|Year : 2020 | Volume
| Issue : 2 | Page : 136-142
Green polygalacturonase production by Aspergillus awamori NRC-F18 under solid-state fermentation
Amira M Roshdy, Fatima H Motawe, Mohamed Fadel
Department of Microbial Chemistry, Genetic Engineering and Biotechnology Research Division, National Research Centre, Dokki, Giza, Egypt
|Date of Submission||17-Nov-2019|
|Date of Decision||22-Dec-2019|
|Date of Acceptance||28-Dec-2019|
|Date of Web Publication||30-Jun-2020|
Research Professor Head of Microbial Chemistry Department Genetic Engineering and Biotechnology Division National Research Center El-Behos Street, Dokki, Giza
Source of Support: None, Conflict of Interest: None
Background and objective Pectin-degrading enzymes applied in the food industries are preferred to be obtained from fungal origin because fungi are potent producers of pectic enzymes and the optimal pH of fungal enzymes is very close to the pH of many fruit juices, ranging from pH 3.0 to 5. Pectinolytic enzymes, have several industrial applications in different industries such as food industries to concentrate fruit juices. They increased the yield of fruit juice from the pulp and removal of haze from juices to get a clear product. Research to find microorganisms to produce high-quality polygalacturonase (PGase) and technical constraint include supply of cheap and pure raw materials needed to produce inexpensive enzymes. Utilization of agroindustrial residues offers potential benefits for solid-state fermentation, which is attractive for the implementation of sustainable bioprocesses.
Materials and methods Fungal strain screening for PGase production was studied in 250 ml Erlenmeyer flasks containing 5 g of tested substrate moistened to 50% (v/w, ml/g) with distilled water. One milliliter of spore suspension (106 spores) from each fungus was used as inoculum. The cultures were incubated at 30°C for 4 days. At the end of the incubation period, 100 ml of distilled water was added to each flask, blended by shaking at 150 rpm for 30 min, and harvested by filtration. The filtrates were saved as sources of crude enzyme. The selected fungal strain and substrate were incubated for 120 h at 30°C and culture was taken at 24 h intervals to detect the optimum incubation period. Sugar beet pulp was moistened to different moisture levels, that is, 1 : 1, 1 : 2, 1 : 3, and 1 : 4 (v/w) under the optimum incubation period to determine the more suitable moisture content for enzyme production. Sodium phosphate buffer at 0.1 M was used for adjusting the initial pH of fermentation medium to different values from 3.5 to 7.5 to study the effect of pH on enzyme secretion. The fungus was incubated under different temperatures, that s, 20, 25, 30, 35, and 40°C to study the temperature effect on enzyme production. Inorganic nitrogen sources (at level 0.92 mg N/g solid substrate) were applied in the fermentation medium to study their effect on enzyme yield.
Results and conclusion Aspergillus awamori NRC-F18 showed promising PGase’ production activity than other fungi screened. The study showed that the fungus gave promising results when the moisture content was adjusted to 70% (v/w), initial pH value 5.0, and incubation temperature at 35°C for 72 h. The enzyme activity was increased when urea was the sole nitrogen source at a level of 0.92 mg/g solid substrate supplemented to fermentation medium. Under the above conditions, 396.4 U/g original substrate was obtained. A study on the obtained PGase revealed that it has an optimum pH that ranged from 4.5 to 5.5, as well as it gave the highest activity when incubated at 50°C. Eighty percent ammonium sulfate (w/v) was applied to precipitate enzyme protein, as 24% of total protein involved 61% of total PGase activity obtained with a specific activity of 36.94 U/mg protein against 12.8 U/mg protein in the crude culture supernatant. After fermentation the mixture from unutilized raw material and fungal biomass after enzyme elution was 76% (w/w) from the original substrate dry weight involving 14.2% protein related to 9.2% before beet pulp fermentation which could be utilized as feedstuff component in rations for ruminant feed.
