No description available.

No description available.

No description available.

No description available.

No description available.

In this study a library of EcoRI fragments encompassing the entire TOL region of the Pseudomonas putida TOL plasmid pDK1 was constructed in the Escheria coli cloning vector pBR325.

Submitted in complete fulfillment for the Degree of Master of Science (Applied Science), Durban University of Technology, Durban, South Africa, 2016. / β-xylosidases catalyse the hydrolyses of xylooligosaccharides into the monosaccharide sugar, xylose. In this study we report the production of xylose under different conditions in Pichia pastoris and Saccharomyces. cerevisiae, and its conversion to bioethanol using Pichia stipitis. The aim of this study was to change the characteristics of the A. niger 90196 β-xylosidase through random mutagenesis and increase expression under the control of different promoter systems in yeasts P. pastoris and S. cerevisiae. The recombinant library created through random mutagenesis was screened for changes in activity and subsequently pH and temperature stability. One variant showed an increase in enzyme expression, thermostability, and a change in amino acid sequence at residue 226. The enzyme was then cloned, expressed and characterized in P. pastoris GS115 and S. cerevisiae. β-xylosidase was constitutively expressed in P. pastoris using the GAP promoter and the inducible AOX promoter. In S. cerevisiae the enzyme was expressed using the constitutive PGK promoter and inducible ADH2 promoter systems. Enzyme functionality with the different expression systems was compared in both hosts. The GAP system was identified as the highest-producing system in P. pastoris, yielding 70 U/ml after 72 hours, followed by the PGK system in S. cerevisiae, with 8 U/ml. A 12% SDS-PAGE gel revealed a major protein band with an estimated molecular mass of 120 kDA, and the zymogram analysis revealed that this band is a fluorescent band under UV illumination, indicating enzyme activity. Stability characteristics was determined by expressing the enzyme at different pH and temperatures. Under the control of the GAP promoter in P. pastoris, enzyme activity peaked at pH4 while retaining 80% activity between pH 3 – 5. Highest activity of 70 U/ml xylosidase was recorded at 60ºC. Due to the high enzyme production in P. pastoris, the co-expression of this enzyme with a fungal xylanase was evaluated. The xylanase gene from Thermomyces lanuginosus was cloned with the GAP promoter system and expressed together with the β-xylosidase recombinant in P. pastoris. Enzyme activities of the co-expressed recombinant revealed a decrease in enzyme activity levels. The co-expressed xylanase production decreased by 26% from 136 U/ml to 100 U/ml while the xylosidase expression decreased 86% from 70 U/ml to 10 U/ml. The xylose produced from the hydrolysis of birchwood xylan was quantified by HPLC. The monosaccharide sugar was used in a separate saccharification and fermentation strategy by P. stipitis to produce bioethanol, quantified by gas chromatography. Bioethanol production peaked at 72 h producing 0.7% bioethanol from 10 g/l xylose. In conclusion a β-xylosidase from Aspergillus niger was successfully expressed in P. pastoris and was found to express large quantities of xylosidase, that has not been achieved in any prior research to date. The enzyme was also successfully co-expressed with a Thermomyces xylanase and is now capable of bioethanol production through xylan hydrolysis. This highlights potential use in industrial applications in an effort to reduce the world dependence on petroleum and fossil fuels. However the technical challenges associated with commercialization of bioethanol production are still significant. / M

