Rational design and immobilization of a recombinant sucrose: Sucrose 1-fructosyltransferase on Sepabeads® and ReliZyme™ supports for short-chain fructooligosaccharides production

Abstract

The recombinant sucrose:sucrose 1-fructosyltransferase from Schedonorus arundinaceus (Sa1-SSTrec) produces short-chainfructooligosaccharides (scFOS). In this work, Sa1-SSTrec was covalently or hydrophobic immobilized on Sepabeads® and ReliZyme™ supports. The possible clusters and the parameters related to the immobilization process were predicted by rational design of immobilized derivatives (RDID). Immobilization was performed at pH 7.0 and the three immobilized derivatives with higher experimental enzymatic activity (one of each epoxy-activated, amino-activated and hydrophobic supports: Sepabeads® EC-HFA/S, ReliZyme™ EA403/M and Sepabeads® EC-BU/M, respectively) were selected. The mismatch between some predictions and experimental results were explained by probable limitations of the RDID strategy. The thermodynamic parameters determined at 40, 50 and 60ºC, under not reactive conditions (i.e. in absence of sucrose), showed high thermal stability of Sa1-SSTrec after immobilization on the Sepabeads®EC-HFA/S. Batch experiments with this support, at 45ºC reached similar total scFOS (1-kestotriose and 1,1-kestotetraose) than the soluble enzyme, but scFOS productivity was higher for the immobilized biocatalyst. Operational stability of the immobilized biocatalyst after 4 cycles dropped to about 60% and 25% of its initial value at 30 and 45ºC, respectively, probably by denaturation at these temperatures. The obtained results suggest that the immobilized enzyme could be effectively exploited for industrial production of 1-kestotriose.