Functional production and secretion of the Gluconacetobacter diazotrophicus fructose-releasing exo-levanase (LsdB) in Pichia pastoris

The gene encoding the fructose-releasing exo-levanase (LsdB; EC 3.2.1.65) from Gluconacetobacter diazotrophicus SRT4 was expressed in Pichia pastoris using either the methanol-inducible AOX1 or the constitutive GAP promoter. In both systems, the recombinant LsdB was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae alpha-factor signal peptide. The levanase activity reached 21.1 U ml−1 in the culture supernatant of methanol-induced cells grown for 96 h to a final density of 115.2 g l−1 (dry weight) under fed-batch conditions. The GAP promoter-driven expression of the lsdB gene did not cause cell toxicity and provided for a higher LsdB yield (26.6 U ml−1; 0.46 g l−1 ) despite the fermentation time was only 39 h and the biomass reached 59.7 g l−1 (dry weight). The constitutively produced LsdB containing a C-terminal His6-tag fusion was purified to homogeneity from the culture supernatant by nickel affinity chromatography with a process recovery of 77.3%. The purified enzyme, which was not glycosylated at its single potential N-glycosylation site, showed a maximal specific activity (58 U mg−1) for the substrate levan at pH 5.0 and 30 °C. The enzyme also hydrolyzed inulin, raffinose, and sucrose, but not melezitose. The reaction on levan and inulin resulted in the successive release of free fructose without the formation of intermediate oligofructans. We conclude that the P. pastoris GAP promoter based 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.