Production, Purification and Characterisation of a Purified Low Molecular Weight and Thermo-alkaline Tolerance Xylanase by Aspergillus brasiliensis

Abstract

Aims: Xylanase is an enzyme which has been used extensively in many different industrial processes. Xylanase also known as endo-1,4-β-xylanase is a glycosidase that catalyses the conversion of xylan to xylose through endohydrolysis of 1,4-β-D-xylosidic linkage which is commonly found in various agro-industrial wastes such as wheat bran. Thus, the objectives of the present study were to produce, purify and characterise cost-effective xylanase from Aspergillus brasiliensis ATCC 16404. Methodology: Wheat bran as the major sustainable low cost agro-industrial residual was utilised as the sole carbon source for the production of xylanase by A. brasiliensis in submerged fermentation (SmF). Subsequently, a two-step column chromatography was used to purify xylanase followed by step-wise manner of characterisation study on the purified xylanase. Results: Based on the results, the maximum xylanase production of 7.72 U/mL with spore count of 8.33 × 104 spores/mL at medium pH 6.42 was obtained at 48 h of SmF. Xylanase extracted from A. brasiliensis was then purified with DEAE Sepharose and Sephadex G-75 column chromatography. At the end of purification, xylanase was purified up to 3.6-fold with its recovery yield of 1.68% and specific activity of 116.64 U/mg. Additionally, the purified xylanase was detected to be a low molecular weight protein. Indeed, molecular weight of 36 kDa of the purified xylanase was visualized on SDS-PAGE. The purified xylanase was then subjected to the step-wise manner of characterisation study. Based on the results obtained, xylanase was found to be thermo-tolerance from 40°C to 60°C. In fact, the purified xylanase was detected to be most stable at 50°C whereby 98.33% of its activity was retained even after 3 h of incubation. Furthermore, xylanase from A. brasiliensis was also found to be most active at 50°C where its relative activity increased from 95.24% at 45°C to the maximum activity of 20.51 U/mL at 50°C. Besides that, the pH stability of xylanase was appreciable from pH range of pH 4 to 8. Notably, the purified xylanase showed the highest stability at pH 5 as 94.87% of its activity was retained after 3 h of incubation. Additionally, the activity of the purified xylanase remained relatively higher in pH buffer ranging from pH 3 to 9. Indeed, the purified xylanase reached its maximum activity of 22.18 U/mL at pH 5. In addition, xylanase activity was detected to be the highest, producing 24.46 U/mL when 1% beechwood xylan was used as the optimised substrate for the incubation period of 30 min at 50°C. Besides that, metal ions such as Cu2+, Mn2+ and Zn2+ were identified to enhance xylanase activity, whereas Al3+, Ca2+, K+ and Mg2+ performed otherwise. In particular, Cu2+ was identified as the strongest activator while Al3+ was found to be the toughest inhibitor of xylanase activity. Nonetheless, chelating agent of EDTA inhibited the xylanase activity marginally. Furthermore, the non-ionic detergent of Tween 80 was detected to be a weak enhancer whereas the ionic detergent of SDS was a strong inhibitor of xylanase activity. On the other hand, xylanase activity showed a better tolerance towards glycerol with 51.2% and acetone with 33.9% compared to ethanol with 17.9%. Conclusion: In a nutshell, some of the characteristics of the purified xylanase by A. brasiliensis in this study revealed its enormous potential as a thermo-alkaline tolerance enzyme in xylan degradation process that is applicable and useful in the manufacturing of animal feeds, fruit juice and paper pulping.