Yuan Zhao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 89-96). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgements --- p.iv / Declaration --- p.v / Abbreviations --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xii / List of Figures --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Lignocelluloses --- p.2 / Chapter 1.1.1 --- component of lignocellulose --- p.2 / Chapter 1.1.2 --- Xylans --- p.3 / Chapter 1.2 --- Degradation of lignocellulose --- p.9 / Chapter 1.3 --- "Endo-β-1,4- xylanase" --- p.12 / Chapter 1.3.1 --- Structure of xylanase --- p.12 / Chapter 1.3.2 --- Mode of action --- p.17 / Chapter 1.3.3 --- Appications of xylanase --- p.20 / Chapter 1.4 --- Aims of my study --- p.24 / Chapter Chapter 2 --- Materials and Methods --- p.25 / Chapter 2.1 --- Cloning of xylanase genes --- p.26 / Chapter 2.1.1 --- Materials --- p.26 / Chapter 2.1.1.1 --- Bacterial and fungal strains --- p.26 / Chapter 2.1.1.2 --- Growth media --- p.26 / Chapter 2.1.1.3 --- Vector --- p.26 / Chapter 2.1.1.4 --- Reagents for agarose gel electrophoresis --- p.27 / Chapter 2.1.1.5 --- Reagents for preparation of competent cells --- p.27 / Chapter 2.1.2 --- Methods --- p.29 / Chapter 2.1.2.1 --- Isolation of chromosomal DNA --- p.29 / Chapter 2.1.2.2 --- Amplification of exons of xylanase genes --- p.29 / Chapter 2.1.2.3 --- Agarose gel electrophoresis of DNA --- p.37 / Chapter 2.1.2.4 --- DNA recovery from agarose gel --- p.37 / Chapter 2.1.2.5 --- Assemble and amplify the full length genes --- p.38 / Chapter 2.1.2.6 --- Restriction endonuclease digestion --- p.39 / Chapter 2.1.2.7 --- Ligation of purified DNA fragment into vector --- p.39 / Chapter 2.1.2.8 --- Transformation --- p.40 / Chapter 2.1.2.9 --- Methods for making competent cells --- p.40 / Chapter 2.1.2.10 --- Plasmid DNA preparation --- p.40 / Chapter 2.1.2.11 --- DNA sequencing --- p.41 / Chapter 2.2 --- Mutagenesis of xylanase --- p.43 / Chapter 2.2.1 --- Amplification of xylanases genes --- p.47 / Chapter 2.2.2 --- DNA random mutagenesis --- p.48 / Chapter 2.2.2.1 --- DNase digestion --- p.48 / Chapter 2.2.2.2 --- Reassembly of DNA fragments --- p.48 / Chapter 2.2.2.3 --- Amplification of full-length genes --- p.48 / Chapter 2.2.2.4 --- Construction of library --- p.49 / Chapter 2.2.3 --- Screening of mutants --- p.49 / Chapter 2.2.3.1 --- Preparation of RBB-xylan --- p.49 / Chapter 2.2.3.2 --- Plate assay for screening of mutants --- p.50 / Chapter 2.3 --- Expression of xylanase genes --- p.51 / Chapter 2.4 --- Enzyme assays --- p.52 / Chapter 2.4.1 --- Xylanase assay with RBB-xylan --- p.52 / Chapter 2.4.2 --- Xylanase assay with DNS-method --- p.52 / Chapter 2.4.2.1 --- Reagents --- p.53 / Chapter 2.4.2.2 --- Xylose standard curve --- p.53 / Chapter 2.4.2. 3 --- Activity assay --- p.54 / Chapter 2.4.2. 4 --- Thermostability assay --- p.54 / Chapter Chapter 3 --- Results --- p.55 / Chapter 3.1 --- Cloning of xylanase genes --- p.56 / Chapter 3.2 --- Mutagenesis of xylanase --- p.59 / Chapter 3.2.1 --- DNA random mutagenesis --- p.59 / Chapter 3.2.2 --- Screening of mutants --- p.67 / Chapter 3.3 --- Enzyme assays --- p.69 / Chapter Chapter 4 --- Discussions --- p.76 / Chapter 4.1 --- Gene shuffling --- p.77 / Chapter 4.2 --- Screening method and activity assay --- p.78 / Chapter 4.3 --- Sequence analysis --- p.80 / Chapter 4.4 --- Future work --- p.88 / Bibliography --- p.89

