Submitted in fulfilment for the requirement of a Degree of Doctor of Technology: Biotechnology, Durban University of Technology, 2010. / The aim of this study was to evaluate T. lanuginosus for the production of hemicellulases, its yield enhancement using mutagenesis and application of a selected xylanase on bagasse pupl to assess the improvement of pulp properties. The objectives were: To determine the localization of hemicellulases in T. lanuginosus strains, To develop high yielding strains of T. lanuginosus through mutagenensis, To investigate the synthesis of xylanase by T. lanuginosus MC134, To optimize the medium components and cultural conitions of T. lanuginosus MC134 strain, To study the influence of agitation and aeration on the production of xylanase by T. lanuginosus MC134 in a fermenter, To evaluate the bleach boosting abilities of T. lanuginosus xylanase on bagasse pulp, To evaluate simultaneous xylanase production and biobleaching potential of T. lanuginosus.

Submitted in complete fulfilment for Masters Degree in Technology: Biotechnology, Durban University of Technology, 2013. / A comparative thermostability analysis of different partially purified xylanases from Rhodothermus marinus, Bacillus halodurans, Thermomyces lanuginosus and Pulpzyme HC was studied using differential scanning fluorometry (DSF), fluorescence spectroscopy and circular dichroism (CD). The R. marinus xylanase was found to have an optimum temperature and pH of 90oC and 6 respectively while the B. halodurans xylanase was optimally active at 70oC and a broad range of alkaline pH of 8 - 10. The commercially available xylanase from T. lanuginosus showed optimal activity at 50oC and pH 7 while the Novozyme xylanase Pulpzyme HC showed optimal activity at 60oC and pH 7. Fluorescence spectroscopy monitored the microenvironment and fluorescence emission of Trp residues. In their native folded state, Trp are generally located in the core of the protein but during unfolding they become exposed. The fluorescence changes as the enzyme undergoes denaturation due to conformational changes and exposure of Trp residues. Differential scanning fluorometry (DSF) monitors thermal unfolding of proteins in the presence of a fluorescent dye such as Spyro Orange. A wide range of buffers were tested for their ability to increase the xylanase stability. T. lanuginosus had the greatest increase in melting temperature with 0.73M Bis Tris pH 6.5 and peaked highest at 78°C. The B. halodurans xylanase exhibited high pH stability (pH 4-10) and exhibited very little change in melting temperature, from 74°C-77°C over the twenty four different conditions. The R. marinus xylanase had no increase in melting temperature showing a maximum melting temperature of 90oC. Circular dichroism (CD) measures unequal absorption of right- and left-handed circularly polarized light by the molecule. The xylanase from R. marinus exhibited the lowest ΔG of 34.71kJ at 90°C as was expected. The B. halodurans xylanase showed a much higher ΔG of -52.71 at its optimum temperature of 70°C when compared with the xylanases from R. marinus and T. lanuginosus. When comparing the three xylanases activities at 70°C, it can be seen that the B. halodurans xylanase exhibited a lower relative activity then both R. marinus and T. lanuginosus xylanases. All three techniques offered different information on the structure and function relationship. Fluorescence spectroscopy, the change in conformation due to fluorescence emission as a result of increased temperature and salt concentrations. DSF, optimal conditions for increased stability and activity at higher temperatures and CD, conformational changes, the fraction of folded protein and change in Gibbs free energy over a range of temperature. / National Research Foundation

