Characterization of the physicochemical properties of cross-linked levanase aggregates for levan-type fructooligosaccharides production

Levan-type fructooligosaccharide (L-FOS) are oligosaccharides that is in high demand in food-based and pharmaceutical industries and it can be produced from the levan hydrolysis. Recombinant levanase from Bacillus lehensis G1 (rlevblg1) is an enzyme that specifically converts levan to L-FOS. However, the use of free rlevblg1 presents a lack of stability and reusability, thus hinder the synthesis of L-FOS for continuous reactions. A carrier-free immobilization of cross-linked enzyme aggregates (CLEAs) were developed to overcome these drawbacks. However, low number of lysine residues of rlevblg1 may reduce cross-linking efficiency to form a stable and active biocatalyst. This issue can be solved by enzyme co-aggregation using additives. Moreover, the formation of CLEAs is also influenced by mass diffusion limitation as the degree of molecular cross-linking attained, significantly affects substrate accessibility especially at higher substrate concentrations. To address this problem, macromolecular cross-linker was used in the formation of CLEAs. In this study, formation of cross-linked levanase aggregates (CLLAs) was performed to improve stability and reusability of free rlevblg1. An active CLLAs using glutaraldehyde (CLLAs-GA), and with bovine serum albumin (CLLAs-GA-BSA) were obtained, and the factors affecting the formation of CLLAs were investigated. The highest activity recovery of CLLAs-GA (92.8 %; 169.5 U/mg) and CLLAs-GA-BSA (121.2 %; 221.3 U/mg) was achieved at optimized conditions. The optimum temperature of CLLAs-GA and CLLAs-GA-BSA increased to 35 °C and 40 °C, respectively, from 30 °C in its free rlevblg1. At high temperature (50 °C), the half-life of CLLAs-GA-BSA was higher than that of free rlevblg1 and CLLAs-GA. The reusability of CLLAs for 8 cycles was retained more than 50 % activity. The Vmax value of CLLAs-GA-BSA (21.97 U/mg) was increased by 14.3 % from the free rlevblg1 (19.23 U/mg). Dialdehyde starch-tapioca (DAST) was successfully developed and used to cross-link levanase to form CLLAs-DAST and CLLAs-DAST-BSA which showed activity recovery of 65.6 % (119.8 U/mg) and 81.6 % (149.0 U/mg), respectively. After DAST cross-linking, the pH and thermal stability increased, and the tolerance in organic solvents improved which resulted in an activation of CLLAs. A kinetic study revealed that CLLAs-DAST (16.72 mg/mL) and CLLAs-DAST-BSA (16.58 mg/mL) had higher affinity (Km) toward levan than that of CLLAs-GA (20.52 mg/mL) and CLLAs-GA-BSA (18.20 mg/mL). Thus, improving substrate accessibility with higher effectiveness factors especially at higher levan concentrations (10-12 mg/mL). The highest total L-FOS was achieved by CLLAs-DAST-BSA (78.9 % (w/v)), followed by CLLAs-DAST (62.4 %(w/v)), free rlevblg1 (51.2 % (w/v)), CLLAs-GA-BSA (50.1 % (w/v)) and CLLAs-GA (35.6 % (w/v)), after 3 h reaction. Although CLLAs formation using glutaraldehyde has produced an active and stable CLLAs, diffusion limitation at higher substrate concentrations reduced the L-FOS synthesis. In conclusion, DAST as a cross-linker may have application prospects as a promising and green biocatalyst for product formation such as L-FOS