytic production using pectin as inducer (Aguilar & Huitron, 1993). During the raw agriculture material processing for food, a bulk quantity of agro wastes generated. Thus, agro residues of coffee, orange, rice and sugarcane provide a suitable platform for bio-production of different enzymes using the fermentation process, thereby making valuable of these waste products (Giese, Dekker, & Barbosa, 2008). Orange bagasse contains large amounts of soluble carbohydrates, particularly fructose, glucose, sucrose, and pectin, as well as insoluble cellulose, and has been used as a fermentation products including enzymes (Rosales, Couto, & Sanroman, 2002). Microorganisms have noteworthy collaboration in food (dairy and meat) and alcoholic beverages industries (Buyukkileci, Tari, & Fernandez-Lahore, 2011). The usage of bio-produced food and additives makes them more appropriate rather than the synthetically produced ones (Susana and Sanroman, 2006). Citrus peel is known as the major source of pectinases in several industries instead of apple pomace. The potential applications of pectinases in different industries craving us to conduct the present study.
Q1
2.
Materials and methods
2.1.
Chemicals, microorganism and substrate
The pure culture of Aspergillus niger was available in our lab as previously reported by Ahmed, Zia, Iftikhar, and Iqbal (2011). Fungal spore suspension was prepared by using the method of Dhillon et al. (2004). In brief; Spores of A. niger were cultured in 250 mL flask holding 30 mL of Potato Dextrose broth at 30 ± 1 C for 4 days. To achieve the maximum concentration of spores in the solution, we used saline solution (0.9% w/v) on the fungal spores and shaken flask to accelerate the release of spores into the surrounding saline solution. Orange peel waste was obtained from local fruit market Faisalabad, Pakistan. The substrate was dried (60 C), ground and kept in air tight plastic jars to lessen the moisture contents.
2.2.
Fermentation methodology
Substrate screening was done by taking 4 g orange peel waste into 250 mL flasks of three replicates and inoculated with spore suspension of A. niger. All the experimental flasks were incubated at 30 ± 1 C in a shaking incubator at 180 rpm for 5 days of fermentation period. The ingredients of the flasks were filtered and clear supernatant having enzyme solution was subjected to further analytical studies.
2.3.
Chemical analysis of the substrate
2.3.1.
Determination of % moisture content
The moisture contents of sour orange peel was determined by taking 2 g sample in a sterilized bottle (W1) and dried at 100 C for 2 h. The sample was cooled and weighed in desiccators, after that sample again dried until constant weight (W2). The moisture percentage was calculated by using the following formula: ð%Þ Moisture ¼
2.3.2.
ðW1Þ ðW2Þ 100 ðW1Þ
(E1)
Determination of % ash content
Pre-weight crucible containing 5 mL HNO3 and 2 g of substrate (W1) was used to heat the material until char forming. After that sample put in muffle furnace for 6 h at 555 C and weighed (W2) to determine the percentage ash contents of sample. ð%Þ Ash ¼
2.3.3.
ðW1Þ ðW2Þ 100 Weight of sample
(E2)
Determination of crude % fat content
5 g sample (W1) was added in a pre-weighed thimble and keep in an extraction apparatus (Soxhelt) for 16 h and extraction was done with hexane. Subsequently, thimble with dried sample weighed to calculate the fat contents (W2). The crude %fat content of sample was calculated by using the following formula: ð%Þ Crude Fat ¼
2.3.4.
ðW1Þ ðW2Þ 100 ðW1Þ
(E3)
Determination of % crude fiber content
For the determination of crude fiber contents, 2 g of dry material (W1) after extraction with ether was boiled with 200 mL of H2SO4 containing 1.25 g of H2SO4 per 100 mL for 30 min with 200 mL of NaOH solution containing 1.25 g carbonate free NaOH per 100 mL and filtered. The material was dried in an oven and weighed (W2). The decrease in weight was considered as crude fiber.
ð%Þ Crude Fiber ¼
2.3.5.
ðW1Þ ðW2Þ 100 Weight of sample
(E4)
Determination of % lignin content
In brief, 2 g (W1) sample was added in 10 mL of 70% (w/w H2SO4) solution and boiled for 4e5 h. The cellulose and hemicellulose contents were hydrolyzed after the above treatment and remaining suspension was filtered with hot water. Afterward, addition of 30 mL of 70% H2SO4 into the mixture was carried out and kept in oven for 24 h at 105 C and weighed (W2). Then treated sample was moved into preweighed dry porcelain crucible for high temperature treatment (650 C) for 4 h. The sample was allowed to cool down and lignin content (%) was calculated by using the following formula. ð%Þ Lignin ¼ ðW1Þ ðW2Þ
(E5)
Please cite this article in press as: Ahmed, I., et al., Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger; its purification and characterization, Journal of Radiation Research and Applied Sciences (2015), http://dx.doi.org/10.1016/ j.jrras.2015.11.003
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2.4.
Analytical methods
2.4.1.
Total sugar estimation
In order to measure total sugar in the medium standard titration method was used. Briefly, a mixture of 100 mL water and 25 g of sample and 2 mL of Lead acetate solution was added and stayed for 10e15 min. The following formula was intended to measure %total sugar in the sample. Total Sugar ð%Þ ¼
2.4.2.
Factor 100 Dilution Titer 1000
Reducing sugar estimation
Protein determination
For determination of protein, Lowry, Rosebrough, Farr, and Randall (1951) method was carried out using bovine serum albumen as standard.
2.4.4.
Determination of pectinase activity
Enzyme activity was measured by dinitrosalicylic acid reagent (DNS) method (Miller, 1959). In brief, a mixture containing 2.0 mL sodium citrate buffer (pH ¼ 5.0), 1 mL enzyme extract and 0.2 mL of pectin solution (1%) was incubated for 25 min at 35 C ± 1 C. After incubation time, 1 mL of incubated solution was withdrawn and mixed with 0.5 mL of sodium carbonate (1 M) solution in a test tube. Addition of 3 mL DNS reagent in each test tube and shaken for 10 min dynamically to mix the contents completely. Samples were heated for 10 min and cooled down at room temperature before adding the 20 mL distilled water. The enzyme activity was measured spectrophotometrically by taking the absorbance at 570 nm.
2.5.
Optimization parameters
Different parameters were investigated for optimization including fermentation period (24e144 h), Substrate concentration (1e5%), Inoculum size (2e10 mL), pH (4e6.5) and Temperature (25e45 C) to achieve maximum yield of pectinase enzyme. All the parameters were run in triplicates.