Submitted in fulfilment of the requirements for the degree of Master of Engineering, Durban University of Technology, Durban, South Africa, 2015. / Fermentation processes are vital for the production of numerous bioproducts. Fermentation being the mass culture of micro – organisms for the production of some desired product, is an extensive field, with immense prospects for study and improvement. Enzyme production is of significance as these proteins are biological catalysts, finding niches in numerous industries, xylanase for example is utilized in the pulp and paper, animal feed, biofuel and food production processes. During enzyme production, a critical step is biomass separation, whereby the valuable product, the enzyme, is removed from the broth or micro – biological culture before it is denatured. This is typically achieved via centrifugation. The aim of this study was to develop and evaluate a submerged membrane fermenter system with the specific outcome of increasing the rate of production of xylanase, from the thermophilic fungal species Thermomyces lanuginous DSM 5826. Preliminary shake flask experiments were performed to determine the optimal production conditions, followed by partial characterization of the enzyme. A bioreactor was then fabricated to include a flat sheet membrane module, with outlets for permeate and broth withdrawal and inlets for feed and sterile air input. Experiments were conducted to determine the optimal dilution rate for maximum volumetric productivity. Results from the shake flask experiments indicated that the best conditions for xylanase production, yielding xylanase activity of 5118.60 ± 42.76 U.mL-1 was using nutrient medium containing beechwood xylan (1.5 % w/v), yeast extract (1.5 % w/v), potassium dihydrogen phosphate (0.5 % w/v), adjusted to a pH of 6.5 and inoculated with 1.0 mL of spore solution, rotating in a shaking incubator set to 150 rpm at 50 °C. Apart from analysis of the effect of the carbon source on xylanase activity, coarse corn cobs were used in the shake flask experiments as a cost saving initiative. The pH optima was determined to be 6.5 while the temperature optima of the enzyme was 70 °C. SDS PAGE analysis revealed that the molecular weight of the enzyme was between 25 and 35 kDa and qualitative analysis via a zymogram revealed clear zones of hydrolysis on a xylan infused agarose gel. During short run membrane fermenter experiments the percentage increase in enzyme activity between the batch operation (610.58 ± 34.54 U.mL-1) and semi – continuous operation (981.73 ± 55.54 U.mL-1) with beechwood xylan nutrient replenishment was 60.78 %. The maximum volumetric productivity achieved with beechwood supplementation after 192 hours in semi – continuous operation (5.32 ± 0.30 U.mL-1.hr-1) was 2.1 times greater than that of batch operation (2.54 ± 0.14 U.mL-1.hr-1) which equates to an increase of 110.28 % in productivity measured at its peak. The increase in total activity between batch (610 576.92 U) and beechwood xylan medium supplemented semi – continuous mode (1 184 937.50 U) resulted in a 94.07 % increase. During long run experimental periods, the increase in production of xylanase between the batch (873.26 ± 61.78 U.mL-1) and the xylan medium membrane system (1522.41 ± 107.65 U.mL-1) was determined to be 74.34 % while an overall average increase in productivity between the batch and xylan fed membrane system was 43.25%. The total enzyme activity with in membrane mode with beechwood xylan nutrient medium feed was 160 % greater than the batch process offering a 2.6 – fold increase. Experiments where de – ionized water was alternated with beechwood xylan nutrient medium had no significant impact on the productivity or enzyme activity. The optimal dilution rate for maximum volumetric productivity as determined to be 0.0033 hr-1. The results are indicative of the potential viability of such a design, yielding the desired outcome of a membrane integrated system to significantly increase the production of enzymes during fermentation.

Submitted in fulfillment of the requirements of the degree of Master of Technology (Biotechnology), Durban University of Technology, Durban, South Africa, 2014. / Microbial enzymes are gaining worldwide attention due to their potential industrial applications. Microorganisms producing thermostable -xylanase and their associated hemicellulases have significant application in the paper and pulp, food, animal feed, and textile industries. The potential of partially purified xylanase from Thermomyces lanuginosus MC 134, Luminase PB 100, Luminase PB 200 (a commercial xylanase) and T. lanuginosus DSM 5826 (Sigma Aldrich) was evaluated in bleaching of bagasse pulp. The temperature and pH optima for all the enzymes were 60°C and pH 6, respectively. The temperature (50- 80°C) and pH (5-8) stability of the enzymes were also assessed. All the enzymes were relatively stable at 60°C and pH 6 for 180 min. T. lanuginosus MC 134 retained 80% of its activity at 60°C and pH 6 for 180 min and PB 200 retained 75% of its activity at 80°C for 180 min. T. lanuginosus MC 134 also exhibited good alkaline stability at pH 8. The commercial xylanases Luminase PB 100, Luminase PB 200, T. lanuginosus DSM 5826 (Sigma Aldrich) were purified to homogeneity using a gel filtration column packed with sephadex G-100 and characterized for Km and Vmax. However extracellular crude xylanases from T. lanuginosus MC 134 was purified to homogeneity using (N )2S04 precipitation and gel filtration column, packed with sephadex G-100. The purified xylanases exhibited a molecular mass of- 26 to 24 kDa, given range as determined by SDS page. The Km and Vmax values of Luminase PB 100, Luminase PB 200, T. lanuginosus MC 134, and T. lanuginosus DSM 5826, xylanases were determined by the Michaelis-Menten equation using birchwood xylan as the substrate. The Km value for Luminase PB 100, Luminase PB 200, T. lanuginosus DSM 5826 and T. lanuginosus MC 134 were, 8.1 mg/mL, 11.7 mg/mL and 14.3 mg/mL respectively. The Vmax for Luminase PB 100, Luminase PB 200, T lanuginosus DSM 5826 and T lanuginosus MC 134 were 232.6, 454.6 and 74.6 !Jl11ol/min/mg. Biobleaching conditions of the xylanases were also optimised and the release of reducing sugars and lignin derived compounds showed that an enzyme dosage of 50U/g of pulp was ideal for biobleaching at pH 6 and 60°C for 180 min. This brightness for T lanuginosus MC 134, Luminase PB 200, Luminase PB 100 was 45.5 ± 0.11%, 44.1 ± 0.007% and 42.7 ± 0.03% respectively at pH 6, compared to untreated samples. Reducing sugars and UV-absorbing lignin-derived compound values were considerably higher in xylanase-treated samples. All the enzymes analysed exhibited similar trends in the release of lignin derived compounds and reducing sugars which indicated their potential in the pulp and paper industry. / PDF Full-text unavailable. Please refer to hard copy for Full-text / M

