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Research Article Open access CC BY 4.0

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

Hooi Ling Ho, Li Sha Soh, Soon Hwa Ong

Microbiology Research Journal International · pp. 1–25 · Published 16 Oct 2015

10.9734/BMRJ/2016/20766

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.

Xylanase wheat bran Aspergillus brasiliensis submerged fermentation (SmF) production purification characterization

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