African Journal of Botany

African Journal of Botany ISSN: 3519-3824 Vol. 8 (3), pp. 001-016, March, 2020. © International Scholars Journals

Full Length Research Paper

Purification and characterization of a xylanase from the endophytic fungus Alternaria alternata isolated from the Thai medicinal plant, Croton oblongifolius Roxb.

Nichawee Wipusaree¹, Prakitsin Sihanonth², Jittra Piapukiew³, Polkit Sangvanich⁴ and Aphichart Karnchanatat⁵*

¹Biotechnology Program, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand.

²Department of Microbiology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand.

³Department of Botany, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand.

⁴Department of Chemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand.

⁵Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand.

Accepted 16 November, 2019

Abstract

Xylanases are a class of enzymes that degrade b-1, 4-xylan, a linear polysaccharide found as hemicellulose in plant cell walls, in xylose. They are one of the most important enzyme groups used in industry and agriculture. Fifty-four endophytic fungi were isolated and examined for xylanase production. Xylanase activity was found in thirty of the isolates in primary screening by growing on solid xylan agar plates. After secondary screening for xylanase activity in xylan liquid culture, the isolate that yielded the highest xylanase-production (PTRa9) was selected for further evaluation. Optimal xylanase production was achieved after 4 days of culture with 2% (w/v) rice bran and 0.1% (w/v) ammonium sulfate as the carbon and nitrogen source respectively. This xylanase was enriched 60.8-fold to apparent homogeneity by sequential ammonium sulphate precipitation, Diethylaminoethyl (DEAE)-cellulose ion exchange and Superdex 75 gel filtration chromatography. The resultant 54.8 kDa protein had a specific activity of 161.1 U/mg protein, an optimal temperature of 45°C, with >90% activity from -20 to 45 ^oC, a broad pH range of 3.0 to 11.0 (optimal at pH 5.0), and was sensitive to most divalent cations but especially by Hg²⁺, Cu²⁺ and EDTA. From the kinetic analysis, it had a K_m of 0.421 mg/ml and a V_max of 0.826 U/mg protein.

Key words: Xylanase, endophytic fungi, Thai medicinal plant.