Baboo, JZ; (2012) Automated high-throughput approaches for the development and investigation of novel oxidative biocatalytic processes. Doctoral thesis , UCL (University College London).

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Abstract

Oxidative biocatalysts have a vast industrial and biotechnological potential in areas such as fine chemical and antibiotic synthesis. They offer an environmentally compatible and sustainable route to catalysis, often simpler and more specific than chemical alternatives. However, the routine use of biocatalysts in biopharmaceutical manufacture has been hindered by biocatalyst complexity and the experimental burden necessary for implementation. This thesis aims to investigate, using automated microscale technologies, how oxidative biocatalytic bioprocesses can be designed and developed at a reduced cost and timeframe compared to conventional laboratory scale experimentation. A robotic platform was used with 96-Deep square well microtiter plates to develop an effective bioprocess for investigating cyclohexanone monooxygenase (CHMO). E. coli cultivations for CHMO production, bioconversion, liquid-liquid metabolite extraction and analytic techniques were conducted using the developed microscale automated approach. Each step allowed rapid and reproducible collection of quantitative kinetic data over multiples runs achieving ‘walk away operation’. Whole bioprocess evaluation was achieved, whereby linking multiple unit operations enabled rapid assessment of process interactions. Factors influencing CHMO activity and bioconversion yields were investigated along with alternative bioconversion substrates. From identified limitations of the CHMO system an optimised process was developed where the processing time was almost halved and CHMO activity increased 5-fold. Two novel self-sufficient cytochrome P450 systems, P450SU1 and P450SU2 were investigated using an automated approach where factors limiting bioconversion were identified. Implementation of the required improvements resulted in a 5-fold improvement in enzymatic expression and 5-fold and 1.5-fold increase in product formation from cytochrome P450SU1 and P450SU2, respectively. A matched oxygen transfer coefficient approach was used for predictive scale-up. The optimised microscale CHMO and P450 processes were scaled to 75 L and 7.5 L bioreactor scale, respectively. Growth and bioconversion kinetics were found to be identical between scales for the CHMO system whereas differences were observed for the P450 systems. Results described in this thesis have demonstrated the benefits of microscale automated methodologies for the creation, investigation and predictive scale-up of oxidative biocatalytic bioprocesses. The established strategies evaluated in this work contribute to meeting the current demand to decrease developmental costs and timelines.

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