Keywords: Aspergillus awamori NRC-F18, polygalacturonase production, solid-state fermentation, sugar beet pulp
|How to cite this article:|
Roshdy AM, Motawe FH, Fadel M. Green polygalacturonase production by Aspergillus awamori NRC-F18 under solid-state fermentation. Egypt Pharmaceut J 2020;19:136-42
|How to cite this URL:|
Roshdy AM, Motawe FH, Fadel M. Green polygalacturonase production by Aspergillus awamori NRC-F18 under solid-state fermentation. Egypt Pharmaceut J [serial online] 2020 [cited 2020 Aug 12];19:136-42. Available from: http://www.epj.eg.net/text.asp?2020/19/2/136/288657
| Introduction|| |
Pectin-degrading enzymes applied in food industries are preferred to be obtained from fungal origin because fungi are potent producers of pectic enzymes and the optimal pH of fungal enzymes is very close to the pH of many fruit juices, which ranged from pH 3.0 to 5.5 . Pectinolytic enzymes have several industrial applications in different industries such as fruit juice concentration and food industries . They increased the yield of fruit juice from the pulp and removal of haze from juices to get a clear product  Research to find microorganisms to produce high-quality polygalacturonase (PGase) as well as technical constraint include supply of cheap and pure raw materials needed to produce inexpensive enzymes. Utilization of agroindustrial residues offers potential benefits for solid-state fermentation (SSF), which is attractive for the implementation of sustainable bioprocesses. Further advantages of SSF processes are lower energy requirement and higher product yields as well as less wastewater production and lesser risk of bacterial contamination. Many factors have a critical influence on the process development in SSF, such as selection of microorganism, substrate and optimum physicochemical and biological process parameters . Filamentous fungi were found to be good producers for pectinolytic enzymes. They are capable of synthesizing and secreting large quantities of certain proteins into the extracellular medium. Agroindustrial wastes are used as a low-cost carbon source for the cultivation of Aspergillus species for microbial enzyme production in SSF processes . Several agroindustrial wastes and by-products such as orange bagasse , sugar-cane bagasse , wheat bran , and other food processing waste  are effective substrates for depolymerizing enzyme production by SSF. Green production of enzymes or other microbial metabolites means that no wastes that cause pollution are released to the environment at the end of the production process.
| Materials and methods|| |
Six fungal strains namely Aspergillus oryzae FK-923, Aspergillus niger F-258, Aspergillus awamori NRC-F18, A. fumigatus F-993, Tricoderma viride F-107, and T. viride F-321 were obtained from the Microbial Chemistry Department, National Research Center, Dokki, Giza, Egypt, and were maintained on potato dextrose agar slants at 30°C for 72 h.
Inoculum was made from 3-day-old potato dextrose agar cultures. The inoculum (containing 106–107 spores/ml) was suspended in 0.05 M phosphate buffer. The suspension, when necessary, was diluted with sterile 0.05 M sodium phosphate buffer (PBS) (pH 5.0) to give a spore count within the predetermined range. A volume of 1 ml of inoculum was added to each 5 g of sugar beet pulp (SBP) in 250 ml capacity conical flasks. The remaining liquid needed to obtain the desired moisture content in the SBP was tap water (pH 5), which was mixed with the substrate before sterilization. This procedure was repeated for each container as requested.
SSF and enzyme production by different fungi cultivated on various substrates: Fungal strain screening for PGase production was studied in 250 ml Erlenmeyer flasks containing 5 g of tested substrate moistened to 50% (v/w, ml/g) with distilled water. A volume of 1 ml of spore suspension (106 spores) from each fungal was used as an inoculum. The cultures were incubated at 30°C for 4 days. At the end of the incubation period, 100 ml of distilled water was added to each flask, blended by shaking at 150 rpm for 30 min, and harvested by filtration. The filtrates were saved as sources of crude enzyme. The selected fungal strain and substrate were incubated for 120 h at 30°C and culture was taken at 24 h intervals to detect the optimum incubation period. SBP was moistened to different moisture levels, that is, 1 : 1, 1 : 2, 1 : 3, and 1 : 4 (v/w) under the optimum incubation period to determine the more suitable moisture content for enzyme production. Sodium phosphate buffer at 0.1 M was used for adjusting the initial pH of fermentation medium to different values from 3.5 to 7.5 to study the effect of pH on enzyme secretion. The fungus was incubated under different temperatures, that is, 20, 25, 30, 35, and 40°C to study the temperature effect on enzyme production. Inorganic nitrogen sources (urea, ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium oxalate, diammonium hydrogen citrate, sodium nitrate, and potassium nitrate) at level 0.92 mg N/g solid substrate were applied in the fermentation medium to study their effect on enzyme yield.
