Partial purification and characterisation of Phialophora alba xylanases and its application to pretreated sugarcane bagasse.

Abstract

Xylan is the major component of hemicellulose and its degradation can be achieved through the hydrolytic action of microbial xylanases. Xylanases have an array of applications one being bioethanol production. The lack of thermophilic xylanases has prompted the search for new enzymes with increased thermostability. Previous work on the crude enzyme of Phialophora alba has demonstrated optimal activity (39 U/μg) at a pH of 4 and two temperature optima of 50°C and 90°C. These desirable properties highlighted the need for further research on the purified enzyme. In the present study P. alba was identified as a thermophilc Ascomycete that forms conidia and chlamydospores during the asexual and sexual stages of its life cycle, respectively. The various isozymes present in the crude enzyme extract were subsequently detected by zymogram analysis. Up to six xylanase isozymes ranging from 90-210 kDa in size were detected. The crude enzyme was subsequently purified by precipitation and ion exchange chromatography (IEX). Protein precipitation methods, desalting methods, IEX resins, elution buffers and NaCl gradients were optimized. The 31-70% ammonium sulphate precipitate had the highest levels of xylanase activity. Separation of proteins with the anion exchanger, HiTrap Q sepharose fast flow column and a linear gradient of 0-2.5 M NaCl in phosphate buffer (50 mM, pH 7) yielded a partially pure xylanase isozyme with molecular weight of 210 kDa. A final yield of 1.4% and purification fold 10.6 was obtained after ion exchange chromatography. The specific activity of the xylanase was 21 IU/μg. At optimum pH (pH 4) and temperature (50°C) a combined xylanase activity of 32 IU.ml⁻¹ was detected. The partially pure xylanase was stable from pH 4-6 with 86% of xylanase activity retained for 90 minutes. Thermostability was observed from 40-70°C with 95% of activity retained for 90 minutes at optimum temperature. The ability of the partially pure xylanase and crude enzyme to hydrolyze untreated and pretreated (alkali and temperature/pressure) sugarcane bagasse was tested at a constant enzyme loading rate of 15 IU/g. Overall, maximum hydrolysis was achieved with the alkali pretreatment and saccharification with the crude enzyme: approximately, 2.4 g/ml of reducing sugars were liberated over a 48 hours. The partially pure xylanase liberated a maximum amount of 2.3 g/ml reducing sugars after 48 hours. The results obtained highlight the desirable characteristics of the partially pure enzyme and its applicability to bioethanol production.