Immobilization and Characterization of E. gracilis Extract with Enriched Laminaribiose Phosphorylase Activity for Bienzymatic Production of Laminaribiose

Abstract

Immobilization methods and carriers were screened for immobilization of Euglena gracilis extract with laminaribiose phosphorylase activity. The extract was successfully immobilized on three different carriers via covalent linkage. Suitable immobilization carriers were Sepabeads EC-EP/S and ECR 8209M with epoxy groups and ECR 8309M with amino groups as functional units. Immobilization on Sepabeads EC-EP/S resulted in highest retained activity (65%). The immobilizates were characterized for pH, temperature, and buffer molarity preferences. The immobilized enzyme lost 48% of its activity when used seven times. Together with sucrose phosphorylase, laminaribiose phosphorylase was successfully applied for bienzymatic production of laminaribiose from sucrose and glucose with a final laminaribiose concentration of 14.3 ± 2.1 g/L (20% yield).

Continuous enzymatic production and adsorption of laminaribiose in packed bed reactors

“…LP and SP were immobilized through a hybrid-immobilization procedure developed in previous studies following a two-pearl concept [6,7,10]. In this procedure, a covalent linkage on Sepabeads (EC-EP/S with 0.1-0.3 mm particle size) was performed first.…”

“…But glucose is needed for start of the reaction and kinetics requirements and for that reason, glucose is added at the beginning of the reaction [5]. Therefore, a bienzymatic reaction system using LP and SP immobilized on Sepabeads and encapsulated in chitosan with sucrose and glucose as substrates was applied to produce laminaribiose under mild conditions [6,7]. Although diffusion in chitosan is significantly slower than free diffusion [8], the impact on overall reaction rate is small [9] and thermal stability of chitosan-based hybrid immobilizates is increased by a factor of 10 compared to native enzymes [7].…”

Glycan Phosphorylases in Multi-Enzyme Synthetic Processes

“…Apart from the one-pot enzymatic approach in solution, immobilised-enzyme combinations can also be explored as an alternative for the use of disaccharide phosphorylases as biocatalysts. One recent example shows that production of laminaribiose could be accomplished by a bi-enzymatic system containing individually immobilised Euglena gracilis protein extract with laminaribiose phosphorylase activity (LP, EC 2.4.1.31) and recombinant SP onto Sepabeads (Figure 6C ) [ 42 ]. The advantages of enzyme immobilisation are the extension of operational life time and reusability of the enzymes, which were explicitly demonstrated in Müller’s work, where the immobilised laminaribiose phosphorylase retained more than 50% activity when used seven times, providing a proof-of-concept that could potentially be implemented for other phosphorylases.…”

“…BiGLNBP = GNB/LNB phosphorylase from Bifidobacterium infantis ; BiGalK = galactokinase from Bifidobacterium infantis ; NmCSS = CMP-Sia synthetase from Neisseria meningitides ; PmST1 = α-(2→3)-sialyltransferase 1 from Pasteurella multocida ; Pd2,6ST = α-(2→6)-sialyltransferase from Photobacerium damselae. C ) Combination of immobilised LP from Euglena gracilis and SP can be used for the production of laminaribiose using sucrose as a starting material [ 42 ]. SP = sucrose phosphorylase; Pi = inorganic phosphate; LP = laminaribiose phosphorylase.…”

Diffusion Studies of Glucose and Sucrose in Chitosan Membranes and Beads for Enzymatic Production Processes

“…It has also been used for the encapsulation of sucrose phosphorylase (SP) and laminaribiose phosphorylase (LP) in chitosan hydrogels to produce the rare sugar laminaribiose . There, the encapsulation of SP and LP in chitosan after an immobilization on Sepabeads increased the stability of the enzymes by one order of magnitude compared to the immobilization on Sepabeads only , . This increase in stability of enzymes is a well‐known advantage of encapsulation in chitosan together with its antibacterial activity .…”