Cheung, Thomas Kwun Long; (2025) Evaluation of enzyme addition on sucrose extraction during the sugar beet diffusion process. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Due to removal of the EU sugar quota, the UK sugar industry is currently interested in maximising the efficiency of their existing process chains in order to remain competitive in global markets. One opportunity is improvement of the existing diffusion process to increase the yield of sucrose produced per beet, reducing the amount of sugar lost to pulp. This thesis aims to investigate whether pectinases and cellulase have the potential to enhance sucrose extraction from sugar beets when applied to the existing diffusion process. To achieve this, this study focuses on the characterisation of several commercial enzymes in order to evaluate compatibility with the diffusion process, the development of a scale-down diffuser model to simulate industrial counter-current diffusion conditions, the expression of a novel, thermostable pectate lyase and the application of these enzymes in the diffusion process with the ultimate objective of enhancing sucrose extraction. Initial characterisation studies were performed on seven commercially available enzyme preparations, evaluating invertase activity, temperature activity and thermostability. From these studies, and in combination with industry data and literature reviews, Rohament CL, a broad-range fungal cellulase and Rohapect SY+, a pectin lyase, were shortlisted for experiments within the sugar beet diffusion process. These two enzymes did not contain any measurable invertase activity and, of the enzymes investigated, displayed the best activity and stability under the diffusion conditions. A counter-current, multi-stage diffusion model was then designed, built and tested to measure the effect of enzymes on the sugar beet diffusion process. This could accurately mimic performance of the standard diffusion process in terms of operating in a counter-current manner, however initial trials suggest no significant increase to the rate of sucrose extraction when Rohament CL, Rohapect SY+ or both enzymes were added to the diffusion process. Pre-treatment of sugar beet cossettes, via various processes, also did not enable any enzymatic enhancement to be observed. Due to the poor thermostability of the commercial enzymes, a thermostable pectate lyase, TMA14, was subsequently expressed and characterised for application in the diffusion process. This enzyme displays an optimum temperature activity profile at 90°C with significant activity at 70°C, and retains 99% stability over a 2 h duration at 70°C. Trials with TMA14 in the counter-current diffuser demonstrate no significant increase to rate of extraction, extraction ratio or pressability. In conclusion, the potential for existing commercially available enzymes to enhance sucrose extraction appears to be limited due to the already high efficiency of the existing diffusion process. Future work needs to address the development of enzymes with the required specificities and thermostabilities to work under industrial diffusion process conditions.

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