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

by Leung Sau-mei. / With: Two articles bound together subsequent to publication. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 101-108. / Abstract --- p.i / Acknowledgements --- p.iv / List of Abbreviations --- p.v / Table of content --- p.vi / Chapter Section 1 --- Introduction / Chapter 1.1 --- The phenomenon of host controlled restriction and modification in bacteria --- p.2 / Chapter 1.2 --- Classification of restriction and modification systems --- p.3 / Chapter 1.3 --- The nomenclature system for restriction and modification systems --- p.8 / Chapter 1.4 --- Variety of type II restriction and modification systems --- p.9 / Chapter 1.5 --- Properties of type II restriction endonucleases --- p.11 / Chapter 1.5.1 --- Biological function --- p.11 / Chapter 1.5.2 --- Protein structure --- p.12 / Chapter 1.5.3 --- Genetics --- p.16 / Chapter 1.5.4 --- Cleavage mechanism --- p.18 / Chapter 1.5.5 --- Factors affecting optimal activity --- p.21 / Chapter 1.6 --- Aim of study --- p.27 / Chapter Section 2 --- Materials and methods / Chapter 2.1 --- Screening for type II restriction endonucleases --- p.28 / Chapter 2.1.1 --- Sources --- p.28 / Chapter 2.1.2 --- Preparation of crude enzyme extract --- p.29 / Chapter 2.1.3 --- Assay of restriction enzyme activity --- p.30 / Chapter 2.1.4 --- Characterization of strains --- p.31 / Chapter 2.2 --- Purification of restriction endonucleases --- p.31 / Chapter 2.2.1 --- Preparation of column materials --- p.32 / Chapter 2.2.2 --- Purification of BcoI --- p.33 / Chapter 2.2.3 --- "Purification of Bcol0278I, BspI, Bsu8646I and PvuHKU" --- p.33 / Chapter 2.2.4 --- Purification of Bsu8565I and Pei9403I --- p.33 / Chapter 2.2.5 --- Purification of Sol3335I --- p.34 / Chapter 2.3 --- Characterization of discovered restriction endonucleases --- p.34 / Chapter 2.3.1 --- Determination of recognition specificity --- p.34 / Chapter 2.3.2 --- Determination of cleavage specificity of BcoI --- p.35 / Chapter 2.3.3 --- Unit definition --- p.36 / Chapter 2.3.4 --- Assays for ionic requirement and optimal temperature --- p.37 / Chapter 2.3.5 --- Heat inactivation --- p.37 / Chapter 2.4 --- Construction of Bacillus coagulans SM1 genomic library --- p.38 / Chapter 2.4.1 --- Preparation of chromosomal DNA --- p.38 / Chapter 2.4.1.1 --- Restriction digestion of chromosomal DNA --- p.40 / Chapter 2.4.2 --- Large scale preparation of vector pBR322 DNA --- p.43 / Chapter 2.4.2.1 --- Restriction digestion of vector DNA --- p.44 / Chapter 2.4.2.2 --- Preparation of lambda insert DNA for control tests --- p.45 / Chapter 2.4.3 --- Ligation of insert and vector DNA --- p.46 / Chapter 2.4.4 --- Transformation --- p.46 / Chapter 2.4.4.1 --- Preparation of electro-competent cells --- p.46 / Chapter 2.4.4.2 --- Electro-transformation --- p.47 / Chapter 2.4.5 --- Rapid screening for the presence of plasmid --- p.49 / Chapter Section 3 --- Results / Chapter 3.1 --- Presence of type II restriction endonucleases --- p.50 / Chapter 3.2 --- Strain identification --- p.50 / Chapter 3.3 --- Purification and properties of the discovered restriction endonucleases --- p.52 / Chapter 3.3.1 --- BcoI --- p.55 / Chapter 3.3.2 --- "Bco10278I, BspI, Bsu8646I and PvuHKUI" --- p.59 / Chapter 3.3.3 --- Bsu8565I and Pei9403I --- p.65 / Chapter 3.3.4 --- Sol3335I --- p.70 / Chapter 3.4 --- Construction of Bacillus coagulans SM1 genomic library --- p.73 / Chapter 3.4.1 --- Preparation of chromosomal DNA --- p.73 / Chapter 3.4.1.1 --- Generation of 4-10 kb insert fragments --- p.73 / Chapter 3.4.2 --- Preparation of plasmid DNA --- p.75 / Chapter 3.4.3 --- Ligation of insert and vector DNA --- p.76 / Chapter 3.4.4 --- Rapid screening for the presence of plasmid --- p.77 / Chapter Section 4 --- Discussion / Chapter 4.1 --- Screening of type II restriction endonucleases --- p.79 / Chapter 4.1.1 --- Methods for screening of type II restriction endonucleases --- p.79 / Chapter 4.1.2 --- Factors affecting the detection of restriction endonucleases --- p.83 / Chapter 4.2 --- Purification of the discovered restriction endonucleases --- p.87 / Chapter 4.3 --- Characterization of discovered restriction endonucleases --- p.91 / Chapter 4.3.1 --- Determination of recognition site --- p.91 / Chapter 4.3.2 --- Determination of cleavage specificity --- p.93 / Chapter 4.4 --- Characteristics of the discovered restriction endonucleases --- p.95 / Chapter 4.5 --- Molecular cloning of BcoI --- p.96 / Chapter 4.6 --- Future prospects --- p.99 / References --- p.101 / Appendix I --- p.109

