Vinyl sulfone-activated silica for efficient covalent immobilization of alkaline unstable enzymes: Application to levansucrase for fructooligosaccharide synthesis

[EN] Most methodologies for covalent immobilization of enzymes usually take place at high pH values to enhance the nucleophilicity of protein reactive residues; however, many enzymes inactivate during the immobilization process due to their intrinsic instability at alkaline pH values. Vinyl sulfone (VS)-activated carriers may react with several protein side-chains at neutral pHs. In this work, levansucrase-an alkaline unstable enzyme of technological interest because it forms fructooligosaccharides (FOS) and levan from sucrose-was covalently attached to VS-activated silica at pH 7.0 in a short time (5 h). Theoretical immobilization yields were close to 95% but the apparent activity did not surpass 25%, probably due to random attachment with unproductive orientations and rigidification of the enzyme structure. Due to diffusional hindrance and/or local microenvironmental effects caused by the silica surface, the immobilized levansucrase was unable to produce levan but synthesized a similar amount of FOS than the free enzyme [95 g L in 28 h, with a major contribution of FOS of the β(2 → 1) type]. The VS-activated biocatalysts showed a notable operational stability in batch reactors.
Spanish Ministry of Economy and Competitiveness (BIO2013-48779-C4-1-R). We thank the support of COST-Action CM1303 on Systems Biocatalysts. P. S.-M. thanks the Spanish Ministry of Education for FPU Grant (FPU13/01185).