The PGase activity was assayed by estimating the amount of reducing sugar released under assay conditions. Activity was measured by determining the amount of reducing groups released according to the DNS method described by Miller . The substrate used for assay was 1% polygalacturonic acid ‘PGA,’ in 0.1 M citrate buffer pH 5.0. The assay mixture was prepared with the following components: 0.1 ml enzyme and 0.9 ml of 1% PGA solution. Blank was prepared for each sample by boiling the reaction mixture before addition of substrate. Tubes were incubated at 50°C for 30 min. The amount of reducing sugars released per ml per minute was calculated from the standard curve of galacturonic acid. One unit of enzyme activity is defined as the enzyme that releases 1 µmol/ml/mingalacturonic acid ‘PGA’ under standard assay conditions.
Enzyme protein was determined by the method described by Lowry et al.  and crude protein in the residual fermented substrate was according to micro-Kjeldahl method of the AOAC .
Partial purification of enzyme
The crude enzyme filtrate was recovered (eluted) from the fungal SSF culture and centrifuged at 6000 rpm for 20 min under refrigerated condition. Solid ammonium sulfate salt was slowly added to the cold supernatant of crude enzyme preparation with continuous shaking on a rotary shaker (200 rpm) until it reached 60% w/v saturation. The shaking was continued until complete ammonium sulfate solubilization and then kept (left) overnight at 4°C in a refrigerator. The precipitated protein was separated by centrifugation at 6000 rpm for 30 min at 4°C. The precipitated protein was dissolved in sodium acetate buffer (0.1 M pH 5) and subjected to further studies.
Characterization of PGase: Temperature stability of the enzyme was estimated by incubating the enzyme for 4 h at temperatures from 35 to 80°C in assay buffer and then by measuring the remaining activity by standard assay to determine the inactivation temperature. The pH stability of the enzyme was evaluated by varying the pH of the reaction mixture to between 3.0 and 8.0 at an increment of 0.5. Activity was then assessed under standard conditions.
| Results|| |
Screening and selection of fungal strain and suitable substrate for producing polygalacturonase
Selection of microorganism and suitable substrate are required to obtain inexpensive enzymes with high activity. [Table 1] presents the data gained from the cultivation of five nontoxic fungal strains tested for producing PGase. A. awamori NRC-F18 produced the more promising enzyme activity at 258.6 U/g from original substrate SBP, so it was chosen for further studies to optimize the culture conditions and characterization of PGase enzyme.
|Table 1 Screening of fungal strains for polygalacturonase production on different substrates at a moisture ratio of 1 : 1 v/w under solid-state fermentation technique|
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Controlling temperature is an important factor for enzyme formation when SSF is applied. Maximum enzyme production by A. awamori NRC-F18 (232.4–264.8 U/g) when fungus was grown at temperature ranges of between 25 and 35°C [Table 2] and is affected negatively when incubated at 40°C.
|Table 2 Effect of temperature on exopolygalacturonase production from Aspergillus awamori NRC-F18 from sugar beet pulp under solid-state fermentation|
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[Table 3] shows the data obtained when the initial pH of fungus cultivation was adjusted between pH 3.5 and 7.5. The fungus produced promising enzyme production (286.4 U/g) and released protein (20.2 mg/g initial substrate and the specific activity reached 13.96 U/mg protein) when initial pH of fermentation was at pH 5.5. Enzyme synthesis was affected negatively after pH 6.0.
