Levan-type fructooligosaccharides synthesis by a levansucrase-endolevanase fusion enzyme (LevB₁SacB)

We describe here the enzymatic production of levan type-fructooligosaccharides (L-FOS) with a DP from 2 to 10, through simultaneous synthesis and hydrolysis reactions. This was accomplished by LevB₁SacB, a new enzyme resulting from the fusion of SacB, a levansucrase from Bacillus subtilis and LevB₁, an endolevanase from B. licheniformis. In the fusion enzyme, SacB retains its catalytic behavior with a decrease in kcat from 164 to 108s^-1. LevB₁ in LevB₁SacB kinetic behavior improves considerably reaching saturation with levan and following Michaelis-Menten kinetics, quite differently from the previously reported first order kinetic behavior. We also report that LevB₁SacB or both enzymes (LevB₁ & SacB) at equimolar concentrations in simultaneous reactions result in an optimal, wide and diverse L-FOS profile, including 6-kestose, levanbiose and blastose among other L-FOS and 1-kestose, which accumulates as by-product of SacB levan synthesis. Yields of around 40% (w/w) were obtained from 600g/l sucrose with either LevB₁SacB or LevB₁ & SacB. The reaction was successfully scaled up to a stirred 2l bioreactor.