초록
<P><B>Abstract</B></P> <P>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 LevB<SUB>1</SUB>SacB, a new enzyme resulting from the fusion of SacB, a levansucrase from <I>Bacillus subtilis</I> and LevB<SUB>1</SUB>, an endolevanase from <I>B. licheniformis.</I> In the fusion enzyme, SacB retains its catalytic behavior with a decrease in kcat from 164 to 108s<SUP>−1</SUP>. LevB<SUB>1</SUB> in LevB<SUB>1</SUB>SacB 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 LevB<SUB>1</SUB>SacB or both enzymes (LevB<SUB>1</SUB> & 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 LevB<SUB>1</SUB>SacB or LevB<SUB>1</SUB> & SacB. The reaction was successfully scaled up to a stirred 2l bioreactor.</P> <P><B>Highlights</B></P> <P> <UL> <LI> SacB synthesizes a fructan polymer which may be simultaneously hydrolyzed by LevB1. </LI> <LI> SacB and LevB1 behave efficiently as a new enzyme in the single protein LevB1SacB. </LI> <LI> Levan FOS structures with DP from 2 to 10 are synthesized by the new fusion enzyme. </LI> <LI> The levan-type FOS include levanbiose, blastose and 6-kestose as main products. </LI> </UL> </P>