|Table 3 Production of polygalacturonase by Aspergillus awamori NRC-F18cultivated on sugar beet pulp under solid-state fermentation at different initial pH values|
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Moisture content is a limiting factor for the success of SSF technique and consequently enzyme production. The promising PGase activity was 368.2 U/g and specific activity was 16.78 U/mg protein/g substrate (23.2 mg protein excreted/g substrate was attained at an initial moisture content of 70%, v/w, adjusted before fermentation) ([Table 4]).
|Table 4 Polygalacturonase production by Aspergillus awamori NRC-F18 cultivated on sugar beet pulp adjusted to different moisture levels by solid-state fermentation technique|
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[Table 5] shows that the maximum PGases activity (286.2 U/g) was achieved after 96 h, and the protein released was 23.5 mg/g substrate with a specific activity of 16.5 U/mg protein.
|Table 5 Production of polygalacturonase by Aspergillus awamori NRC-F18 cultivated on sugar beet pulp under solid-state fermentation technique at different incubation periods|
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[Table 6] shows that the supplementation of nitrogen source in the fermentation medium resulted in enzyme enhancement compared with control, and enzyme activity (396.2 U/g) was seen by supplementation urea as sole nitrogen in fermentation medium followed by ammonium sulfate, diammonium phosphate, and ammonium nitrate (386.2, 362.2, and 342.8 U/g, respectively).
|Table 6 Polygalacturonase production from Aspergillus Awamori NRC-F18 cultivated on sugar beet pulp with different nitrogen sources, under solid-state fermentation technique|
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Partial purification of enzyme
Sixty percent ammonium sulfate (w/v) was applied to precipitate enzyme protein, as 24% of total protein involved 61% of total PGase activity, with a specific activity of 36.94 U/mg protein against 12.8 U/mg protein in the crude culture supernatant.
Characterization of enzyme
The enzyme was found with complete original activity when incubated at 50°C for 4 h, and have nearly 92 and 84% of its activity when incubated at 55 and 60°C, respectively, after the period ([Figure 1]). On the other hand, the present study stated that enzyme was stable between pH 4.5 and 5.5 and at higher pH (over pH 6.5) it markedly decreased ([Figure 2]).
|Figure 1 Activity of polygalacturonase produced by Aspergillus awamori NRC-F18 cultivated on sugar beet pulp, under solid-state fermentation method (70% w/v, moisture) at different degrees of temperature, after 96 h incubation period at 35°C and initial pH 5.5.|
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|Figure 2 Activity of polygalacturonase produced by Aspergillus awamori NRC-F18 cultivated on sugar beet pulp, under solid-state fermentation method (70%, w/v, moisture content) at different pH values, after 96 h incubation period at 35°C.|
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Feed evaluation of fermented substrate after enzyme elution
The data shows ([Table 7]) upgrading in feed quality for SBP after the fermentation process for the production of lignocellulose-degrading enzymes. The improvement is seen in increasing of crude protein from 9.4 to 13.7%, increasing in TDN from 67.0 to 69.0%, and performance in gross energy from 3995 to 4384 kcal/kg. On the other hand, there was a decrease in crude fiber, acid detergent fiber (ADF), and neutral detergent fiber (NDF).
