Partial purification and characterisation of Phialophora alba xylanases and its application to pretreated sugarcane bagasse.
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Date
2013
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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.
Description
Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.
Keywords
Xylanases., Phialophora., Hemicellulose., Microbial biotechnology., Theses--Microbiology.