PHYSIOCHEMICAL AND PROTEIN INTERACTION STUDIES OF ENDOLEVANASE MAGNETIC CROSS-LINKED ENZYME AGGREGATES FOR LEVAN-TYPE FRUCTOOLIGOSACCHARIDES PREPARATION

Abstract

The efficiency of cross-linked enzyme aggregates (CLEAs) is mainly affected by the strength and binding site of the formed linkages between the enzyme and cross- linker. Therefore, this study investigated the impact of intermolecular interaction and attachment site of different macromolecular cross-linkers, in generating CLEAs of endolevanase from Bacillus lehensis G1 (rlevblg1) for levan-type- fructooligosaccharides (L-FOS) production, through the combination of computational and experimental analysis. Due to the distanced bonding of dextran from the active site, rlevblg1 cross-linked with dextran (rlevblg1-dex-CLEAs) exhibited the highest binding affinity (-7.1 kcal/mol) and activity recovery compared to six other cross-linkers. The activity recovery of rlevblg1-dex-CLEAs continued to increase and reached 90.5% at the optimum condition. Subsequently, rlevblg1-dex- CLEAs was characterized and displayed higher thermal stability after 1 hour of incubation as well as greater storage stability over 180 days at 4 °C (60.9%) in comparison to the free enzyme. Moreover, rlevblg1-dex-CLEAs demonstrated higher affinity towards substrate with lower Km value (13.79 mM) than free enzyme (14.80 mM) and produced longer L-FOS (levanpentaose (DP5)). Meanwhile, the reusability of rlevblg1-dex-CLEAs was limited to five cycles due to enzyme leaching during separation process, which could be avoided by the formation of magnetic-CLEAs (M- CLEAs). However, undesirable interactions of the cross-linker and magnetic nano- particles (MNPs) on the enzyme surface would inhibit the recovery activity of M- CLEAs. Therefore, in silico analysis was exploited to predict the attachment site and design dextran derivatives with two different functional groups to perform site- directed cross-linking. Dextran with aldehyde group has attached far from the active site of rlevblg1 compared to dextran with acetyl functional group. Oxidized dextran (Odex) was developed and used to coat the MNPs externally. The coated-MNPs (OdexM) exhibited super-paramagnetic behavior (78.9 emu/g) and was applied to develop M-CLEAs of rlevblg1. The addition of OdexM offered easy separation of the aggregates from the reaction medium and activity recovery of 74.7%. Moreover, rlevblg1-OdexM-CLEAs exhibited acceptable mechanical stability and potential reusability for 10 cycles with more than 50% recovered activity up to the 5th cycle. The optimum temperature of rlevblg1-OdexM-CLEAs has shifted from 30 to 40 °C resulting in a significant improvement in the thermal stability which was 7.5-fold higher than free rlevblg1 after 1 h of incubation at 40 °C. After 8 min of incubation at 45 °C, the activity recovery of free rlevblg1 was completely lost, whereas rlevblg1- OdexM-CLEAs retained almost 20% activity recovery. In addition, compared to rlevblg1-dex-CLEAs, rlevblg1-OdexM-CLEAs demonstrated better structural flexibility, higher substrate affinity with lower Km value (7.32 mM), greater effectiveness factor especially at high concentration of levan as well as higher production of L-FOS. In conclusion, the use of computational analysis for suitable polysaccharides functionalization to coat the MNPs has proven to be a promising tool to construct mechanically stable M-CLEAs and develop robust biocatalysts for product synthesis.