|Table 7 Chemical composition of sugar beet pulp after and before fermentation|
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| Discussion|| |
It has been reported that a large number of fungal strains and many substrates were reported for PGase production. Previous researches have reported that fungi such as Penicillium spp. and Aspergillus were utilized as explants for PGase production of microorganisms . High PGase activity was produced by a thermophilic Aspergillus fumigatus isolated from decomposing orange peel . In addition, high PGase yields were produced when agricultural and agro-industrial residues were utilized as a carbon source from solid-state culture of Aspergillus sojae , gamma-irradiated Penicillium citrinum , Mucor circinelloides ITCC 6025 , Enterobacte raerogenes NBO2 , and A. niger CSTRF . On the other hand, numerous low-cost substrates were utilized for PGase production such as fruit-processing wastes  (agricultural residues and agro-industrial by-product , orange wastes , and grape pomace . Controlling temperature is an important factor for enzyme formation when SSF is applied . Incubation temperature of microorganism has an effect on protein released in the cultivation medium as well as specific activity . A. fumigatus can be produced maximally when cultivated on temperatures adjusted to 50°C . Freitas et al.  reported that a temperature of 45°C was the best for PGases production by Monascus spp. N8 and Aspergillus spp. N12. Thakur et al.  studied the same for M. circinelloides and the optimum temperature was found to be 35°C for A. niger CSTRF.The pH value of the culture medium was reported as affecting the permeability of cells as well as stability of enzyme. Different fungi secrete PGase in acidic media from pH 4.0 to 5.0 ,,,,,,,,,,,. A. niger CSTRF was reported to give high PGase activity at pH 4.5 . The moisture causes swelling of the substrate and facilitates utilization of the nutrients by the organism. Previous studies have reported that if the substrate is too moistened, the substrate porosity decreases, which reduces oxygen diffusion and accessibility for microorganisms, whereas, at low moisture levels, growth of the microorganism affected negatively as the nutrient is difficult to release owing to fungus mycelium penetration through the substrate . Moisture contents limit the availability of oxygen for fungus as well as feasibility to utilized nutrients from the substrate. A promising enzyme is produced at a certain point of moisture and then leveled off. A. sojae ATCC 20235 produced the best enzyme activity when applying SBP as an inducer substrate when fermented with wheat bran as the medium moistened to 80%, v/w with 0.2 M HCl. Previous studies on this aspect revealed various results for different strains. Maximum PGases activity was reported on the fifth day for Penicillium spp.  and on the fourth day for Alternaria alternata . Generally, enzyme activity increases with an increase in incubation time and reached its maximum after 96 h, which agrees with the present study. Highest enzyme yield was achieved by A. sojae ATCC 20235 after 8 days. The present finding agreed with that previously reported by Gokhale et al. , which found that urea prevents the drop in pH in unbuffered fermentation medium. On the other hand, Kuhad et al.  gained optimal PGase production by Streptomyces sp. when urea was added to the basal medium devoid of yeast extract and peptone. Thakur et al.  reported that the enzyme was stable up to the 4th h at 42°C. de Andrade et al.  found that the enzyme gave about 82 and 63% of its original optimum activity when incubated at 60 and 70°C, respectively, for 2 h. Previous works agree and support the present finding as it has been reported that optimum activity for PGase produced by Cylindrocarpon destructans and Thermoascus aurantiacus was pH 5.0 ,,, while the optimum enzyme activity was at pH 5.5 for that produced by M. circinelloides , and enzyme produced by A. niger CSTRF gave its optimum activity at pH 4.5 . Biologically treated SBP by fungi was evaluated as feed mixture rations for growing sheep, goats, and large animals such as lactating cows, lambs, dairy, as buffalo ration and beef cattle ,,,. Biologically treated SBP led to upgrading crude protein to about 19% . They found that feed conversion and cost of feed/kg gain were the best for ration contained 25% biologically treated SBP. The present study results ([Table 7]) indicate that the introduction of biologically treated SBP at a rate of 25% of the concentrate feed mixture could improve feed conversion of growing lambs and was economical .
| Conclusion|| |
SBP was used for PGase production through fermentation by A. awamori NRC-F18 under the SSF system. The study showed that fungus gave promising results when the moisture content was adjusted to 70% (v/w), initial pH value was 5.5, at an incubation temperature of 35°C for 96 h. The enzyme activity was increased when urea was the sole nitrogen source at a level of 0.92 mg/g solid substrate supplemented to the fermentation medium. Under the above conditions, 396.4 U/g original substrate was obtained. Study on the obtained PGase revealed that it has an optimum pH that ranged from 4.5 to 5.5, as well as it gave the highest activity when incubated at 55°C. Sixty percent ammonium sulfate (w/v) was applied to precipitate enzyme protein, as 24% of total protein involved 61% of total PGase activity was obtained at a specific activity of 36.94 U/mg protein against 12.8 U/mg protein in the crude culture supernatant. The mixture from unutilized raw material and fungal biomass after the enzyme elution was 76% (w/w) from the original substrate dry weight involved 14.2% protein related to 9.2% before beet pulp fermentation could be utilized as feedstuff component in rations for ruminant feed.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]