2.6.
chromatography column (2 20 cm) was used to further purification, equilibrated with 0.01 mM TriseHCl buffer (pH 6.0). Both, protein contents and enzyme activity were measured as described in earlier part. For this, partially purified enzyme was subjected to chromatography column at flow rate of 0.5 mL/min to collect 20 fractions of 2 mL each. Molecular mass of purified Pectinase was investigated according to the method of Laemmli (1970) by performing Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE).
(E6)
3,5-dinitrosalicyclic acid (DNS) colorimetric method was followed to measure the reducing sugar of sample with glucose as standard (Miller, 1959). The mixture of 1 mL DNS and 1 mL of water was boiled for 5 min. Color intensity was measured spectrophotometrically at 550 nm.
2.4.3.
3
Enzyme purification
The methodology developed previously by Ahmed et al. (2011) was used for the purpose of purification. In brief, harvested crude enzyme was centrifuged at 10,000 g for 15 min at 4 C and subjected to precipitate by crystals of ammonium sulfate with 5% increasing each time (starting from 30% up to 90%). The resulting precipitate bearing high pectinase activity were suspended in 0.01 M TriseHCl buffer and dialyzed. Before subjected to ammonium sulfate precipitation enzyme activity was measured followed by after precipitation. The lyophilization was carried out after partial purification for further studies e.g. SDS-PAGE (molecular mass determination) and gel filtration chromatography, respectively. Gel filtration column
2.7.
Characterization of pectinase
The characterization study of purified pectinase after DEAEcellulose column chromatography was done to examine the effect of temperature (40e60 C) and pH (4e8).
3.
Results and discussion
3.1.
Chemical composition of substrate
Chemical composition of orange peel waste was determined through physiochemical analysis for pectinase production. The data in (Fig. 1) indicates the proximally analyzed orange peel waste contained moisture (40.7%), ash (7.39%), fat (1.85%), pectin (7.0%), lignin (6.4%), crude fiber (7.8%), total sugar (14.08%), reducing sugars (10.70%) and non-reducing sugar (3.70%). Almost similar results of ash and fat contents reported the Oluremi et al. (2007). The total amount of pectin present in the orange peel waste was 7% i.e. 70 mg/g according to our investigations. The present reported findings are in accordance to the earlier reported data from different research scientists Wang, Chuang, and Hsu (2008), Kratchanova, Pavlova, and Panchev (2004) and Liu et al. (2001). Q2
3.2.
Optimization of incubation time
To check the effect of different time periods on pectinase production from A. niger on physiochemical analyzed orange peel waste by submerged fermentation, experiments were conducted at different time periods from 24 to 144 h. Data analyzed showed that pectinase production was increased gradually during the fermentation period and reached to its maximum value (77.2 ± 1.42 mM/mL/min) after 5th day of initial incubation (Table 1). Further increase in fermentation period decreased the production of pectinase. This phenomenon was also observe by Mrudula and Anitharai (2011) during the pectinase and polygalacturonase production by Penbicillium sp. Production of pectinase increases with increase in fermentation period due to increase in activity of pectin methyl esterase (PME) and it is also dependent on the type of fermentation including concentration of nutrients in the medium, organisms, and physiological conditions (Joshi, Parmar, & Rana, 2006).
3.3.
Optimization of substrate concentrations
The obtained results of different substrate concentration (1e5%) on production of pectinase by A. niger are summarized in Table 2. The maximum enzyme activity (99.4 ± 1.1 mM/mL/
Please cite this article in press as: Ahmed, I., et al., Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger; its purification and characterization, Journal of Radiation Research and Applied Sciences (2015), http://dx.doi.org/10.1016/ j.jrras.2015.11.003
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J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s x x x ( 2 0 1 5 ) 1 e7
Fig. 1 e Chemical composition of orange peel waste.
min) was observed under optimum (4%) substrate concentration. After careful analyzing during the experiment we observed that under enhanced carbon source concentration, more inducers available to A. niger that in response enhance the production of pectinase. The amount of reducing sugars produced by pectinase in fermentation media was (2.92 ± 0.09 mg/mL). In earlier study, Giese et al. (2008) reported similar results while on contrary to Giese et al. (2008) and our present investigations, previously, Naidu and Panda (1998) reported that high concentration of carbon sources inhibits enzyme synthesis. During the trial it was observed that A. niger used reducing sugars present in the filtrate for its functional activity to produce potential enzymes. Moreover,
Table 1 e Activities of pectinase produced by Aspergillus niger. Fermentation periods
24 48 72 96 120 144
Filtrate characteristics pH 4.7 ± 4.6 ± 4.5 ± 4.8 ± 4.9 ± 4.2 ±
Reducing sugars (mg/mL)
0.20 0.24 0.10 0.14 0.41 0.29
3.10 2.90 2.82 3.41 3.36 3.28
± 0.22 ± 0.10 ± 0.53 ± 0.9 ± 2.00 ± 1.50
Enzyme activity (mm/mL/min) 11.4 21.7 41.6 55.4 77.2 52.4
± 1.34 ± 1.99 ± 1.41 ± 3.15 ± 1.42 ± 2.14
culture organism achieved maximum yield of pectinase by hydrolyzing polysaccharides. As a result the amount of reducing sugar tends to increase when A. niger used as an enzyme producing microorganism (Draginia, Madarev, Radulovic, & Skrinjar, 2007).
3.4.
Optimization of inoculum size
Maximum pectinase activity (103.2 ± 1.9 mm/mL/min) was observed with 8% inoculum size while the amount of reducing sugars produced and pH of the medium was 3.2 ± 0.11 mg/mL and 4.3 ± 0.3 respectively (Table 3). Optimum inoculum density is an important consideration for fermentation process since accumulation of spores can inhibit growth and development of the culture organism (Nighoskar, Phanse, Sinha, Nighojkar, & Kumar, 2006). Presently it was observed that the presence of large amount of reducing sugars has inhibitory effect on the pectinase production.
3.5.
Optimization of pH
The effect of initial medium pH for pectinase enzyme production from 5 to 8 was investigated and maximum activity (117.1 ± 3.4 mM/mL/min) was found (Table 4). The decline in enzyme activity was observed as the pH level increased from 5.5. The amount of reducing sugars in the medium released
Table 2 e Activities of pectinase produced by Aspergillus niger.
