- Title
- Bio-prospecting a Soil Metagenomic Library for Carbohydrate Active Esterases
- Creator
- Shezi, Ntombifuthi
- ThesisAdvisor
- Pletschke, Brett
- ThesisAdvisor
- Rashamuse, Konanani
- ThesisAdvisor
- Mathiba, Kgama
- Date
- 2016
- Type
- Thesis
- Type
- Masters
- Type
- MSc
- Identifier
- vital:4172
- Identifier
- http://hdl.handle.net/10962/d1021266
- Description
- Lignocellulosic biomass is a promising renewable resource on earth. Plant biomass contains fermentable sugars and other moieties that can be converted to biofuels or other chemicals. Enzymatic hydrolysis of these biopolymers is significant in the liberation of sugars for fermentation into desired products. Owing to its complex structure, synergistic action of enzymes is required for its degradation. Enzymes that are involved in biomass degradation include cellulases, hemicellulases and the accessory enzymes acetyl xylan esterases and ferulic acid esterases. Ferulic acid esterases (FAEs, EC 3.1.1.73), represent a subclass of carboxylester hydrolases (EC 3.1.1.-) that catalyse the release of hydroxycinnamic acids (such as ferulic acid, p-coumaric, ferulic, sinapic and caffeic acid) that are generally found esterified to polysaccharides, such as arabinoxylans. Hydroxycinnamic acids have widespread potential applications due to their antimicrobial, photoprotectant and antioxidant properties, as well as their use as flavour precursors. Therefore, this interesting group of FAEs has a potentially wide variety of applications in agriculture, food and pharmaceutical industries. In the search for novel biocatalysts, metagenomics is considered as an alternative approach to conventional microbe screening, therefore, searching for novel biocatalysts from a soil metagenome that harbours a unique diversity of biocatalyst is significant. The aim of this study was to extract DNA from soil associated with cattle manure and construct a soil metagenomic library using a fosmid based plasmid vector and subsequently functionally screen for ferulic acid esterases using ethyl ferulate as a model substrate. A total of 59 recombinant fosmids conferring ferulic acid esterase phenotypes were identified (Hit rate 1:3122) and the two fosmids that consistently showed high FAE activities were selected for further study. Following nucleotide sequencing and translational analysis, two fae encoding open reading frames (FAE9 and FAE27) of approximately 274 and 322 aa, respectively, were identified. The amino acid sequence of the two ORFs contained a classical conserved esterase/lipase G-x-S-x-G sequence motif. The two genes (fae9 and fae27) were successfully expressed in Escherichia coli BL21 (DE3) and the purified enzymes exhibited respective temperature optima of 50 °C and 40 °C, and respective pH optima of 6.0 and 7.0. Further biochemical characterisation showed that FAE9 and FAE27 have high substrate specificity, following the fact that EFA is the preferred substrate for FAE9 (kcat/Km value of 128 s−1.mM-1) and also the preferred substrate for FAE27 (kcat/Km value of 137 s−1.mM-1). This work proves that soil is a valuable environmental source for novel esterase screening through functional based metagenomic approach. Therefore, this method may be used to screen for other valuable enzymes from environmental sources using inexpensive natural sources to encourage the screening of specific enzymes. Biochemistry of the two isolated enzymes makes these enzymes to be useful in industrial applications due to broad substrate activity that could replace the specialised enzymes to complete plant biomass degradation.
- Format
- 126 leaves, pdf
- Publisher
- Rhodes University, Faculty of Science, Biochemistry and Microbiology
- Language
- English
- Rights
- Shezi, Ntombifuthi
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RU Department of Biochemistry and Microbiology (2015-)
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