Show simple item record

dc.contributor.authorPillay, Sarveshni
dc.date.accessioned2008-07-16T12:46:52Z
dc.date.available2008-07-16T12:46:52Z
dc.date.issued2007
dc.identifier.other308426
dc.identifier.urihttp://hdl.handle.net/10321/310
dc.descriptionThesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 vi, 92 leavesen_US
dc.description.abstractInterest 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.en_US
dc.language.isoenen_US
dc.subjectBiotechnologyen_US
dc.subjectXylanases--Biotechnologyen_US
dc.subjectEnzymes--Industrial applicationsen_US
dc.subjectProtein engineeringen_US
dc.subjectPolymerase chain reactionen_US
dc.subjectDNA polymerasesen_US
dc.subjectBiotechnology--Dissertations, Academicen_US
dc.titleImprovement of thermostability of a fungal xylanase using error-prone polymerase chain reaction (EpPCR)en_US
dc.typeThesisen_US


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record