Abstract
The effective and sustainable use of plant biomass for second generation biofuels is of vital importance for reducing dependence on fossil fuels. Carbohydrate-active enzymes (CAZymes) that degrade lignocellulosic plant cell wall material are an important part of this effort. CAZymes have multiple biological roles in plants and in addition to biofuel production they are extensively used in other industrial processes including in detergents, textiles, paper and the food industry. A vast repertoire of CAZymes exists in nature but there is a growing disparity between our ability to putatively identify new CAZymes and our ability to empirically characterise their activities. This is a serious hindrance for the optimal exploitation of their diversity and there is therefore a pressing need for the development of new high-throughput technology for CAZyme screening.
Here we describe the development of a new method for assessing CAZyme activities that is based on combining the multiplexing capacity of carbohydrate microarrays with the specificity of monoclonal antibodies and carbohydrate binding modules. The results presented in this thesis demonstrate that this new high-throughput semi-quantitative screening technique is sensitive, reproducible and robust. The possibilities of the method, regarding both substrates and enzymes, have been extensively explored, demonstrating its broad versatility. The catalytic activities of single enzymes, enzyme mixtures and crude culture broths can be assessed against single defined polysaccharides, mixtures of defined polysaccharides and complex biomass extracts. Furthermore, the capacity of the technique to analyse endo- and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases and lytic polysaccharide monooxygenases has been confirmed.
The applicability of the method to identify the substrate specificities of purified uncharacterised enzymes as well as for screening CAZyme activities in complex enzyme mixtures, such as crude culture broths and plant extracts, is shown by examples presented in this thesis.
We envisage that the method will contribute to both the discovery of CAZymes and the empirical characterisation of their activities, thus aiding their industrial utilisation and biological understanding
Here we describe the development of a new method for assessing CAZyme activities that is based on combining the multiplexing capacity of carbohydrate microarrays with the specificity of monoclonal antibodies and carbohydrate binding modules. The results presented in this thesis demonstrate that this new high-throughput semi-quantitative screening technique is sensitive, reproducible and robust. The possibilities of the method, regarding both substrates and enzymes, have been extensively explored, demonstrating its broad versatility. The catalytic activities of single enzymes, enzyme mixtures and crude culture broths can be assessed against single defined polysaccharides, mixtures of defined polysaccharides and complex biomass extracts. Furthermore, the capacity of the technique to analyse endo- and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases and lytic polysaccharide monooxygenases has been confirmed.
The applicability of the method to identify the substrate specificities of purified uncharacterised enzymes as well as for screening CAZyme activities in complex enzyme mixtures, such as crude culture broths and plant extracts, is shown by examples presented in this thesis.
We envisage that the method will contribute to both the discovery of CAZymes and the empirical characterisation of their activities, thus aiding their industrial utilisation and biological understanding
| Original language | English |
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| Publisher | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
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| Number of pages | 176 |
| Publication status | Published - 2015 |