Optimization of cellulase CelA2 with improved performance in high ionic strength for the production of biofuels
Year: 2013
Type: dissertation
Abstract: Limited reserves of fossil fuels and environmental impact on CO2 emissions motivate the research and development of fuel alternatives produced from renewable bioresources. A key-step in biofuel production is the chemo-enzymatic depolymerization of cellulose in order to release fermentable sugars under mild conditions. However, physico-chemical pretreatments must be applied to increase the solubility of cellulose prior to enzyme degradation. The use of ionic liquids (IL) was reported to decrease the cellulose crystallinity thus increasing the availability of cellulose for enzymatic degradation. First generation of ionic liquids were expensive, not biodegradable and/or have a high environmental factor, thus deep eutectic solvents (DES) have emerged as an alternative solvent to ILs. DES show similar properties as ILs, with the advantage of being biodegradable and cost-effective. Furthermore, process economics can be improved by using non-purified effluents (e.g. concentrated seawater) for pretreatment in biorefineries. Regardless of the salts used (e.g. from seawater, ILs, or DES) it is challenging to find cellulases that operate efficiently under high salt conditions. Cellulase CelA2 was isolated from a biogas plant metagenome library and revealed an outstanding endoglucanase performance in ionic liquids. Therefore CelA2 was chosen as a suitable candidate for further improving the resistance and activity in salty effluents by a directed evolution campaign. For directed cellulase evolution, a novel fluorescence based microtiter plate screening system was developed. CelA2 variants with improved performance were identified from random mutagenesis libraries (epPCR), and further analysis showed that amino acid position 288 was the key residue for increased resistance and specific activity. In a subsequent directed evolution campaign, a novel CelA2 variant was identified, which showed a marginal activity compared to wild type, which dramatically increased by adding IL, DES or NaCl. Based on Molecular Dynamics analysis a potential mechanism for this salt-activation was proposed. Furthermore, a novel method for directed evolution was established, which inverts the classic MEGAWHOP strategy by introducing random mutations in the desired expression vector (epMEGAWHOP). This method can be used as a rapid and straightforward alternative for increasing protein expression in iterative rounds of epMEGAWHOP which is not limited by the enzyme type or expression vector.
Source: RWTH Publications (RWTH Aachen)
Authors: Christian Lehmann, Ulrich Schwaneberg
Language: English
Subfield: Biomedical Engineering
Field: Engineering
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W2265063942