Table 3 e Activities of pectinase produced by Aspergillus niger.
Substrate conc. (%)
Inoculum size (%)
1 2 3 4 5
Filtrate characteristics pH 4.6 ± 4.4 ± 3.9 ± 4.5 ± 4.8 ±
0.20 0.15 0.29 0.14 0.23
Reducing sugars (mg/mL) 0.79 1.70 2.67 2.92 3.39
± 0.22 ± 0.05 ± 0.19 ± 0.09 ± 0.11
Enzyme activity (mm/mL/min) 66.2 ± 81.9 ± 89.5 ± 99.4 ± 76.4 ±
1.9 1.7 1.3 1.1 1.8
2 4 6 8 10
Filtrate characteristics pH 4.8 4.4 4.7 4.3 4.6
± 0.12 ± 0.20 ± 0.70 ± 0.30 ± 0.34
Reducing sugars (mg/mL) 2.36 ± 2.79 ± 2.92 ± 3.21 ± 2.80 ±
0.41 0.39 0.21 0.11 2.15
Enzyme activity (mm/mL/min) 62.9 ± 1.2 77.0 ± 1.4 94.5 ± 1.1 103.2 ± 1.9 75.6 ± 2.1
Please cite this article in press as: Ahmed, I., et al., Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger; its purification and characterization, Journal of Radiation Research and Applied Sciences (2015), http://dx.doi.org/10.1016/ j.jrras.2015.11.003
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Table 4 e Activities of pectinase produced by Aspergillus niger. pH
Filtrate characteristics pH
4 4.5 5 5.5 6 6.5
Reducing sugars (mg/mL)
4.5 ± 0.20 4.8 ± 0.31 4.9 ± 0.17 4.7 ± 0.21 4.6 ± 0.19 4.1 ± 0.11
3.15 ± 3.39 ± 3.31 ± 3.54 ± 3.28 ± 3.15 ±
Enzyme activity (mm/mL/min)
0.02 0.41 0.29 0.09 0.31 0.36
68.5 7666 91.9 117.1 86.2 71.9
± 2.2 ± 3.9 ± 2.3 ± 3.4 ± 2.9 ± 2.3
Table 5 e Activities of pectinase produced by Aspergillus niger. Temperature ( C)
Filtrate characteristics pH 5.2 ± 4.9 ± 4.7 ± 4.5 ± 3.8 ±
25 30 35 40 45
0.41 0.20 0.29 0.19 1.1
Reducing sugars (mg/mL) 3.18 3.36 3.19 2.92 2.54
Enzyme activity (mm/mL/min)
± 0.20 ± 0.39 ± 0.19 ± 0.22 ± 0.85
90.6 126.0 97.1 89.5 67.3
± 2.4 ± 2.8 ± 3.4 ± 2.5 ± 2.2
was (3.54 ± 0.9 mg/mL). According to Spagna, Pifferi, and Gillioli (1995), maximum pectinase activity was under optimum pH 5.0 while, according to Pedrolli, Gomes, Monti, and Carmona (2008) 4.5 pH value was an optimum.
3.6.
Q3
Optimization of temperature
Fig. 2 e Molecular mass determination of purified pectinase by SDS-PAGE [Lane-M, Molecular weights in kDa of standard marker; lane-1, Crude enzyme extract; lane-2 Purified pectinase]. mg. A sudden increase in enzyme purity was observed after ion exchange chromatography, such as enzyme purity and specific activity were jumped up to 5.59 fold and 97.2 U/mg, respectively. Electrophoresis studies revealed that purified pectinase enzyme produced from A. niger has 30 kDa molecular mass (Fig. 2). Various studies revealed different molecular mass of pectinase enzyme produced from different microbial species (Oyede, 1998). Different microbial sources exhibited extensive divergence in molecular mass of exo-PG i.e. Aspergillus japonicum (38.0 and 65.0 kDa) (Semenova, Grishutin, Gusakov, Okunev, & Sinitsyn, 2003), Aspergillus kawachii (60.0 kDa) (Esquivel & Voget, 2004). The difference has been associated with the substrate employed, type and nature of microorganism, host cell wall and analytical methods (Oyede, 1998).
To study the effect of temperature on the pectinase enzyme production, shake flask experiments were run at various temperature between 25 and 40 C. Maximum enzyme activity (126.0 ± 2.8 mm/mL/min) and reducing sugar (3.36 ± 3.9 mg/mL) was observed in batch culture fermented at 30 C (Table 5). Further increase in temperature suppressed the enzyme activity. The attained results are correlated to the data earlier reported by Pedrolli et al. (2008) and Nighoskar et al. (2006). Thermo-stable strains of Aspergillus aculeatus, Fusarium oxysporum species produced enhanced yield of pectinase even at higher temperature i.e. 60e65 C. The difference between temperature ranges may be due to the genetic difference of various species (Table 6).
3.8.
Characterization of pectinase
3.7.
3.8.1.
Effect of pH on activity & stability
Purification and SDS-PAGE
Purified pectinase after ammonium sulfate precipitation exhibited protein (120 mg/mL) and specific activity of 23.6 U/
The obtained results demonstrated that maximum activity of pectinase enzyme produced from A. niger was observed at pH 5 (Fig. 3). The optimum pH of our findings is comparable to the
Table 6 e Pectinase purification summary. Purification steps
Crude enzyme (NH4)2SO4 Precipitation Dialysis Sephadex-G-100
Volume (mL)
Enzyme activity (U)
Protein content (mg)
Specific activity (U/ mg)
Purification fold
% Yield
100 20
13,500 2840
775 120
17.4 23.6
1 1.36
100 15.11
47 18
46.9 97.2
2.87 5.59
16.33 12.96
15 10
2205 1750
Please cite this article in press as: Ahmed, I., et al., Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger; its purification and characterization, Journal of Radiation Research and Applied Sciences (2015), http://dx.doi.org/10.1016/ j.jrras.2015.11.003
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biosynthesized enzyme useful in textile as well as food processing industrial sectors.
Conflict of interest statement Authors are happy to declare that we do not have any conflict of interest in any capacity.
Acknowledgments Fig. 3 e Effect of pH on purified pectinase activity.
