Cross-linked enzyme aggregates (combi-CLEAs) derived from levansucrase and variant inulosucrase are highly efficient catalysts for the synthesis of levan-type fructooligosaccharides

Abstract

Efficient and convenient access to short to medium chain length levan-type fructooligosaccharides (LFOS) is needed in order realise the nutritional potential of this class of bioactive oligosaccharides. While LFOS are synthesised by fructansucrase enzymes, these reactions are routinely associated with high molecular weight fructan polymer formation. Recent studies have shown that FOS production can be enhanced by the combination of levansucrase and inulosucrase in a one-pot reaction. In the present study, the novel mixed enzyme cross-linked enzyme aggregates (combi-CLEAs) based on levansucrase (Lev) and N543A variant inulosucrase (Inu) were prepared by ammonium sulfate precipitation, followed by glutaraldehyde cross-linking. The effect of Lev and Inu ratio on the activity of combi-CLEAs was explored. The results showed that >70% of total sucrase activity was recovered after immobilization and that the combi-CLEAs produced high amounts of LFOS (degree of polymerisation 3 to 21), while high molecular weight polysaccharide production was much reduced. Biochemical characterisation indicated that the optimum pH and temperature of combi-CLEAs (pH 5.5 and 50ᵒC, respectively) were comparable to those of free enzyme; however, the stability of the enzyme was improved. In addition, these combi-CLEAs have operational stability for several reaction cycles, which makes them very attractive for biotechnology applications.