Levan-type FOS production using a Bacillus licheniformis endolevanase

• β2-6 FOS are produced by endolevanase hydrolysis of enzymatically synthesized levan.
• Levanase from B. licheniformis has a low free fructose release from levan.
• β2-6 FOS include molecules from DP2 to 8, with levanobiose as the main product.
• β2-6 FOS are used effectively as carbon source by selected probiotic bacteria.

In the present work we describe an enzymatic production method to obtain β2-6 fructose oligosaccharides (levan-type FOS) through a sequential reaction in which a bacterial endolevanase is applied to levan produced from sucrose by bacterial levansucrases. A putative gene encoding an endolevanase, designated as LevBl, was identified through a bioinformatics search, isolated from a strain of Bacillus licheniformis IBt1 from our own collection and expressed in Escherichia coli. LevB1 showed a specific activity of 1.8 U/mg protein at 35 °C in 50 mM phosphate buffer pH 6.0. A first order kinetic behavior was found when up to 150 g/L of low molecular weight levan (8.3 kDa) was used as the substrate. The product profile was determined by HPAEC-PAD and consisted of levan-type FOS with a polymerization degree between 2 and 8, with levanbiose as the major product after long reaction times. Yields of 97% of levan-type FOS were obtained when 1.0 U/mL of LevB1 reacted with 100 g/L of levan produced by the levansucrase from Bacillus subtilis. Finally, it was observed that levan-type FOS are efficiently fermented by probiotic lactic acid bacteria.