The preparation of two immobilized levansucrase biocatalysts and their application for the synthesis of lactosucrose. (November 2022) - Bahlawan, Rami
Abstract: The synthesis of novel prebiotics has gained attention due to a greater interest in intestinal health. In this study, lactosucrose was produced by levansucrase (LS, EC 2.4.1.10) catalyzing the transfructosylation of sucrose and lactose with whey (WP) and milk permeate (MP) as lactose sources. Selected functionalized supports were investigated for the immobilization of LSs from Gluconobacter oxydans (LS1) and Vibrio natriegens (LS2) by heterofunctional attachment. The highest immobilization protein yields of 94 % and 87 % and retained activities of 55 % and 98 %, respectively, were achieved upon the immobilization of LS1 and LS2 on Relizyme TM EP403/S functionalized with iminodiacetic acid (IDA)-cupric ions (Cu2+). The immobilization of LSs enhanced the reaction selectivity towards transfructosylation. Moreover, post-immobilization treatments (alkaline pH incubation, polyethylenimine (PEI) cross-linking) impacted the enzyme activity of the immobilized enzymes resulting in lower retained activities (11–74 %) but allowed them to achieve a higher thermal stabilization (up to 53 times increase), particularly with the high pH treated immobilized V. natriegens LS2. The identified optimal conditions for lactosucrose synthesis with high selectivity by free and immobilized Relizyme TM EP403/IDA-Cu immobilized LS2 resulted in 117 and 101 g/L, respectively, using WP as a lactose source. Lastly, the Relizyme TM EP403/IDA-Cu immobilized LS2 was successfully reused 3 consecutive times. Graphical Abstract: ga1 Highlights: Levansucrases (LSs) showed higher efficiency for transfructosylation bioconversion upon immobilisation. Relizyme™ EP403 support functionalized with IDA-Cu provided the highest immobilization efficiency of LSs. Post-immobilization treatments (high pH & PEI crosslinking) provided further thermal stabilization of LSs. The highest bioconversion of dairy by-products into lactosucrose was achieved with immobilized V. natriegens LS2. Reaction time and substrate ratio were significant in modulating bioconversion of whey permeate into lactosucrose.