Sucrose is still widely used as a sweetener, but its high caloric value and caries-inducing effect have evoked a shift in consumer demand for healthier natural sweeteners. In such a direction, several sugar-processing companies in Asia, Europe and North America are actively marketing short fructans with a low degree of polymerisation (DP) commonly known as FOS (fructooligosaccharides). Fructans with a low DP (FOS) are natural low-caloric and non-cariogenic sweeteners with organoleptic properties very similar to sucrose. On the other hand, aspartame, saccharine and other artificial intense sweeteners, besides their undesirable cooling effect in mouth, have raised public concerns about their safety.

FOS, as other fructans, are prebiotic products since they are insensitive to digestive enzymes and are selective carbon substrates for Bifidobacteria, and most probably also Lactobacilli, the main representatives of the beneficial microflora resident in the intestinal tract of humans and animals. The proliferation of Bifidobacteria results in the competitive exclusion of the colonic pathogenic bacteria Escherichia coli, Clostridium ssp. and Salmonella ssp. In the gut, Bifidobacteria convert fructans into short chain fatty acids and lactic acids allowing important associated benefits to human and animal health such as: prevention of constipation, reduction of serum cholesterol, increase of calcium and magnesium absorption, prevention of colon cancer and production of B-vitamins. Commercial FOS consists essentially of 1-kestose (GFF, where G = glucose and F = fructose), nystose (GFFF) and fructosyl nystose (GFFFF). Of these,1-kestose has the highest sweetening strength. To date, they are produced on a commercial scale in industrial reactors from sucrose using a fungal fructosyl transferase.

On the other hand, bacterial polyfructans are mostly called levan (DP>10000) and, due to their high DP and good solubility in water, are specially preferred as emulsifiers or encapsulating agents in a wide range of industrial products as biodegradable plastics, cosmetics, glues, textile coatings and detergents. In addition, levan is attractive as a blood plasma volume extender. Finally it is a preferred substrate for the production of High Fructose Syrups (HFS), because of the very low glucose content. However, despite all these advantages levan is not yet commercialised at a significant scale since its industrial production from sucrose remains still very costly.

Gluconacetobacter diazotrophicus levansucrase (LsdA) catalyzes the synthesis of both, oligo and polyfructans by transferring fructosyl moieties from sucrose-containing saccharides to acceptor molecules. During the course of sucrose transformation fructooligosaccharides are accumulated particularly kestose and kesto­tetraose (55% of fructose transferred by the enzyme is accumulated as kestotriose and kestotretraose) however, the efficiency of this process is limited by two main issues: 1) the production of levan (10% of the sucrose are consumed in levan production (polymerization)) and 2) the transfer of fructosil residue to water (hydrolisis).

LsdA high yield of FOS, mainly 1-kestose and nystose, from its reaction on sucrose is commercially atractive. However, the secretion levels of this exoenzyme in the wild type or a genetically modified G. diazotrophicus strain remained rather low after optimization of culture conditions. Also, the presence of polysaccharides in culture supernatants discourages industrial-scale LsdA production using the natural host. The availability of this enzyme could be improved by using recombinant sources. LsdA was expressed in an active form in E. coli, but the protein accumulated intracellularly, hampering its purification. The methylotrophic yeast Pichia pastoris constitutes an excellent recombinant expression system. Most important among the advantages of using this yeastas a host for the production of fructosyltransferases are the absence of any sucrolytic activity, the availability of a strong methanol-induced alcohol oxidase 1 (AOX1) promoter, and the existence of an efficient protein secretion system which contrast with low levels (0.5%) of secreted native proteins. A P. pastoris strain harboring one copy of the lsdA expression cassette integrated in the genome resulted in the production and secretion of active enzyme.

Recombinant LsdA produced in yeast was glycosylated and displayed optimal pH and temperature for enzyme activity similar to native natural G. diazotrophicus levansucrase, however, thermal stability was increased. Neither fructosylpolymerase activity nor FOS production was affected.

Since there is not actually an industrial process for high scale FOS production based on a bacterial fructosyltransferase produced in yeast, in this paper we studied the influence of N-glycosylation on the kinetic and catalytical properties of LsdA produced in Pichia pastoris. We studied substrate specificity, fructoligosacharide production and transfructosylation reactions of the yeast expressed enzyme under different pH, temperatures and substrate concentrations. The equilibrium between hydrolase and synthase activities was determined by progressively substituting water by addition of NaCl or different organic solvents into the enzymes reaction mixture, at different temperatures and substrate concentrations. As a result we concluded that it is possible to increase polymerization rates in water restricted environnments increasing the yield of the desired products such as FOS or levan.