Theses and dissertations (Applied Sciences)
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Item Directed evolution of B-xylanase from Thermomyces lanugtnosus(2000) Stephens, Dawn Elizabeth; Permaul, Kugen; Prior, A.; Singh, SurenMost natural enzymes may be unsuitable for biotechnological processes since they have evolved over millions of years to acquire their specific biological functions. Such enzymes are often genetically altered to suit the rigours of industrial processes. Directed evolution is one such strategy and makes use of iterative rounds of random mutagenesis, screening and recombination to enhance the existing properties of enzymes. Thermomyces lanuginosus is a thermophilic fungus that produces high levels of a thermostable xylanase. The xylanase gene from T lanuginosus DSM 5826 (xynA) was functionally expressed in E. coli as a LacZ-fusion protein (Schlacher et al., 1996) and later crystallized (Gruber et al., 1998). In this study, it was undertaken to improve the thermo stability and catalytic activity of xynA using error-prone PCR with different concentrations of MnCh. The first step prior to mutagenesis was to determine the levels of xylanase that could be attained by the wild type XynA, both in the presence and absence of an inducer. IPTG, a lactose analogue, was used since xynA was expressed with a lac promoter. High amounts of IPTG were found to adversely affect xylanase production, whilst a low amount (0.1 mM) enhanced xylanase production. This amount was used to later induce xylanase production by the variants obtained after mutagenesis. IPTG was found to increase the rate and production of xylanase. After random mutagenesis of xynA, transformed colonies were first selected for xylanase production on 0.4% Remazol Brilliant Blue xylan and then screened at different temperatures for improved stability and activity. After the first round of screening, four variants, viz., IB5, IB7, IBLl and ID2, showed slight improvement in both stability and activity and were subjected to further mutagenesis, using low concentrations of MnCh. Three variants, viz., 2B7-1O, 2B7-6 and 2BIl-16, with markedly enhanced stability, were obtained. Variant 2B7-10 exhibited a five fold higher activity (3430 nkat/ug total protein) than the wild type XynA (657 nkatl ug total protein). It retained 71% of its activity after treatment at 80°C for 60 min and had a t1/2of 215 min at 70°C, which is higher than that attained by XynA. Long-term thermo stability screening at 70, 80, 90 and 100°C revealed that variants 2B7-6 and 2B11-16 were, however, the most stable enzymes generated in this study, although their activities were lower or almost comparable with their parents. Sequence analysis of variant ID2 revealed 4 amino acid substitutions within the a-helix of the protein. This region was strongly conserved with the more stable variant xylanases generated in this study. The most profound mutation seen with variant 2B7-10 was the disruption of the disulphide bridge. Most of the mutants obtained in this study displayed a trade-off between stability and activity, the exception being mutant 2B7-10. Currently, DNA shuffling techniques are being used to recombine these traits in a single xylanase.Item Protein engineering of fungal xylanase(2007) Stephens, Dawn ElizabethProtein engineering technologies, such as directed evolution and DNA recombination, are often used to modify enzymes on a genetic level for the creation of useful industrial catalysts. Pre-treatment of paper pulps with xylanases have been shown to decrease the amounts of toxic chlorine dioxide used to bleach pulp. This study was undertaken to improve the thermal and alkaline stabilities of the xylanase from the fungus Thermomyces lanuginosus using ep-PCR and DNA shuffling.