Fructooligosaccharide production by a truncated Leuconostoc citreum inulosucrase mutant

Abstract

Site-directed mutagenesis was performed on IslA4, a truncated form of inulosucrase (IS) derived from Leuconostoc citreum IS that contains only the IS catalytic domain. This truncated form is more hydrolytic than the wild-type enzyme and produces both high-molecular-weight inulin and fructooligosaccharides (FOS). Among the various mutants obtained from IslA4 by following strategies designed for SacB (the levansucrase from Bacillus subtilis), S425A no longer produces inulin, but instead produces FOS exclusively and hydrolyzes sucrose. Reaction conditions were explored to increase FOS productivity by reducing the hydrolysis, resulting in 65% conversion from a 0.67 M sucrose solution. S425A displays complex kinetic behavior in which the transfructosylation rate is described by first-order kinetics, while sucrose hydrolysis follows Michaelis–Menten behavior. This combined model correctly describes both the overall initial reaction rate as well as the reaction evolution for FOS synthesis. S425A IS may be useful for the synthesis of FOS from sucrose.

Keywords::

• Leuconostoc citreum
• fructosyltransferases
• fructooligosaccharides
• inulosucrase
• site-directed mutagenesis

Acknowledgements

This project was financed by Consejo Nacional de Ciencia y Tecnología (CONACyT – Morelos, FOMIX MOr-2007-C01-80360 and Ciencia Básica no. 81637). We thank Fernando gonzález and Mitsue León for technical assistance and Alfonso Miranda for support in NMR analysis of the fructosides.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This paper was first published as an Early Online Article on 24 November 2009.