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by Ka-shing Cheung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 112-114). / Abstract --- p.i / Acknowledgments --- p.iii / Table of contents --- p.v / Abbreviations --- p.x / List of figures --- p.xi / List of tables --- p.xiii / Chapter 1. --- Introduction / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Purpose of study --- p.3 / Chapter 2. --- Literature review / Chapter 2.1 --- Cellulose: properties and degradation --- p.4 / Chapter 2.2 --- Cellulase system / Chapter 2.2.1 --- Definition and substrate specificity --- p.5 / Chapter 2.2.2 --- Co-operation of cellulases --- p.5 / Chapter 2.2.3 --- Multiplicity of cellulases --- p.6 / Chapter 2.2.4 --- Regulation of cellulase synthesis --- p.6 / Chapter 2.2.5 --- Architecture of cellulase protein --- p.8 / Chapter 2.3 --- Molecular biology of fungal cellulase genes / Chapter 2.3.1 --- Structural organization of fungal cellulase genes --- p.15 / Chapter 2.3.1.1 --- Promoter and regulatory sequence --- p.15 / Chapter 2.3.1.2 --- Sequence at transcriptional start point (tsp) --- p.16 / Chapter 2.3.1.3 --- Signal peptide --- p.18 / Chapter 2.3.1.4 --- Intron --- p.18 / Chapter 2.3.1.5 --- General sequence homology --- p.21 / Chapter 2.3.2 --- Regulation of cellulase production at molecular level --- p.23 / Chapter 2.3.3 --- Multiplicity of cellulase gene --- p.24 / Chapter 2.3.4 --- Tactics to clone fungal cellulase genes --- p.25 / Chapter 2.3.4.1 --- Past experience --- p.25 / Chapter 2.3.4.2 --- Present approach --- p.28 / Chapter 2.3.5 --- The importance of cellulase gene cloning --- p.29 / Chapter 2.4 --- Cellulolytic microorganisms / Chapter 2.4.1 --- Ecological roles and diversity --- p.31 / Chapter 2.4.2 --- "Biology of the straw mushroom, Volvariella volvacea" --- p.31 / Chapter 3. --- Materials and methods / Chapter 3.1 --- Recipes of media and solutions / Chapter 3.1.1 --- Culture media and microbial-growth related chemicals --- p.34 / Chapter 3.1.2 --- Solutions --- p.36 / Chapter 3.2 --- Bacterial and fungal strains and the growth and storage of mycelium / Chapter 3.2.1 --- Bacterial and fungal strains --- p.42 / Chapter 3.2.2 --- Growth and storage of mycelium --- p.42 / Chapter 3.3 --- Extraction of DNA from mycelium --- p.43 / Chapter 3.4 --- Degenerate polymerase chain reaction (PCR) / Chapter 3.4.1 --- Primers --- p.45 / Chapter 3.4.2 --- Amplification conditions of degenerate PCR --- p.46 / Chapter 3.5 --- Cloning of PCR products / Chapter 3.5.1 --- Ligation --- p.47 / Chapter 3.5.2 --- Transformation --- p.47 / Chapter 3.5.3 --- Screening by blue/white selection --- p.47 / Chapter 3.5.4 --- Screening by PCR --- p.48 / Chapter 3.6 --- Plasmid extraction by alkaline lysis / Chapter 3.6.1 --- Midi-preparation of plasmid by Qiagen column --- p.51 / Chapter 3.6.2 --- Preparation of plasmid using Promega's Wizard minipreps DNA purification system --- p.51 / Chapter 3.7 --- Sequencing analysis of cloned PCR products / Chapter 3.7.1 --- Growth and titering of helper phage R408 --- p.53 / Chapter 3.7.1.1 --- Plate elution method --- p.53 / Chapter 3.7.1.2 --- Liquid culture method --- p.53 / Chapter 3.7.1.3 --- Titering of R408 --- p.53 / Chapter 3.7.2 --- Rescue of single-stranded DNA from pCR-Script phagemid --- p.54 / Chapter 3.7.3 --- Sequencing by chain-termination reaction --- p.54 / Chapter 3.7.4 --- Preparation of polyacrylamide gel for DNA sequencing --- p.56 / Chapter 3.7.5 --- Running a sequencing gel --- p.57 / Chapter 3.7.6 --- "Fixation, exposure and development of sequencing gel and X-ray film" --- p.57 / Chapter 3.7.7 --- Sequence analysis --- p.58 / Chapter 3.8 --- Digestion of DNA with restriction enzymes --- p.59 / Chapter 3.9 --- Agarose gel electrophoresis --- p.60 / Chapter 3.10 --- Purification of DNA from agarose gel by Qiaex --- p.61 / Chapter 3.11 --- Southern hybridization / Chapter 3.11.1 --- Southern blotting and DNA immobilization --- p.62 / Chapter 3.11.2 --- Random-labelling of DNA probe and removal of unincorporated nucleotides --- p.63 / Chapter 3.11.3 --- Pre-hybridization and hybridization --- p.63 / Chapter 3.11.4 --- Exposure and development --- p.64 / Chapter 3.11.5 --- Determination of molecular weight of hybridization signals --- p.65 / Chapter 4. --- Results / Chapter 4.1 --- Extraction of DNA from the straw mushroom mycelium --- p.66 / Chapter 4.2 --- Amplification of V. volvacea genomic DNA using degenerate primers --- p.70 / Chapter 4.3 --- Cloning of PCR products using pCR-Script SK (+) cloning kit / Chapter 4.3.1 --- Screening by blue/white selection --- p.77 / Chapter 4.3.2 --- Screening by PCR --- p.77 / Chapter 4.4 --- Plasmid extraction by alkaline lysis --- p.80 / Chapter 4.5 --- Preparation of single-stranded DNA template for sequencing / Chapter 4.5.1 --- Growth and titering of helper phage R408 --- p.82 / Chapter 4.5.2 --- Rescue of single-stranded DNA from pCR-Script phagemid --- p.82 / Chapter 4.6 --- Sequencing of cloned PCR products / Chapter 4.6.1 --- The choice of template --- p.84 / Chapter 4.6.2 --- DNA and translated amino acid sequence of PCR clones --- p.84 / Chapter 4.6.3 --- Alignment of DNA sequences against other fungal cellulase genes --- p.93 / Chapter 4.6.4 --- Alignment of translated amino acid sequences against other fungal cellulase --- p.96 / Chapter 4.7 --- Purification of DNA from agarose gel by Qiaex --- p.98 / Chapter 4.8 --- Southern hybridization / Chapter 4.8.1 --- Restriction digestion of genomic DNA --- p.101 / Chapter 4.8.2 --- Hybridization --- p.104 / Chapter 5. --- Discussion / Chapter 5.1 --- Extraction of DNA from V. volvacea mycelium --- p.107 / Chapter 5.2 --- Rationales of designing degenerate primers from heterologous amino acid sequence --- p.107 / Chapter 5.3 --- Amplification of V. volvacea DNA using degenerate primers --- p.110 / Chapter 5.4 --- Cloning of PCR products using pCR-Script system --- p.111 / Chapter 5.5 --- The precaution of using Qiaex-purified DNA --- p.112 / Chapter 5.6 --- Sequencing analysis / Chapter 5.6.1 --- DNA sequence analysis --- p.113 / Chapter 5.6.2 --- Protein sequence analysis --- p.114 / Chapter 5.7 --- Southern hybridization --- p.116 / Chapter 6. --- Conclusion and further analysis --- p.117 / Chapter 7. --- References --- p.119