by Wong Mee-wa. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 100-104. / Contents / Abstract / Abbreviations / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Biological Functions and Structure of GH --- p.1 / Chapter 1.3 --- Molecular Cloning of GH cDNA and its Expression --- p.3 / Chapter 1.4 --- Molecular Cloning of the Genomic Sequence of Growth Hormone --- p.5 / Chapter 1.4.1 --- The Isolation of Mammalian GH gene --- p.5 / Chapter 1.4.2 --- Molecular Cloning of Rainbow Trout Growth Hormone Gene --- p.6 / Chapter 1.4.3 --- Molecular Cloning of Atlantic Salmon Growth Hormone Gene --- p.9 / Chapter 1.4.4 --- Molecular Evolution of GH gene --- p.9 / Chapter 1.4.5 --- Control Elements on GH gene --- p.12 / Chapter 1.4.6 --- Production of Transgenic Fish --- p.14 / Chapter 1.5 --- Purpose of present Study --- p.15 / Chapter Chapter 2 --- Construction of a Genomic Library --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Strategy --- p.19 / Chapter 2.3 --- Materials and Methods --- p.20 / Chapter 2.3.1 --- Materials --- p.20 / Chapter 2.3.2 --- Procedure --- p.24 / Chapter 2.3.2.1 --- Extraction of Total Genomic DMA --- p.24 / Chapter 2.3.2.2 --- Southern Blotting and Hybridization --- p.25 / Chapter 2.3.2.3 --- Extraction of the GH Enriched DNA Fraction --- p.28 / Chapter 2.3.2.4 --- Cloning into Phage Vector Lambda GT11 --- p.29 / Chapter 2.3.2.5 --- Studies on the Enriched Genomic Library --- p.31 / Chapter 2.4 --- Result --- p.33 / Chapter 2.5 --- Discussion --- p.43 / Chapter Chapter 3 --- Screening --- p.46 / Chapter 3.1 --- Strategy --- p.46 / Chapter 3.2 --- Materials and Methods --- p.47 / Chapter 3.2.1 --- Materials --- p.47 / Chapter 3.2.2 --- Procedure --- p.48 / Chapter 3.2.2.1 --- Primary Screening --- p.48 / Chapter 3.2.2.2 --- Purificaiton of Positive Signal --- p.49 / Chapter 3.2.2.3 --- DNA Extraction from Positive Clone --- p.49 / Chapter 3.3 --- Result --- p.50 / Chapter 3.4 --- Discussion --- p.58 / Chapter Chapter 4 --- Studies on the Positive Clone --- p.59 / Chapter 4.1 --- Introduction --- p.59 / Chapter 4.2 --- Materials and Methods --- p.60 / Chapter 4.2.1 --- Materials --- p.60 / Chapter 4.2.2 --- Procedure --- p.62 / Chapter 4.2.2.1 --- Insert DNA Preparation --- p.62 / Chapter 4.2.2.2 --- Single Enzyme Digestion --- p.62 / Chapter 4.2.2.3 --- Double Enzyme Digestion --- p.62 / Chapter 4.2.2.4 --- Preparation of Position Specific Probes --- p.62 / Chapter 4.2.2.5 --- Hybridization with Position Specific Probes --- p.64 / Chapter 4.2.2.6 --- Preparation of Competent Cells --- p.65 / Chapter 4.2.2.7 --- Subcloning into pUC18 --- p.65 / Chapter 4.2.2.8 --- Plasmid Preparation --- p.66 / Chapter 4.2.2.9 --- Sequencing --- p.67 / Chapter 4.3 --- Result --- p.69 / Chapter 4.4 --- Discussion --- p.78 / Chapter Chapter 5 --- General Discussion --- p.80 / Appendix A --- p.83 / Appendix B --- p.93 / References --- p.100