Development and Characterization of an Enzyme Membrane Reactor for Fructo-Oligosaccharide Production

Abstract

Fructo-oligosaccharides (FOS) are linear fructans comprising 2–5 fructose units linked to a terminal glucose residue. They are widely used as food and feed additives due to their sweetness, low calorific value, and prebiotic properties. Here we describe the synthesis of FOS catalyzed by a cell-free crude enzyme solution containing recombinant fructosyltransferase (1-FFT) produced in the yeast Kluyveromyces lactis. During the enzyme catalysis, glucose accumulates as a by-product and eventually inhibits FOS production. We therefore used an enzyme membrane reactor (EMR) to achieve the continuous removal of glucose and the simultaneous replenishment of sucrose. We observed a loss of flux during the reaction with the characteristics of complete pore blocking, probably caused by a combination of proteins (enzyme molecules) and polysaccharides (FOS). Such complex fouling mechanisms must be overcome to achieve the efficient production of FOS using EMR systems.

Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing

“…But this represents a decrease of 26.5% when both CDWs are taken into account, suggesting that both enzymes in the crude enzyme solution show transferase and hydrolysis activity. Because higher glucose concentrations can inhibit the transferase activity of the enzymes, systems such as enzyme membrane reactors may be needed for further process development so that the residence time of the enzymes can be adjusted to further optimize the transferase/hydrolysis ratio (Erdos et al, 2017;Burghardt et al, 2019b;Fan et al, 2020).…”

Process Design for the Production of Prebiotic Oligosaccharides in an Enzyme Membrane Bioreactor: Interaction between Enzymatic Reaction and Membrane Filtration

“…In an EMBR system, this parameter is limited by membrane filtration performance, which is highly dependent on the choice of membrane and operational conditions. Our previous investigations showed that the efficiency of the EMBR system gradually declined during the long‐term production of FOS, reflecting the loss of enzyme activity due to enzyme leakage through the membrane, deactivation of the enzyme by shear stress, and the formation of sugar‐protein complexes via the Maillard reaction 16, 17. These issues were addressed in the current investigation by selecting an appropriate membrane and suitable operational conditions.…”

“…The protein composition of the feed and permeate solutions was determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) as previously described in [16].…”

“…The concentration of each component in the FOS solution was determined by ultra-high-performance liquid chromatography (UHPLC) as previously described in [16].…”

Purification of Crude Fructo-Oligosaccharide Preparations Using Probiotic Bacteria for the Selective Fermentation of Monosaccharide Byproducts

“…The general formula is GF n and specific examples include 1-kestose (GF 2 ), nystose (GF 3 ), and 1F-fructofuranosylnystose (GF 4 ; Kovács et al, 2014 ; Ur Rehman et al, 2016 ). FOS can be obtained directly from natural sources like asparagus, garlic, chicory, onion and banana, or can be enzymatically produced from sucrose using fructosyltransferases ( Jaime et al, 2001 ; Singh et al, 2017 ; Burghardt et al, 2019a , b ). Enzymatically produced FOS are preferred by industry because the chain length can be controlled precisely by regulating the reaction conditions and duration.…”