Production of pectinase by locally-isolated fungus, aspergillus fumigatus R6 in solid state fermentation for use in kenaf bioretting

• Main Author: Wong, Li Yin
• Format: Thesis
• Language: English
• Published: 2016
• Online Access: http://psasir.upm.edu.my/id/eprint/69011/1/FBSB%202016%2016%20IR.pdf; http://psasir.upm.edu.my/id/eprint/69011/

Kenaf fibres were produced by removing the pectin, a cementing material that binds the fibres together through the retting process. The traditional method of water retting requires long times and caused water pollution, while dew retting is weather and geographical dependence and produced fibres with low tensile strength. Retting using microbial pectinase is a promising approach as it reduces the times and produces fibres with high tensile strength, furthermore, it is environmental-friendly. The main objective of this study is to determine the efficacy of pectinase produced from a locally isolated fungus strain in kenaf bioretting. Pectinolytic fungi were isolated from the local sources and screened for their pectinase activity qualitatively and quantitatively. The selected pectinolytic fungus with the highest pectinase activity was identified by amplification of ITS region. The cultural conditions for maximum pectinase production from the selected strain were optimised using response surface methodology in solid state conditions,followed by purification using ammonium sulphate precipitation and gel chromatographic methods in order to characterise the pectinase produced. The pectinase produced was applied in kenaf bioretting to evaluate the possibility and efficiency of using pectinase in kenaf retting. A potential pectinolytic strain had been successfully isolated from the kenaf retting tank and was identified as Aspergillus fumigatus R6. Two types of pectinase were produced by A. fumigatus R6, which were polygalacturonase and pectin lyase using rice bran as the substrate in solid state conditions. The optimised cultural conditions for maximum production of polygalacturonase by A.fumigatus R6 was at an initial moisture level of 49.6%, 33°C, and 129 h of incubation time.A. fumigatus R6 polygalacturonase was purified using 60 – 80% ammonium sulphate precipitation and gel filtration chromatographic methods with a purification fold of 2.54 and polygalacturonase yield of 59.64%. The purified PgPl showed two bands on SDSPAGE with a molecular weight of around 34 kDa and 95 kDa. The purified PgPl had an optimum temperature of 65°C. Two peaks were observed for pH optimal at pH 5 and pH 7, respectively. Pectinase produced by A. fumigatus R6 was stable at 40°C and covered a wide range of pH (pH 4 – 11). A 32 h treatment with A. fumigatus R6 crude pectinase solution produced the kenaf bast fibres with the highest tensile strength (459 ± 166 MPa). Scanning electron microscopy (SEM) micrograph showed that the fibres fineness decreased and the surface of the kenaf fibres became smoother with a longer exposure of treatment. Crude pectinase produced from A. fumigatus R6 consisted of polygalacturonase with some pectin lyase and xylanase activities and low cellulase activity. Kenaf bast fibres tensile properties can improve by further optimisation of the enzyme formulation. A ratio of 3: 1 (v/w) of pectinase solution to kenaf bast produced kenaf fibres with the highest tensile strength. The enzyme formulation that produced kenaf bast fibres with the highest tensile strength was at 2 U/mL polygalacturonase activity supplemented with 50 mM of ethylenediaminetetraacetic acid (EDTA). In conclusion, A. fumigatus R6 pectinase shows potential to be used in kenaf bast bioretting process to produce strong kenaf fibres.