The present work was carried out under the supervision of Dr. Muhammad Anjum Zia at Enzyme Biotechnology Lab, Department of Biochemistry UAF Pakistan. On providing collaborative help for the present study, Dr. Azin Nowrouzi is thankfully acknowledged.
references
Fig. 4 e Effect of temperature on purified pectinase activity.
pectinase of Penicillium varidictum and Penicillium oxalicum (Silva, Martins, Silva, & Gomes, 2002; Zhang et al., 2009), respectively. Freitas et al. (2006) reported that optimum pH (5.5) for pectinase activity from thermo-tolerant Aspergillus sp.
3.8.2.
Effect of temperature on activity & stability
To find out the thermo stability of pectinase, enzyme was incubated at various temperatures ranging from 30 to 60 C. The results (Fig. 4) revealed that 50 C was optimum temperature of purified pectinase whereas, enzyme activity suppressed at temperature higher than 50 C. Similar results were reported by Esquivel and Voget (2004), Chellegatti, Fonseca, and Said (2002) for polygalacturonase. Exo polygalacturaonase from Aspergillus species and endo polygalacturonase from Mucour rouxxi exhibited maximum activity at 50 and 35 C respectively (Freitas, Martin, Silva, da Silva, & Gomes, 2006; Saad, Briand, & Gardarin, 2007).
4.
Conclusions
In conclusion, the presently investigated indigenous strain A. niger showed incredible potential for pectinase synthesis. After optimization of conditions, the purification fold, specific activity and % recovery were respectively high when compared to the initial yields. Therefore, the results direct the scope of A. niger for maximum pectinase enzyme production in the presence of cheaper substrate makes this
Aguilar, G., & Huitron, C. (1993). Conidial and mycelial-bound exopectinase of Aspergillus sp. FEMS Microbiology Letters, 108, 127e134. Ahmed, I., Zia, M. A., Iftikhar, T., & Iqbal, H. M. H. (2011). Characterization and detergent compatibility of purified protease produced from Aspergillus niger by utilizing agro wastes. BioResources, 6(4), 4505e4522. Alkorta, I., Garbisu, C., Llama, M. J., & Serra, J. L. (1998). Industrial applications of pectic enzymes: a review. Process Biochemistry, 33(1), 21e28. Buyukkileci, A. O., Tari, C., & Fernandez-Lahore, M. (2011). Enhanced production of exo-polygalacturonase from agrobased products by Aspergillus sojae. BioResources, 6(3), 3452e3468. Chellegatti, M. A. S. C., Fonseca, M. J. V., & Said, S. (2002). Purification and partial characterization of exopolygalacturonase I from penicillium frequentans. Microbiological Research, 157, 19e24. Draginia, M. P., Madarev, S. Z., Radulovic, L. M., & Skrinjar, M. (2007). Production of exo-pectinase by penicillium roqueorti using pumpkin oil cake. Proceedings of the National Academy of Sciences, 13, 313e320. Esquivel, J. C. C., & Voget, C. E. (2004). Purification and partial characterization of an acidic polygalacturonase from Aspergillus kawachii. Journal of Biotechnology, 110, 21e28. Freitas, P. M., Martin, N., Silva, D., da Silva, R., & Gomes, E. (2006). Production and partial characterization of polygalacturonases produced by thermophilic Monascus sp. N8 and by thermotolerant Aspergillus sp. N12 on solid state fermentation. Brazilian Journal of Microbiology, 37, 302e306. Giese, E. C., Dekker, R. F. H., & Barbosa, A. M. (2008). Oranges Bagasse as substrate for the production of pectinase and laccase by Botryosphaeria Rhodina MAMB 05 in submerged and solid state fermentation. BioResources, 3(2), 335e345. Joshi, V. K., Parmar, M., & Rana, N. S. (2006). Pectin esterase production from apple pomace in solid-state and submerged fermentations. Food Technology and Biotechnology, 44(2), 253e256. Kratchanova, M., Pavlova, E., & Panchev, I. (2004). The effect of microwave heating of fresh orange peels on the fruit tissue and quality of extracted pectin. Carbohydrate Polymers, 56, 181e185. Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature, 227, 680e685.
Please cite this article in press as: Ahmed, I., et al., Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger; its purification and characterization, Journal of Radiation Research and Applied Sciences (2015), http://dx.doi.org/10.1016/ j.jrras.2015.11.003
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Liu, L. Z., Ahmad, H., Lue, Y., Gardiner, D. T., Gunasekera, R. S., & Mckeehan, W. L. (2001). Citrus pectin; characterization and inhibitory effect on fibroblast growth factor receptor. International Journal of Agricultural and Food Chemistry, 49, 3051e3057. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with folin phenol reagent. Journal of Biological Chemistry, 193, 265e275. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426e428. Mrudula, S., & Anitharai, R. (2011). Pectinase production in solid state fermentation by Aspergillus niger using orange peel as substrate. Global Journal of Biotechnology and Biochemistry, 6, 64e71. Naidu, G. S. N., & Panda, T. (1998). Application of response surface methodology to evaluate some aspects of pectolytic enzymes from Aspergillus niger. Biochemical Engineering Journal, 2, 71e77. Nighoskar, S., Phanse, Y., Sinha, D., Nighojkar, A., & Kumar, A. (2006). Production of polygalacturonase by immobilized cells of Aspergillus niger using orange peel as inducer. Process Biochemistry, 41, 1136e1140. Oyede, M. A. (1998). Studies on cell wall degrading enzymes associated with degradation of cassava (Manihot esculenta) tubers by some phytopathogenic fungi. PhD Thesis (p. 205). Nigeria: OAU. Pedrolli, D. B., Gomes, E., Monti, R., & Carmona, E. C. (2008). Studies on productivity and characterization of polygalacturonase from Aspergillus giganteus submerged culture using citrus pectin and orange waste. Applied Biochemistry and Biotechnology, 144, 191e200.