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

Bu Su. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 139-158). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Peijun Zuo. / "November 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 188-212). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 vi, 92 leaves / Interest in xylanases from different microbial sources has increased markedly in the past decade, in part because of the application of these enzymes in a number of industries, the main area being the pulp and paper industry. While conventional methods will continue to be applied to enzyme production from micro-organisms, the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis and this knowledge is now being applied both in the laboratory and commercially. In this study, a directed evolution strategy was used to select an enzyme variant with high thermostability. This study describes the use of error-prone PCR to modify the xylanase gene from Thermomyces lanuginosus DSM 5826, rendering it tolerant to temperatures in excess of 80°C. Mutagenesis comprised of different concentrations of nucleotides and manganese ions. The variants were generated in iterative steps and subsequent screening for the best mutant was evaluated using RBB-xylan agar plates. The optimum temperature for the activity of xylanases amongst all the enzyme variants was 72°C whilst the temperature optimum for the wild type enzyme was 70°C. Long term thermostability screening was therefore carried out at 80°C and 90°C. The screen yielded a variant which had a 38% improvement in thermostability compared to the wild type xylanase from pX3 (the unmutated gene). Successive rounds of error-prone PCR were carried out and in each round the progeny mutant displayed better thermostability than the parent. The most stable variant exhibited 71% residual activity after 90 minutes at 80˚C. Sequence analysis revealed four single amino acid residue changes that possibly enhanced their thermostabilities. This in vitro enzyme evolution technique therefore served as an effective tool in improving the thermostable property of this xylanase which is an important requirement in industry and has considerable potential for many industrial applications.

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 vi, 92 leaves / Interest in xylanases from different microbial sources has increased markedly in the past decade, in part because of the application of these enzymes in a number of industries, the main area being the pulp and paper industry. While conventional methods will continue to be applied to enzyme production from micro-organisms, the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis and this knowledge is now being applied both in the laboratory and commercially. In this study, a directed evolution strategy was used to select an enzyme variant with high thermostability. This study describes the use of error-prone PCR to modify the xylanase gene from Thermomyces lanuginosus DSM 5826, rendering it tolerant to temperatures in excess of 80°C. Mutagenesis comprised of different concentrations of nucleotides and manganese ions. The variants were generated in iterative steps and subsequent screening for the best mutant was evaluated using RBB-xylan agar plates. The optimum temperature for the activity of xylanases amongst all the enzyme variants was 72°C whilst the temperature optimum for the wild type enzyme was 70°C. Long term thermostability screening was therefore carried out at 80°C and 90°C. The screen yielded a variant which had a 38% improvement in thermostability compared to the wild type xylanase from pX3 (the unmutated gene). Successive rounds of error-prone PCR were carried out and in each round the progeny mutant displayed better thermostability than the parent. The most stable variant exhibited 71% residual activity after 90 minutes at 80˚C. Sequence analysis revealed four single amino acid residue changes that possibly enhanced their thermostabilities. This in vitro enzyme evolution technique therefore served as an effective tool in improving the thermostable property of this xylanase which is an important requirement in industry and has considerable potential for many industrial applications.