Hu, Heng; (2024) Evaluation of the Performance of Immobilised Enzymes in Batch and Continuous Reactions. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Immobilised enzymes present a promising solution to enhance the stability and reusability of enzymes, streamline downstream processes, and minimise product contamination. Despite the existence of various methods for enzyme immobilisation, a comprehensive characterisation of these methods has been notably absent. This project seeks to address this gap by thoroughly comparing diverse methods and materials for enzyme immobilisation, establishing a framework for optimal technique selection. Evaluation encompassed five materials: PVA hydrogel, sol-gel, alginate, chitosan, and Ni-NTA agarose beads, assessing their physical and chemical characteristics. The performance of immobilised enzymes was scrutinised through two model reactions: inulin hydrolysis catalysed by inulinase and L-erythrulose (ERY) synthesis catalysed by transketolase (TK), both in batch and continuous reactions. The study determined the physical properties of various immobilisation materials to understand their resistance to external forces. Alginate beads emerged as the stiffest material,which showed a three times higher Young’s modulus than PVA and chitosan beads. Surface roughness analysis revealed PVA hydrogel beads had the highest and most variable porous struc- ture, while chitosan beads exhibited a uniform surface, and alginate beads presented a rougher texture compared to sol-gel. Assessment of immobilised enzyme performance in batch reactions included measurements of initial retained activity, storage and operational stability, and the influence of changes in reaction conditions. The results indicated that PVA hydrogel is the preferred material for inulinase immobil- isation, restoring 91.11 % of enzyme activity post-immobilisation, followed by chitosan at 89.56 %, which is 6-fold and 9-fold higher than sol-gel and al- ginate immobilised inulinase activity, respectively. Chitosan immobilised inulinase demonstrates good storage stability for 25 days, while PVA and sol-gel immobilised inulinase were inactive after two weeks of storage. Despite some enzyme leakage with chitosan beads, sol-gel is not recommended due to lower enzyme activity, poor storage stability, and non-recyclability. In TK- catalysed ERY, PVA immobilised TK outperforms Ni-NTA and chitosan, with an 85.93 ± 0.97 % yield and 42.36 ± 3.44 % retained activity, surpassing Ni- NTA and chitosan. Both PVA and chitosan exhibited similar 10-day storage stability and equal operational stability, with PVA demonstrating higher total production in repeated reactions. Hence, PVA hydrogel demonstrated remarkable efficiency for both inulinase and TK, retaining the highest activity. Despite challenges in maintaining bead shape, chitosan showed potential for long-term storage. In continuous reactions, PVA immobilised inulinase outperformed its counterparts using alginate and chitosan immobilisation, exhibiting higher production (28.98 mg, 5 times higher than alginate, and 9 times higher than chitosan), and the longest operational stability of 6 days. For TK immobilisation in continuous reactions, Ni-NTA agarose bead prefabricated columns achieved the highest production. Whereas PVA immobilised TK demonstrated the longest operational feasibility (72 hours) compared to chitosan (67.5 hours) and Ni-NTA (46 hours). Ni-NTA immobilised TK achieved the highest production (46.18 mM), followed by PVA (19.91 mM), and chitosan (6.26 mM). In conclusion, PVA hydrogel emerged as the preferred material for general enzyme immobilisation for both batch and continuous flow systems, with robust mechanical strength suitable for high-pressure applications, maintaining higher productivity than other materials. When affinity binding is possible for enzyme immobilisation, Ni-NTA affinity binding is the preferred method in continuous flow, leading to higher production.

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