2.0
STUDY OBJECTIVES
The overall objective of the study was to develop an enzyme based technology for controlled reduction of xylan solubility and to demonstrate the technical performance of the insoluble xylan products in selected industrial applications. The specific objectives included:
1. Selective isolation and characterization of xylan from Eucalyptus grandis,
Pinus patula, Bambusa balcooa and sugarcane (Saccharum officinarum)
bagasse found in South Africa by using two selected mild alkali-low temperature isolation protocols.
2. Production and characterization of recombinant α-L-arabinofuranosidase (AbfB) with polymeric xylan substrate specificity expressed without xylanase activity in A. niger microbial system
3. Optimization of selective enzymatic hydrolysis for industrial application of isolated xylans, which included:
a. Assessing degree of selective removal of arabinose and 4-O- methyl glucuronic acid (MeGlcA) side groups by recombinant α-L- arabinofuranosidase (AbfB) of Aspergillus niger and purified α-D- glucuronidase of Schizophyllum commune (AguA) respectively from isolated xylan.
b. Identifying optimal levels of hydrolysis time, temperature and dosage of recombinant α-L-arabinofuranosidase and purified α-D- glucuronidase for maximum removal of arabinose and 4-O-methyl glucuronic acid side chains and the subsequent reduction in solubility of model xylans using response surface methodology.
c. Assessing the effect of individual and synergetic effects of recombinant α-L-arabinofuranosidase and purified α-D-glucuronidase on in situ modification and adsorption of xylans derived from wood and grass sources onto cellulosic material.
32 d. Assessing the morphological features and ability of hydrogels produced from selective hydrolysis of xylans by recombinant α-L- arabinofuranosidase for encapsulation and slow release of horse radish peroxidase.
2.1
RESEARCH FRAMEWORK
The research activities were performed using a multidiscipline approach requiring expertise and knowledge from microbiology, wood science and chemistry and process engineering. The integration of the activities and the required expertise are depicted in Figure 2.1. The expertise from microbiology was relevant for construction of the microbial systems for expression of the side chain removing enzymes, whereas the knowledge from wood science and chemistry was necessary for characterisation of lignocellulosic materials for extraction of xylans. The production of the side chain removing enzymes, the enzymatic modification of the xylans and the investigation of the possible applications of the modified xylans constituted the process engineering component of the research. All activities were performed on the premise of developing an environmentally benign technology for production of biomaterials from renewable natural resources that have similar or improved functionalities compared to materials derived from fossil fuel.
2.2
SCIENTIFIC CONTRIBUTION
The planning, designing and execution of the experiments and interpretation of the results in this study were done by the author. Technical assistance was received from Dr S.H. Rose of Microbiology Department at Stellenbosch University in the cloning of the abfB gene in A. niger as specified in Chapter 4. The study has made scientific contributions in the following areas:
2.2.1 Selective isolation and characterisation of water soluble polymeric xylans from South African feedstocks (Chapter 3)
The study has generated information on the characteristics of South African feedstocks for extraction of unique xylans and for other lignocelluloses processes. In particular, the study has elucidated the structure and chemical composition of Eucalyptus (Eucalyptus grandis) and sugarcane (Saccharum officinarum) bagasse from Mpumalanga Province of South Africa and pine (Pinus patula) and bamboo (Bambusa balcooa) both from Western Cape Province of South Africa. In addition,
33 Figure 2.1: Research framework for the development of enzyme technology for reducing solubility properties of xylans leading to formation of speciality coating material and encapsulation matrix.
IN SITU ENZYME AIDED XYLAN
ADSORPTION ONTO COTTON LINT
IN SITU ENCAPSULATION OF HORSE
RADISH PEROXIDASE ENZYME BASED XYLAN MODIFICATION PROCESS Characterisation WOOD SCIENCE BIOPROCESS ENGINEERING Biomass Feedstock Mild alkali xylan
Extraction Water soluble xylan
MICROBIOLOGY A. niger transformation AbfB gene Screening, characterisation and optimisation
Side chain removing enzymes
34 the extractability and chemical and structural features of the xylans extracted from the South African feedstocks were unveiled. Such information could be used for custom modification of the xylan properties to introduce different functionalities relevant for establishing a lignocelluloses biorefinery.
2.2.2 α-L-Arabinofuranosidase production in recombinant fungal system (Chapter 4)
An improved microbial production system and fermentation protocol for overexpressing recombinant AbfB in A. niger in high concentration and free of xylanase activity has been developed. The development of the recombinant A. niger production system is an advancement that would enhance production of speciality enzymes for novel applications in lignocelluloses processing, in particular, for xylan processing.
2.2.3 Enzymatic modification and industrial utilisation of xylans (Chapters 5-8)
The purified α-D-glucuronidase and the recombinant α-L-arabinofuranosidase (free of xylanase activities) are side chain degrading enzymes, which will advance lignocelluloses processing. The study has demonstrated novel ways of using unique group of α-D-glucuronidase for selective removal of MeGlcA from polymeric xylan. The recombinant α-L-arabinofuranosidase and purified α-D-glucuronidase were used as biological tools for reducing solubility of xylan extracted from the South African feedstocks in order to produce novel nanohydrogels. The xylan nanohydrogels can be used as biodegradable encapsulation, implantation matrix for slow delivery of compounds. In addition, the study has developed an improved enzyme based method for enhancing adsorption of water soluble xylans onto cellulosic materials thus allowing in situ modification (selective removal of side chains) and adsorption of the xylans onto cellulosic materials. Such technology has potential to transform and improve the efficiency of the wet end processes in pulp and paper making. Therefore, the study has novel contribution to advance research in the areas of green engineering, particle engineering, nanotechnology, biomedical engineering e.g. development of targeted and slow delivery systems and tissue replacement) and in surface chemistry.
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