Extracellular pectinase production by Aspergillus niger HFM 8 through solid substrate fermentation using pomelo peels as a substrate
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Date
2014-08
Authors
Salikin, Nor Hawani
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Abstract
The accumulation of pomelo peels (Citrus grandis) as the agro-industrial residues after the fruit processing and manufacturing should be maximally exploited for the microbial bioconversion of the detrimental wastes into a value-added by product such as enzymes. Pomelo peels are authenticated as rich in pectin, acting as the main inducer for pectinase enzyme production by microorganism through solid substrate fermentation system (SSF). Isolate HFM 8 revealed as the best fungal pectinase producer after the implementation of double steps screening processes (primary and secondary screenings) with maximal activity of 112.79 ± 17.2 U/g substrate. Based on the phenotypic features and molecular identification, this novel isolate was denoted as Aspergillus niger HFM 8. The improvement of physicochemical cultural conditions was emphasized in this current research with an attempt to enhance the pectinase productivity by the microorganism. An augmentation of 48.82% on pectinase yield by Aspergillus niger HFM 8 in a shake flask system was recorded after being cultivated in an enhanced physicochemical condition constituted of 0.75 mm particle size of substrate, 60% (v/w) of initial moisture (sterilized distilled water, pH 5.0), inoculum size of 1 x 104 spores/ml, incubation in room temperature (30 ± 2oC), inclusion of 0.1% (w/w) urea as the external nitrogen source, no mixing effect (static condition during fermentation) and utilization of acetate buffer (pH 4.5, 0.1 M) as the extracting solvent during the
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enzyme recovery. A large scale pectinase production by Aspergillus niger HFM 8 was then conducted employing a tray system. Under the optimized physical conditions comprised of 1.0 cm substrate depth equalized to 60 g pomelo peels substrate (0.75 mm of particle size), 40% (v/w) of initial moisture (sterilized distilled water, pH 5.0), inoculum size of 1 x 106 spores/ml, incubation in room temperature (30 ± 2oC), inclusion of 0.1% (w/w) urea as the external nitrogen source, inclusion of mixing effect once for every 24 hours interval and utilization of acetate buffer (pH 4.5, 0.1 M) as the extracting solvent during the enzyme recovery, an amount of 23.00% increment on pectinase productivity was detected. Comparatively, the fungal pectinase production boosted up to 191.41% of increment from a shake flask system to a tray system indicating the potentiality of Aspergillus niger HFM 8 to be industrially employed particularly in pectinase production. Further purification of the crude pectinase was conducted by practising double steps chromatography constituted of anion exchange (DEAE Sephadex) followed by gel filtration (Sephadex G-100). SDS-PAGE procedure determined the molecular weight of the purified pectinase as 55.37 kDa. This enzyme was found optimal at 40oC and pH 4.0 whereby the catalytic reaction was stable between 35oC to 45oC and in condition of pH 3.5 to pH 4.0. The purified pectinase was highly specific on pectin as the substrate whilst no solvents or metal ions found enhancing the catalytic reaction. Inversely, FeCl3 and AgNO3 prohibit the pectinase reaction. Apparently, pomelo peels are markedly applicable for commercial pectinase production employing a solid substrate fermentation system whereby the produced enzyme is highly applicable to be incorporated into the fruit juice extraction and clarification process
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Aspergillus niger HFM 8