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Development of a Cascade of Immobilized Enzymes for the Synthesis of Activated Sugars
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
2018 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Sugar nucleotides are crucial compounds in various biosynthetic pathways. However, their use in industry and research is limited due to high cost and low availability. Developing a process that synthesize sugar nucleotides at low-cost and large quantities is therefore essential. Enzymatic synthesis was shown to be a promising approach. However, enzyme stability and reusability should be improved to accomplish economically viable processes.In this work, sugar nucleotides were synthesized through one-pot multi-enzyme (OPME) regeneration cascades by the use of immobilized enzymes that were produced through epoxy immobilization. This synthesis intends to produce sugar nucleotides with economical and technical efficiency. Therefore, the objective of enzyme immobilization was to utilize low-cost and commercially available supports. Proteins were immobilized from cell lysates to avoid expensive protein purification. Product analysis indicated that side product formation can be neglected. Moreover, immobilized purified proteins achieved much lower productivity, compared to immobilized proteins from cell lysate.Reaction with enzymes immobilized on amino-epoxy support Relizyme HFA 403 achieved the highest productivity of UDP-Glucose compared to other immobilization supports based on UDP-sugar phosphorylase immobilization. The same support was tested on all enzymes from OPME and achieved effective product yield with respect to GlcNAc. In addition, investigation of various immobilization conditions was completed on UDP-sugar phosphorylase that catalyze reaction directly to the product to increase the product yield. Productivity was indeed improved and the same immobilization conditions were applied to other enzymes. However, the same trend was not observed for OPME cascade productivity.The steadiness of immobilized enzymes was tested through storage stability, reusability and thermal stability. After 30 days of storage immobilized enzymes retained 55 % of initial productivity. Their reusability was tested for eight 24 h reaction cycles. After eighth cycle around 10 % of initial activity remained. Thermal stability of immobilized enzymes attained low productivity at 40 ˚C and none at 50 ˚C. Compared to magnetic beads that were also investigated in this work epoxy immobilization exhibited higher productivity. In addition, cost estimation demonstrated that the price of the product synthesized with immobilized enzymes could be lower than the price of commercially available products.

Place, publisher, year, edition, pages
2018.
National Category
Natural Sciences Chemical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-236765OAI: oai:DiVA.org:kth-236765DiVA, id: diva2:1257721
Available from: 2018-10-22 Created: 2018-10-22 Last updated: 2018-10-22Bibliographically approved

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CiteExportLink to record
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  • apa
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