In this study the enzymatic activity of adsorbed Thermomonospora fusca E��� and Trichoderma reesei CBHI cellulases were investigated using fluorescence techniques. In particular, cellulases were allowed to contact hydrophobic polystyrene surfaces under conditions of different solution concentrations, and adsorption times. Each of these variables is known to have a potential effect on enzyme structure and activity at an interface. Enzymatic activity was measured after partial elution of the adsorbed layer with both protein-free buffer and the surfactant, dodecyltrimethylammonium bromide. For E��� at high concentration (0.5 mg/ml), adsorbed enzyme activity decreased about 20% in increasing adsorption time from 0.25 h to 24 h. At low concentration (0.001 mg/ml), adsorbed enzyme activity decreased by one order of magnitude during a 24 h period. CBHI layers lost activity only after a sufficiently long contact time with the surface, and this effect was not strongly dependent on enzyme concentrations in solution. These findings were explained with reference to structural changes undergone by adsorbed enzyme as a function of time and available interfacial area. / Graduation date: 2000

Graduation date: 1999

Lignocellulose is composed of lignin, hemicellulose, and cellulose. Lignocellulose waste is a sustainable and renewable resource available for use in biotechnological applications. Efficient enzyme production and enzymes with high catalytic activity are needed for the use of lignocellulose. The study of cellulases and xylanases that degrade cellulose and xylan into constituent monosaccharides is required to advance industrial application of these enzymes. The use of a traditional pure culture approach to discover and characterize cellulases and xylanases from novel actinomycete isolates and the use of metagenomics to uncover previously unidentified cellulase genes was undertaken. Actinomycetes were cultivated from soil samples and the isolate with the best cellulase and xylanase activity was subjected to strain improvement through protoplast fusion. Enhanced enzymatic activity was found in one fusant. Differential release of sugars from xylan was observed through gas chromatographic analysis between the parental and fusant cultures. Genome shuffling was observed in 16S rRNA genes after protoplast fusion. Finally, one putative endo-β-1,4-glucanase was discovered in a metagenomic library created from cellulose-enriched potting soil. / UOIT

