Optimised Production of Xylanase by Aspergillus brasiliensis under Submerged Fermentation (SmF) and Its Purification Using a Two-step Column Chromatography

摘要

Aims: Carbon and nitrogen source play a significant role in microorganisms growth especially for the production of enzymes. Expensive medium composition creates huge setbacks especially in the production of commercial enzymes such as xylanase. Hence, in this study, wheat bran as the agricultural residue supplemented with yeast extract was used as the sole carbon and nitrogen source to culture Aspergillus brasiliensis ATCC 16404. This cheap alternative carbon source was used comprehensively to replace the expensive medium especially xylan in the production of xylanase. In fact, one of the main factors of using wheat bran is due to its cost effectiveness and availability in current market. Therefore, the main objectives of this study were to elucidate the production of xylanase from A. brasiliensis under submerged fermentation (SmF) using wheat bran as the sole carbon source followed by purification of the xylanase to its homogeneity. SmF was chosen primarily as the mode of fermentation for the production of xylanase since it was easily operated in terms of controlling its parameters including the supply of oxygen and nutrients in a large culture volume for the growth of fungi using the optimised medium composition proposed. Subsequently, the purification of xylanase was performed in an easy two-step column chromatography after the precipitation with ammonium sulphate and dialysis, respectively. Methodology: In this study, the production of xylanase by A. brasiliensis was conducted under SmF using wheat bran as the prime carbon source followed by a two-step column chromatography of diethylaminoethanol (DEAE) Sepharose and Sephadex G-75 for the purification of xylanase before subjected to zymography analysis to detect the biological activity of the purified xylanase based on its substrates hydrolysation. Results: From the results, high production of xylanase was exerted, producing 11.49 U/mL with protein concentration of 2.33 mg/mL at 72-hour of fermentation. The maximum spore count of 7.91 × 105 cells/mL with final pH of 6.9 was able to produce the optimum xylanase activity of 11.49 U/mL. Xylanase secreted from A. brasiliensis was precipitated using ammonium sulphate and purified via DEAE Sepharose and Sephadex G-75 column chromatography. The purified xylanase was illustrated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight of 36 kDa. Approximately a two and the half purification fold with a surprisingly high recovery of 38.15% was obtained. From our study, the purified extracellular xylanase was subjected to the zymography analysis using xylan supplemented potato dextrose agar (PDA) to analyze the biological application of xylanase in hydrolyzing its substrate of xylan. Since xylanase showed its activity only on xylan, therefore, the formation of visible halo ring in the zymography analysis was managed to indicate that this enzyme was an active xylanase after the two-step column chromatography. Conclusion: The purified xylanase that obtained in this study possessed much greater potential in industrial applications since it had successfully demonstrated a visible convincing halo ring as a result of its active functional activity against its substrate of xylan.