Green polygalacturonase production by Aspergillus awamori NRC-F18 under solid-state fermentation

Abstract

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.