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Rosales, E., Couto, S. R., & Sanroman, A. (2002). New uses of food waste: application to laccase production by Trametes hirsute. Biotechnology Letters, 24, 701e704. Saad, N., Briand, M., & Gardarin, C. (2007). Production, purification and characterization of an endopolygalacturonase from mucor rouxii NRRL 1894. Enzyme and Microbial Technology, 42, 800e805. Semenova, M. V., Grishutin, S. G., Gusakov, A. V., Okunev, O. N., & Sinitsyn, A. P. (2003). Isolation and properties of pectinases from the fungus Aspergillus japonicas. Biochemistry, 68, 559e569. Shembekar, V. S., & Dhotre, A. (2009). Studies of pectin degrading microorganisms from soil. Journal of Microbial World, 11(2), 216e222. Silva, D., Martins, E. S., Silva, R., & Gomes, E. (2002). Pectinase production from penicillium viridicatum RFC3 by solid state fermentation using agricultural residues and agro-industrial by products. Brazilian Journal of Microbiology, 33, 318e324. Spagna, G., Pifferi, P. G., & Gillioli, E. (1995). Immobilization of a pectin lyase from A. niger application in food technology. Enzyme and Microbial Technology, 17, 729e738. Susana, R. C., & Sanroman, M. A. (2006). Application of solid-state fermentation to food industry. A review. Journal of Food Engineering, 76(3), 291e302. Wang, Y. C., Chuang, Y. C., & Hsu, H. W. (2008). The Flavonoid, carotenoid and pectin content in peels of citrus cultivated in Taiwan. Food Chemistry, 106, 277e284. Zhang, C. H., Li, Z. M., Peng, X. W., Jia, Y., Zhang, H. X., & Bai, Z. H. (2009). Separation, purification and characterization of endopolygalacturonase, from a newly isolated Penicillium axalicum. Chinese Journal of Process Engineering, 9, 242e249.
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Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger; its purification and characterization Q4
Ishtiaq Ahmed a,*, Muhammad Anjum Zia a, Muhammad Azhar Hussain a, Zain Akram b, Muhammad Tahir Naveed c, Azin Nowrouzi d a
Department of Biochemistry, University of Agriculture Faisalabad, Pakistan Department of Chemistry, University of Sargodha, Pakistan c Center of Agriculture Biochemistry and Biotechnology (CABB), University of Agriculture Faisalabad, Pakistan d Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, Iran b
article info
abstract
Article history:
Agro-industrial residues are primarily composed of complex polysaccharides that
Received 30 August 2015
strengthen microbial growth for the production of industrially important enzymes. Pecti-
Received in revised form
nases are one of the most widely disseminated enzymes in bacteria, fungi and plants.
29 October 2015
Czapeck media supplemented with orange waste peel as carbon source under submerged
Accepted 4 November 2015
fermentation process Aspergillus niger presenting the preeminent enzymatic production.
Available online xxx
On partial optimization culture showed the maximum enzyme yield (117.1 ± 3.4 mM/mL/ min) at 30 C in an orange waste peel medium having pH 5.5 and substrate concentration
Keywords:
(4%) after 5th day of fermentation. The produced enzyme was purified by ammonium
Aspergillus niger
sulfate precipitation and ion exchange chromatography. A purification fold of 5.59 with
Orange waste peel
specific activity and % recovery of 97.2 U/mg and 12.96% was achieved respectively after gel
Pectinases
filtration chromatographic technique. The molecular weight of purified pectinase from A.
Purification
niger was 30 kDa evidenced by SDS-PAGE. Pectinase activity profile showed purified enzyme
SDS-PAGE
was optimally active at pH ¼ 7 and 55 C. The maximum production of pectinase in the presence of cheaper substrate at low concentration makes the enzyme useful in industrial sectors especially for textile and juice industry. Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1.
Introduction
Pectin comprises of D-galacturonic acid occurred in a (1e4)chain, naturally esterifies with methoxy groups and natural
sugars occupy the side chains. Occasionally, rhamnose group present in chain unsettles the chain helix formation (Shembekar & Dhotre, 2009). In the last two decades of twentieth century, an industrial revolution happened in
* Corresponding author. E-mail address: [email protected] (I. Ahmed). Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications. http://dx.doi.org/10.1016/j.jrras.2015.11.003 1687-8507/Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Ahmed, I., et al., Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger; its purification and characterization, Journal of Radiation Research and Applied Sciences (2015), http://dx.doi.org/10.1016/ j.jrras.2015.11.003
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developing countries in citrus producing and processing area (Juices, Oils and other by-products). Pakistan being an agricultural country produces millions of tons of fruits and vegetables annually. Mango, citrus, apple, banana and dates are the highest yielding fruits in Pakistan. The cell wall and middle lamella are enriched of pectin ingredients (Alkorta, Garbisu, Llama, & Serra, 1998). Carbohydrates and proteins are the major but the fat contents are in low amount in dried citrus peel. Significant quantity of pectin in citrus peel urges us to use them as a substrate for microorganism to produce pectinolytic enzymes. Microbial systems enhance pectinolytic production using pectin as inducer (Aguilar & Huitron, 1993). During the raw agriculture material processing for food, a bulk quantity of agro wastes generated. Thus, agro residues of coffee, orange, rice and sugarcane provide a suitable platform for bio-production of different enzymes using the fermentation process, thereby making valuable of these waste products (Giese, Dekker, & Barbosa, 2008). Orange bagasse contains large amounts of soluble carbohydrates, particularly fructose, glucose, sucrose, and pectin, as well as insoluble cellulose, and has been used as a fermentation products including enzymes (Rosales, Couto, & Sanroman, 2002). Microorganisms have noteworthy collaboration in food (dairy and meat) and alcoholic beverages industries (Buyukkileci, Tari, & Fernandez-Lahore, 2011). The usage of bio-produced food and additives makes them more appropriate rather than the synthetically produced ones (Susana and Sanroman, 2006). Citrus peel is known as the major source of pectinases in several industries instead of apple pomace. The potential applications of pectinases in different industries craving us to conduct the present study.
Q1
2.
Materials and methods
2.1.
Chemicals, microorganism and substrate
The pure culture of Aspergillus niger was available in our lab as previously reported by Ahmed, Zia, Iftikhar, and Iqbal (2011). Fungal spore suspension was prepared by using the method of Dhillon et al. (2004). In brief; Spores of A. niger were cultured in 250 mL flask holding 30 mL of Potato Dextrose broth at 30 ± 1 C for 4 days. To achieve the maximum concentration of spores in the solution, we used saline solution (0.9% w/v) on the fungal spores and shaken flask to accelerate the release of spores into the surrounding saline solution. Orange peel waste was obtained from local fruit market Faisalabad, Pakistan. The substrate was dried (60 C), ground and kept in air tight plastic jars to lessen the moisture contents.
2.2.
