Frucooligosaccharides purification: Complexing simple sugars with phenylboronic acid.

Prebiotic fructooligosaccharides (FOS) are currently obtained by enzymatic reaction with fructosyltransferases (FTFs) using sucrose as both donor and acceptor. In these reactions glucose results as the most abundant by-product, arising from each fructosyl transfer event and, together with fructose, because of the inherent hydrolytic activity of the FTFs. As FOS are mainly used as prebiotic in nutraceutical foods, the reduction or total elimination of monosaccharides is required. In this work the selective elimination of monosaccharides from a synthetic FOS mixture was achieved through the selective complexation of glucose and fructose with phenyl boronic acid (PBAc) followed by ethyl-acetate extraction. The process was applied to a complex mixture of FOS obtained in an enzymatic synthesis reaction containing 40% glucose, 15.8% fructose and 35% of FOS, elimination of the sugars was achieved through 3:1 molar reactions, resulting in a levan-type FOS product with 97% purity.

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.

Enzymatic synthesis of poly-L-lactide and poly-L-lactide-co-glycolide in an ionic liquid.

The syntheses of poly-L-lactide (PLLA) and poly-L-lactide-co-glycolide (PLLGA) is reported in the ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate [HMIM][PF(6)] mediated by the enzyme lipase B from Candida antarctica (Novozyme 435). The highest PLLA yield (63%) was attained at 90 degrees C with a molecular weight (M(n)) of 37.8 x 10(3) g/mol determined by size exclusion chromatography. This procedure produced relatively high crystalline polymers (up to 85% PLLA) as determined by DSC. In experiments at 90 degrees C product synthesis also occurred without biocatalyst, however, PLLA synthesis in [HMIM][PF(6)] at 65 degrees C followed only the enzymatic mechanism as ring opening was not observed without the enzyme. In addition, the enzymatic synthesis of PLLGA is first reported here using Novozyme 435 biocatalyst with up to 19% of lactyl units in the resulting copolymer as determined by NMR. Materials were also characterized by TGA, MALDI-TOF-MS, X-ray diffraction, polarimetry and rheology.