Identification and recombinant expression of a bacterial exolevanase useful for the production of high fructose syrups

The sugarcane endophyte Gluconacetobacter diazotrophicus produces levan from sucrose by a secreted levansucrase (LsdA). A levanase-encoding gene (lsdB) was identified starting 52 bp downstream of the lsdA gene. Recombinant expression of the lsdB gene in Escherichia coli and Pichia pastoris resulted in a functional protein capable of hydrolyzing levan and inulin to free fructose without the formation of oligofructans, indicating exo-type activity. LsdB was efficiently secreted into the P. pastoris culture medium driven by the Saccharomyces cerevisiae alphafactor signal peptide using either the methanol-inducible AOX1 or the constitutive GAP promoter. The recombinant protein was not glycosylated at its single potential N-glycosylation site. The GAP promoter-driven expression of the lsdB gene did not cause cell toxicity and provided for a three-fold higher productivity (26.6 U mL -1 ; 39 h fermentation) than the methanol-inducible system (21.1 U mL -1 ; 96 h fermentation). We conclude that the P. pastoris constitutive system provides a convenient alternative for the large-scale production and secretion of LsdB, an enzyme commercially attractive to convert polyfructans into high fructose syrups.