Three thermophilic aerobic bacterial strain, Geobacillus thermoleovorans T4, Bacillus subtilis b5 and Bacillus licheniformis b6 were isolated from the wastewater of a Taiwan Sugar Company sugar refinery in Kaohsiung. All strains were capable of growth in cellulose containing medium. We found out that G. thermoleovorans T4 possessed both of the activity of cellulses and xylanase. This was the first report ever in this bacterial spices. Its endoglucanase was active at temperatures ranging from 30¢J to 100¢J, and pH ranging from 4.0 and 8.0. Its £]-glucosidase activities could be measured at 40¢J - 90¢J and pH 5.0 - 9.0. Its xylanase was active from 30¢J to 90¢J and pH from 3.0 to 10.0, while the exoglucanase was active at temperatures from 30¢J to 90¢J. The pH values of xylasnase was better than G. thermoleovorans K-3d. This enzyme was worth of possible in industry application. Both of B. subtilis b5 and B. licheniformis b6 contained a thermoactive endoglucanase. The endoglucanase of B. subtilis b5 was active at pH values between 4.0 and 7.5, and was thermostable at 100¢J for at least 1 hour ( retaining 60% of the original activity ). The stability of this enzyme was better than all known enzymes extracted from Bacillus spices. The endoglucanase of B. licheniformis b6 was active at pH values between 4.0 and 9.0, and thermostable at 60¢J for least 1 hour.

There are generally four factors recognized as delimiting in the study of lignocelluloses for fuel ethanol production, viz., the source of the cellulase-system and its quality characteristics for cellulose hydrolysis, the substrate and pretreatment method, the process for cellulase production and bioreactor design, and the ability of yeast to ferment mixed hexose and pentose sugars. Wheat straw (WS) and T. reesei mutants were used in the study to evaluate the production of cellulase-systems. Hydrolysis of cellulose revealed the superiority of mild NaOH pretreatment over steam explosion for cellulase production with T. reesei MCG 80 and QMY-1. Response surface models were capable of predicting that NaOH could be used for the pretreatment of WS at 4% (w/w) without urea in the fermentation medium to yield optimum filter paper activity (FPA) of 9.9 IU/mL (247 IU/g WS) and beta-glucosidase activity ($ beta$GA) of 6.4 IU/mL (159 IU/g WS) under solid-state fermentation (SSF) conditions. Multiple regression analysis with multiple coefficients of correlation, R, between 0.957 and 0.99 from the experimental data showed close agreement between the cellulase activities (FPA and $ beta$GA) from the experiments and predicted values. / The superiority of SSF over liquid-state fermentation (LSF) in the production of cellulase-systems was also established, and a prototype pan-bioreactor showed good potential for upgrading cellulase production under SSF conditions. The economics of fuel ethanol production was considered in the optimization model that sought to establish threshold cellulase loadings needed to achieve maximum cellulose hydrolysis for fermentation. High substrate concentrations of up to 7.5% were hydrolyzed with cellulase loadings of 24-30 IU/g and fermented by Pichia stipitis to achieve 90-100% conversion into ethanol. / Crude unextracted cellulase yielded over 90% hydrolysis of delignified wheat straw and proved to be better than extracted cellulase and commercial cellulases for the hydrolysis of pure cellulose and pretreated wheat straw. Studies were also conducted to demonstrate the importance of the ratio of $ beta$GA- to FPA in cellulose hydrolysis which showed that ratios closer to one (1), produced more sugars and lowered the cellobiose content in the hydrolysates. It was also shown that the source of the cellulase is important in eliminating the accumulation of cellobiose during hydrolysis as was demonstrated with cellulase from mixed cultures of T. reesei and Aspergillus phoenicis. Higher $ beta$GA from the latter were implicated since A. phoenicis is a good $ beta$-glucosidase producer. / Delignified wheat straw at 5% concentration when subjected to separate hydrolysis and fermentation and simultaneous hydrolysis and fermentation resulted in similar volumetric productivities (g/L/h) of ethanol.

Thesis (Ph.D.) - University of Queensland, 2006. / Includes bibliography.

Th.--Pharm.--Paris 5, 1984. N°: 106.