Fermentation methodology
Substrate screening was done by taking 4 g orange peel waste into 250 mL flasks of three replicates and inoculated with spore suspension of A. niger. All the experimental flasks were incubated at 30 ± 1 C in a shaking incubator at 180 rpm for 5 days of fermentation period. The ingredients of the flasks were filtered and clear supernatant having enzyme solution was subjected to further analytical studies.
2.3.
Chemical analysis of the substrate
2.3.1.
Determination of % moisture content
The moisture contents of sour orange peel was determined by taking 2 g sample in a sterilized bottle (W1) and dried at 100 C for 2 h. The sample was cooled and weighed in desiccators, after that sample again dried until constant weight (W2). The moisture percentage was calculated by using the following formula: ð%Þ Moisture ¼
2.3.2.
ðW1Þ ðW2Þ 100 ðW1Þ
(E1)
Determination of % ash content
Pre-weight crucible containing 5 mL HNO3 and 2 g of substrate (W1) was used to heat the material until char forming. After that sample put in muffle furnace for 6 h at 555 C and weighed (W2) to determine the percentage ash contents of sample. ð%Þ Ash ¼
2.3.3.
ðW1Þ ðW2Þ 100 Weight of sample
(E2)
Determination of crude % fat content
5 g sample (W1) was added in a pre-weighed thimble and keep in an extraction apparatus (Soxhelt) for 16 h and extraction was done with hexane. Subsequently, thimble with dried sample weighed to calculate the fat contents (W2). The crude %fat content of sample was calculated by using the following formula: ð%Þ Crude Fat ¼
2.3.4.
ðW1Þ ðW2Þ 100 ðW1Þ
(E3)
Determination of % crude fiber content
For the determination of crude fiber contents, 2 g of dry material (W1) after extraction with ether was boiled with 200 mL of H2SO4 containing 1.25 g of H2SO4 per 100 mL for 30 min with 200 mL of NaOH solution containing 1.25 g carbonate free NaOH per 100 mL and filtered. The material was dried in an oven and weighed (W2). The decrease in weight was considered as crude fiber.
ð%Þ Crude Fiber ¼
2.3.5.
ðW1Þ ðW2Þ 100 Weight of sample
(E4)
Determination of % lignin content
In brief, 2 g (W1) sample was added in 10 mL of 70% (w/w H2SO4) solution and boiled for 4e5 h. The cellulose and hemicellulose contents were hydrolyzed after the above treatment and remaining suspension was filtered with hot water. Afterward, addition of 30 mL of 70% H2SO4 into the mixture was carried out and kept in oven for 24 h at 105 C and weighed (W2). Then treated sample was moved into preweighed dry porcelain crucible for high temperature treatment (650 C) for 4 h. The sample was allowed to cool down and lignin content (%) was calculated by using the following formula. ð%Þ Lignin ¼ ðW1Þ ðW2Þ
(E5)
Please cite this article in press as: Ahmed, I., et al., Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger; its purification and characterization, Journal of Radiation Research and Applied Sciences (2015), http://dx.doi.org/10.1016/ j.jrras.2015.11.003
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2.4.
Analytical methods
2.4.1.
Total sugar estimation
In order to measure total sugar in the medium standard titration method was used. Briefly, a mixture of 100 mL water and 25 g of sample and 2 mL of Lead acetate solution was added and stayed for 10e15 min. The following formula was intended to measure %total sugar in the sample. Total Sugar ð%Þ ¼
2.4.2.
Factor 100 Dilution Titer 1000
Reducing sugar estimation
Protein determination
For determination of protein, Lowry, Rosebrough, Farr, and Randall (1951) method was carried out using bovine serum albumen as standard.
2.4.4.
Determination of pectinase activity
Enzyme activity was measured by dinitrosalicylic acid reagent (DNS) method (Miller, 1959). In brief, a mixture containing 2.0 mL sodium citrate buffer (pH ¼ 5.0), 1 mL enzyme extract and 0.2 mL of pectin solution (1%) was incubated for 25 min at 35 C ± 1 C. After incubation time, 1 mL of incubated solution was withdrawn and mixed with 0.5 mL of sodium carbonate (1 M) solution in a test tube. Addition of 3 mL DNS reagent in each test tube and shaken for 10 min dynamically to mix the contents completely. Samples were heated for 10 min and cooled down at room temperature before adding the 20 mL distilled water. The enzyme activity was measured spectrophotometrically by taking the absorbance at 570 nm.
2.5.
Optimization parameters
Different parameters were investigated for optimization including fermentation period (24e144 h), Substrate concentration (1e5%), Inoculum size (2e10 mL), pH (4e6.5) and Temperature (25e45 C) to achieve maximum yield of pectinase enzyme. All the parameters were run in triplicates.
2.6.
chromatography column (2 20 cm) was used to further purification, equilibrated with 0.01 mM TriseHCl buffer (pH 6.0). Both, protein contents and enzyme activity were measured as described in earlier part. For this, partially purified enzyme was subjected to chromatography column at flow rate of 0.5 mL/min to collect 20 fractions of 2 mL each. Molecular mass of purified Pectinase was investigated according to the method of Laemmli (1970) by performing Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE).
(E6)
3,5-dinitrosalicyclic acid (DNS) colorimetric method was followed to measure the reducing sugar of sample with glucose as standard (Miller, 1959). The mixture of 1 mL DNS and 1 mL of water was boiled for 5 min. Color intensity was measured spectrophotometrically at 550 nm.
2.4.3.
3
Enzyme purification
The methodology developed previously by Ahmed et al. (2011) was used for the purpose of purification. In brief, harvested crude enzyme was centrifuged at 10,000 g for 15 min at 4 C and subjected to precipitate by crystals of ammonium sulfate with 5% increasing each time (starting from 30% up to 90%). The resulting precipitate bearing high pectinase activity were suspended in 0.01 M TriseHCl buffer and dialyzed. Before subjected to ammonium sulfate precipitation enzyme activity was measured followed by after precipitation. The lyophilization was carried out after partial purification for further studies e.g. SDS-PAGE (molecular mass determination) and gel filtration chromatography, respectively. Gel filtration column
2.7.
Characterization of pectinase
The characterization study of purified pectinase after DEAEcellulose column chromatography was done to examine the effect of temperature (40e60 C) and pH (4e8).
3.
Results and discussion
3.1.