Submitted in complete fulfillment of the requirements for the Degree of Doctor of Technology: Biotechnology, Durban University of Technology, Durban, South Africa. 2014. / The global demand for green technology has created a need to search for microbes that can play an active role in advancing a greener and cleaner future. Microbial enzymes are nature’s keys to life and their efficiency, specificity and environmental-friendliness has lead to their increased use in industrial processes. Thermomyces lanuginosus is a thermophilic fungus that can degrade plant biomass and produces a variety of enzymes that have industrial application. The fungus T. lanuginosus SSBP has been reported in literature to produce the highest level of xylanase among other Thermomyces strains and some of its enzyme s viz., amylase and lipase are already being used. Because of this ability, it has been identified as one of the organisms that can have various industrial applications. Although a few proteins from this fungus have been cloned and used commercially, the vast majority are still unknown. In order to identify new protein candidates and understand their biochemical interactions, the T. lanuginosus genome (DNA) and the transcriptome (mRNA) were sequenced using 454 Roche and Solexa sequencing platforms. Genome and transcriptome data was assembled using Newbler software forming a genome size of 23.3 Mb contained 30 scaffolds. Protein prediction identified 5105 candidates as protein-coding genes and these gene models were supported by expressed sequence tag and transcriptomic data. The annotated data was assembled into metabolic pathways in order to identify functional pathways and validate the accuracy of the annotation process. T. lanuginosus is usually found in composting plant material thus protein related to plant hydrolysis were analysed. The total number of plant biomass-degrading and related proteins that fall into the carbohydrate-active enzyme (CAZy) family was 224. Most of these proteins were similar to proteins found in other filamentous fungi. Surprisingly, T. lanuginosus contained a single gene coding for xylanase which hydrolyses xylan although this organism is well known for being among the highest producers of this enzyme. An important subset of the above group of proteins is the cellulose degrading-proteins as this can be used in biofuel production. Eight candidates belonging to this group were identified, making this fungus significant in the biofuels. Among the eight cellulase candidates, phylogenetic analysis revealed that three of them were closely related to Trichoderma reesei, a well known industrial cellulase-producer. Utilization of cellulase-related compounds was validated by phenotypic microarray experiments, with cellobiose having inducing biomass in T. lanuginosus. Proteins that are involved in high temperature survival are vital for the survival. of this thermophilic fungus. Interestingly, T. lanuginosus contains 19 heat shocking proteins which are responsible for thermostability. Another adaptation identified in this fungus is the accumulation of trehalose to combat heat stress. Furthermore, T. lanuginosus contains the highest reported number methyltransferases, which have been linked to producing thermostable proteins and higher energy production. Also because of this organism’s ability to grow on composting environments, the assimilation and ability to produce biomass on different carbon sources were analysed using phenotypic microarray technique. The results showed that xylose was the best compound to induce biomass followed by trehalose, maltose and maltotriose. The genomic sequencing of this fungus has provided valuable information that can be used for various biotechnological applications, as well as providing greater insights into its thermostability. Understanding the metabolic pathways involved may allow for manipulation to increase production of these enzymes or cloning into other hosts. This can have an impact in the field of biofuel production and other plant biomass-related processes.

Submitted in fulfillment for the requirement of a Degree of Master of Technology: Biotechnology, in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has provided a key for adapting naturally-occurring enzymes for industrial processes. However, several obstacles have to be overcome after these proteins have been adapted, the main one being finding a suitable host to over-express these recombinant protein. This study investigated Saccharomyces cerevisiae, Pichia pastoris and Escherichia coli as suitable expression hosts for a previously modified fungal xylanase, which is naturally produced by the filamentous fungus, Thermomyces lanuginosus. A xylanase variant, NC38, that was made alkaline-stable using directed evolution was cloned into four different vectors: pDLG1 with an ADH2 promoter and pJC1 with a PGK promoter for expression in S. Cerevisiae, pBGP1 with a GAP promoter for expression in P. pastoris and pET22b(+) for expression in E. Coli BL21 (DE3). S. Cerevisiae clones with the p DLG1-NC38 combination showed very low activity on the plate assay and were not used for expression in liquid media as the promoter was easily repressed by reducing sugars used during production experiments. S. cerevisiae clones carrying pJC1-NC38 were grown in media without uracil while P. Pastoris clones were grown in YPD containing the antibiotic, zeocin and E. Coli clones were grown in LB with ampicillin. The levels of xylanase expression were then compared between P. Pastoris, S. cerevisiae and E. coli. The highest recombinant xylanase expression was observed in P. Pastoris with 261.7U/ml, followed by E.coli with 47.9 U/ml and lastly S. cerevisiae with 13.2 U/ml. The localization of the enzyme was also determined. In the methylotrophic yeast, P. Pastoris, the enzyme was secreted into the culture media with little or no contamination from the host proteins, while the in other hosts, the xylanase was located intracellularly. Therefore in this study, a mutated alkaline stable xylanase was successfully expressed in P. Pastoris and was also secreted into the culture medium with little or no contamination by host proteins, which favours the application of this enzyme in the pulp and paper industry. / National Research Foundation