Chemical composition of substrate
Chemical composition of orange peel waste was determined through physiochemical analysis for pectinase production. The data in (Fig. 1) indicates the proximally analyzed orange peel waste contained moisture (40.7%), ash (7.39%), fat (1.85%), pectin (7.0%), lignin (6.4%), crude fiber (7.8%), total sugar (14.08%), reducing sugars (10.70%) and non-reducing sugar (3.70%). Almost similar results of ash and fat contents reported the Oluremi et al. (2007). The total amount of pectin present in the orange peel waste was 7% i.e. 70 mg/g according to our investigations. The present reported findings are in accordance to the earlier reported data from different research scientists Wang, Chuang, and Hsu (2008), Kratchanova, Pavlova, and Panchev (2004) and Liu et al. (2001). Q2
3.2.
Optimization of incubation time
To check the effect of different time periods on pectinase production from A. niger on physiochemical analyzed orange peel waste by submerged fermentation, experiments were conducted at different time periods from 24 to 144 h. Data analyzed showed that pectinase production was increased gradually during the fermentation period and reached to its maximum value (77.2 ± 1.42 mM/mL/min) after 5th day of initial incubation (Table 1). Further increase in fermentation period decreased the production of pectinase. This phenomenon was also observe by Mrudula and Anitharai (2011) during the pectinase and polygalacturonase production by Penbicillium sp. Production of pectinase increases with increase in fermentation period due to increase in activity of pectin methyl esterase (PME) and it is also dependent on the type of fermentation including concentration of nutrients in the medium, organisms, and physiological conditions (Joshi, Parmar, & Rana, 2006).
3.3.
Optimization of substrate concentrations
The obtained results of different substrate concentration (1e5%) on production of pectinase by A. niger are summarized in Table 2. The maximum enzyme activity (99.4 ± 1.1 mM/mL/
Please cite this article in press as: Ahmed, I., et al., Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger; its purification and characterization, Journal of Radiation Research and Applied Sciences (2015), http://dx.doi.org/10.1016/ j.jrras.2015.11.003
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J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s x x x ( 2 0 1 5 ) 1 e7
Fig. 1 e Chemical composition of orange peel waste.
min) was observed under optimum (4%) substrate concentration. After careful analyzing during the experiment we observed that under enhanced carbon source concentration, more inducers available to A. niger that in response enhance the production of pectinase. The amount of reducing sugars produced by pectinase in fermentation media was (2.92 ± 0.09 mg/mL). In earlier study, Giese et al. (2008) reported similar results while on contrary to Giese et al. (2008) and our present investigations, previously, Naidu and Panda (1998) reported that high concentration of carbon sources inhibits enzyme synthesis. During the trial it was observed that A. niger used reducing sugars present in the filtrate for its functional activity to produce potential enzymes. Moreover,
Table 1 e Activities of pectinase produced by Aspergillus niger. Fermentation periods
24 48 72 96 120 144
Filtrate characteristics pH 4.7 ± 4.6 ± 4.5 ± 4.8 ± 4.9 ± 4.2 ±
Reducing sugars (mg/mL)
0.20 0.24 0.10 0.14 0.41 0.29
3.10 2.90 2.82 3.41 3.36 3.28
± 0.22 ± 0.10 ± 0.53 ± 0.9 ± 2.00 ± 1.50
Enzyme activity (mm/mL/min) 11.4 21.7 41.6 55.4 77.2 52.4
± 1.34 ± 1.99 ± 1.41 ± 3.15 ± 1.42 ± 2.14
culture organism achieved maximum yield of pectinase by hydrolyzing polysaccharides. As a result the amount of reducing sugar tends to increase when A. niger used as an enzyme producing microorganism (Draginia, Madarev, Radulovic, & Skrinjar, 2007).
3.4.
Optimization of inoculum size
Maximum pectinase activity (103.2 ± 1.9 mm/mL/min) was observed with 8% inoculum size while the amount of reducing sugars produced and pH of the medium was 3.2 ± 0.11 mg/mL and 4.3 ± 0.3 respectively (Table 3). Optimum inoculum density is an important consideration for fermentation process since accumulation of spores can inhibit growth and development of the culture organism (Nighoskar, Phanse, Sinha, Nighojkar, & Kumar, 2006). Presently it was observed that the presence of large amount of reducing sugars has inhibitory effect on the pectinase production.
3.5.
Optimization of pH
The effect of initial medium pH for pectinase enzyme production from 5 to 8 was investigated and maximum activity (117.1 ± 3.4 mM/mL/min) was found (Table 4). The decline in enzyme activity was observed as the pH level increased from 5.5. The amount of reducing sugars in the medium released
Table 2 e Activities of pectinase produced by Aspergillus niger.
Table 3 e Activities of pectinase produced by Aspergillus niger.
Substrate conc. (%)
Inoculum size (%)
1 2 3 4 5
Filtrate characteristics pH 4.6 ± 4.4 ± 3.9 ± 4.5 ± 4.8 ±
0.20 0.15 0.29 0.14 0.23
Reducing sugars (mg/mL) 0.79 1.70 2.67 2.92 3.39
± 0.22 ± 0.05 ± 0.19 ± 0.09 ± 0.11
Enzyme activity (mm/mL/min) 66.2 ± 81.9 ± 89.5 ± 99.4 ± 76.4 ±
1.9 1.7 1.3 1.1 1.8
2 4 6 8 10
Filtrate characteristics pH 4.8 4.4 4.7 4.3 4.6
± 0.12 ± 0.20 ± 0.70 ± 0.30 ± 0.34
Reducing sugars (mg/mL) 2.36 ± 2.79 ± 2.92 ± 3.21 ± 2.80 ±
0.41 0.39 0.21 0.11 2.15
Enzyme activity (mm/mL/min) 62.9 ± 1.2 77.0 ± 1.4 94.5 ± 1.1 103.2 ± 1.9 75.6 ± 2.1
Please cite this article in press as: Ahmed, I., et al., Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger; its purification and characterization, Journal of Radiation Research and Applied Sciences (2015), http://dx.doi.org/10.1016/ j.jrras.2015.11.003
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Table 4 e Activities of pectinase produced by Aspergillus niger. pH
Filtrate characteristics pH
4 4.5 5 5.5 6 6.5
Reducing sugars (mg/mL)
4.5 ± 0.20 4.8 ± 0.31 4.9 ± 0.17 4.7 ± 0.21 4.6 ± 0.19 4.1 ± 0.11
3.15 ± 3.39 ± 3.31 ± 3.54 ± 3.28 ± 3.15 ±
Enzyme activity (mm/mL/min)
0.02 0.41 0.29 0.09 0.31 0.36
68.5 7666 91.9 117.1 86.2 71.9
± 2.2 ± 3.9 ± 2.3 ± 3.4 ± 2.9 ± 2.3
Table 5 e Activities of pectinase produced by Aspergillus niger. Temperature ( C)
Filtrate characteristics pH 5.2 ± 4.9 ± 4.7 ± 4.5 ± 3.8 ±
25 30 35 40 45
0.41 0.20 0.29 0.19 1.1
Reducing sugars (mg/mL) 3.18 3.36 3.19 2.92 2.54
Enzyme activity (mm/mL/min)
± 0.20 ± 0.39 ± 0.19 ± 0.22 ± 0.85
90.6 126.0 97.1 89.5 67.3
± 2.4 ± 2.8 ± 3.4 ± 2.5 ± 2.2
was (3.54 ± 0.9 mg/mL). According to Spagna, Pifferi, and Gillioli (1995), maximum pectinase activity was under optimum pH 5.0 while, according to Pedrolli, Gomes, Monti, and Carmona (2008) 4.5 pH value was an optimum.
3.6.
Q3
Optimization of temperature
Fig. 2 e Molecular mass determination of purified pectinase by SDS-PAGE [Lane-M, Molecular weights in kDa of standard marker; lane-1, Crude enzyme extract; lane-2 Purified pectinase]. mg. A sudden increase in enzyme purity was observed after ion exchange chromatography, such as enzyme purity and specific activity were jumped up to 5.59 fold and 97.2 U/mg, respectively. Electrophoresis studies revealed that purified pectinase enzyme produced from A. niger has 30 kDa molecular mass (Fig. 2). Various studies revealed different molecular mass of pectinase enzyme produced from different microbial species (Oyede, 1998). Different microbial sources exhibited extensive divergence in molecular mass of exo-PG i.e. Aspergillus japonicum (38.0 and 65.0 kDa) (Semenova, Grishutin, Gusakov, Okunev, & Sinitsyn, 2003), Aspergillus kawachii (60.0 kDa) (Esquivel & Voget, 2004). The difference has been associated with the substrate employed, type and nature of microorganism, host cell wall and analytical methods (Oyede, 1998).
To study the effect of temperature on the pectinase enzyme production, shake flask experiments were run at various temperature between 25 and 40 C. Maximum enzyme activity (126.0 ± 2.8 mm/mL/min) and reducing sugar (3.36 ± 3.9 mg/mL) was observed in batch culture fermented at 30 C (Table 5). Further increase in temperature suppressed the enzyme activity. The attained results are correlated to the data earlier reported by Pedrolli et al. (2008) and Nighoskar et al. (2006). Thermo-stable strains of Aspergillus aculeatus, Fusarium oxysporum species produced enhanced yield of pectinase even at higher temperature i.e. 60e65 C. The difference between temperature ranges may be due to the genetic difference of various species (Table 6).
3.8.
Characterization of pectinase
3.7.
3.8.1.
Effect of pH on activity & stability
Purification and SDS-PAGE
Purified pectinase after ammonium sulfate precipitation exhibited protein (120 mg/mL) and specific activity of 23.6 U/
The obtained results demonstrated that maximum activity of pectinase enzyme produced from A. niger was observed at pH 5 (Fig. 3). The optimum pH of our findings is comparable to the
Table 6 e Pectinase purification summary. Purification steps
Crude enzyme (NH4)2SO4 Precipitation Dialysis Sephadex-G-100
Volume (mL)
Enzyme activity (U)
Protein content (mg)
Specific activity (U/ mg)
Purification fold
% Yield
100 20
13,500 2840
775 120
17.4 23.6
1 1.36
100 15.11
47 18
46.9 97.2
2.87 5.59
16.33 12.96
15 10
2205 1750
Please cite this article in press as: Ahmed, I., et al., Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger; its purification and characterization, Journal of Radiation Research and Applied Sciences (2015), http://dx.doi.org/10.1016/ j.jrras.2015.11.003
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biosynthesized enzyme useful in textile as well as food processing industrial sectors.
Conflict of interest statement Authors are happy to declare that we do not have any conflict of interest in any capacity.
Acknowledgments Fig. 3 e Effect of pH on purified pectinase activity.
The present work was carried out under the supervision of Dr. Muhammad Anjum Zia at Enzyme Biotechnology Lab, Department of Biochemistry UAF Pakistan. On providing collaborative help for the present study, Dr. Azin Nowrouzi is thankfully acknowledged.
references
Fig. 4 e Effect of temperature on purified pectinase activity.
pectinase of Penicillium varidictum and Penicillium oxalicum (Silva, Martins, Silva, & Gomes, 2002; Zhang et al., 2009), respectively. Freitas et al. (2006) reported that optimum pH (5.5) for pectinase activity from thermo-tolerant Aspergillus sp.
3.8.2.
Effect of temperature on activity & stability
To find out the thermo stability of pectinase, enzyme was incubated at various temperatures ranging from 30 to 60 C. The results (Fig. 4) revealed that 50 C was optimum temperature of purified pectinase whereas, enzyme activity suppressed at temperature higher than 50 C. Similar results were reported by Esquivel and Voget (2004), Chellegatti, Fonseca, and Said (2002) for polygalacturonase. Exo polygalacturaonase from Aspergillus species and endo polygalacturonase from Mucour rouxxi exhibited maximum activity at 50 and 35 C respectively (Freitas, Martin, Silva, da Silva, & Gomes, 2006; Saad, Briand, & Gardarin, 2007).
4.
Conclusions
In conclusion, the presently investigated indigenous strain A. niger showed incredible potential for pectinase synthesis. After optimization of conditions, the purification fold, specific activity and % recovery were respectively high when compared to the initial yields. Therefore, the results direct the scope of A. niger for maximum pectinase enzyme production in the presence of cheaper substrate makes this
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Please cite this article in press as: Ahmed, I., et al., Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger; its purification and characterization, Journal of Radiation Research and Applied Sciences (2015), http://dx.doi.org/10.1016/ j.jrras.2015.11.003
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Please cite this article in press as: Ahmed, I., et al., Bioprocessing of citrus waste peel for induced pectinase production by Aspergillus niger; its purification and characterization, Journal of Radiation Research and Applied Sciences (2015), http://dx.doi.org/10.1016/ j.jrras.2015.11.003
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