Submitted in complete fulfillment for the Degree of Master of Technology (Biotechnology)in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has been a valuable tool in creating enzyme variants that are capable of withstanding the extreme environments of industrial processes. Xylanases are a family of hemicellulolytic enzymes that are used in the biobleaching of pulp. Using directed evolution, a thermostable and alkaline stabl xylanase variant (S340) was created from the thermophilic fungus, Thermomyces lanuginosus. However, a host that was capable of rapid growth and high-level expression of the enzyme in large amounts was required. The insert containing the xylanase gene was cloned into a series a pET vectors in Escherichia coli BL21 (DE3) pLysS and trimmed from 786 bp to 692 bp to remove excess fungal DNA upstream and downstream of the open reading frame (ORF). The gene was then re-inserted back into the pET vectors. Using optimized growth conditions and lactose induction, a 14.9% increase in xylanase activity from 784.3 nkat/ml to 921.8 nkat/ml was recorded in one of the clones. The increase in expression was most probably due to the removal of fungal DNA between the vector promoter and the start codon. The distribution of the xylanase in the extracellular, periplasmic and cytoplasmic fractions was 17.3%, 51.3% and 31.4%, respectively. The modified enzyme was then purified to electrophoretic homogeneity using affinity chromatography. The xylanase had optimal activity at pH 5.5 and 70°C. After 120 min at 90°C and pH 10, S340 still displayed 39% residual activity. This enzyme is therefore well suited for its application in the pulp and paper industry. / National Research Foundation

Submitted in fulfillment for the requirement of a Degree of Master of Technology: Biotechnology, in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has provided a key for adapting naturally-occurring enzymes for industrial processes. However, several obstacles have to be overcome after these proteins have been adapted, the main one being finding a suitable host to over-express these recombinant protein. This study investigated Saccharomyces cerevisiae, Pichia pastoris and Escherichia coli as suitable expression hosts for a previously modified fungal xylanase, which is naturally produced by the filamentous fungus, Thermomyces lanuginosus. A xylanase variant, NC38, that was made alkaline-stable using directed evolution was cloned into four different vectors: pDLG1 with an ADH2 promoter and pJC1 with a PGK promoter for expression in S. Cerevisiae, pBGP1 with a GAP promoter for expression in P. pastoris and pET22b(+) for expression in E. Coli BL21 (DE3). S. Cerevisiae clones with the p DLG1-NC38 combination showed very low activity on the plate assay and were not used for expression in liquid media as the promoter was easily repressed by reducing sugars used during production experiments. S. cerevisiae clones carrying pJC1-NC38 were grown in media without uracil while P. Pastoris clones were grown in YPD containing the antibiotic, zeocin and E. Coli clones were grown in LB with ampicillin. The levels of xylanase expression were then compared between P. Pastoris, S. cerevisiae and E. coli. The highest recombinant xylanase expression was observed in P. Pastoris with 261.7U/ml, followed by E.coli with 47.9 U/ml and lastly S. cerevisiae with 13.2 U/ml. The localization of the enzyme was also determined. In the methylotrophic yeast, P. Pastoris, the enzyme was secreted into the culture media with little or no contamination from the host proteins, while the in other hosts, the xylanase was located intracellularly. Therefore in this study, a mutated alkaline stable xylanase was successfully expressed in P. Pastoris and was also secreted into the culture medium with little or no contamination by host proteins, which favours the application of this enzyme in the pulp and paper industry.

Submitted in complete fulfillment for the Degree of Master of Technology (Biotechnology)in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has been a valuable tool in creating enzyme variants that are capable of withstanding the extreme environments of industrial processes. Xylanases are a family of hemicellulolytic enzymes that are used in the biobleaching of pulp. Using directed evolution, a thermostable and alkaline stabl xylanase variant (S340) was created from the thermophilic fungus, Thermomyces lanuginosus. However, a host that was capable of rapid growth and high-level expression of the enzyme in large amounts was required. The insert containing the xylanase gene was cloned into a series a pET vectors in Escherichia coli BL21 (DE3) pLysS and trimmed from 786 bp to 692 bp to remove excess fungal DNA upstream and downstream of the open reading frame (ORF). The gene was then re-inserted back into the pET vectors. Using optimized growth conditions and lactose induction, a 14.9% increase in xylanase activity from 784.3 nkat/ml to 921.8 nkat/ml was recorded in one of the clones. The increase in expression was most probably due to the removal of fungal DNA between the vector promoter and the start codon. The distribution of the xylanase in the extracellular, periplasmic and cytoplasmic fractions was 17.3%, 51.3% and 31.4%, respectively. The modified enzyme was then purified to electrophoretic homogeneity using affinity chromatography. The xylanase had optimal activity at pH 5.5 and 70°C. After 120 min at 90°C and pH 10, S340 still displayed 39% residual activity. This enzyme is therefore well suited for its application in the pulp and paper industry.