Computational analysis of the fructosyltransferase enzymes in plants, fungi and bacteria.

Fructosyltransferases (FTases) are enzymes produced by plants, fungi, and bacteria, which are responsible for the synthesis of fructooligosaccharides. In this study, we conducted a computational analysis of reported sequences for FTase from a diverse source of organisms, such as plants, fungi, and bacteria. Ninety-one proteins sequences were obtained; all belonging to the glycoside hydrolase 32 (GH32) and 68 (GH68) families. The sequences were grouped in seven clades, five for plants, one for fungi, and one for bacteria. Our findings suggest that FTases from fungi and bacteria likely evolved from dicotyledonous FTases. The analysis of catalytic domains A, D and E, which contain the amino acids involved in the catalytic binding site, allowed the identification of clade-specific conserved characteristics. The analysis of sequence motifs involved in donor/acceptor molecule affinity showed that additional sequences could be responsible for donor/acceptor molecule affinity. The correlation of this large set of FTases allowed to identify additional features that might be used for the identification and classification of new FTases, and to improve the understanding of these valuable enzymes.

Gene cloning and enzyme structure modeling of the Aspergillus oryzae N74 fructosyltransferase.

The fructooligosaccharides (FOS) represent an important source of prebiotic compounds that are widely used as an ingredient in functional foods. Recently, the strain Aspergillus oryzae N74 was reported as a potential microorganism for the industrial production of FOS, due to its high yields of FOS production. In this work, we used a PCR-cloning strategy to clone the A. oryzae N74 ftase gene as a previous step for recombinant enzyme production. Ftase showed a 1630 bp size with a 99% similarity with other A. oryzae strains and between 1 to 68% identities with other Aspergillus strains. This gene encodes for a 525 amino acids protein with 99% similarity with other A. oryzae strains and between 11 to 69% similarities with other Aspergillus strains. Finally, an A. oryzae N74 FTase tertiary structure model was predicted base on its similarity with other glycoside hydrolase 32 family members. The active site was located inside the ß-propeller domain and was formed for non-charged polar and charged amino acids. In summary, these results shows the high level of sequence conservation between A. oryzae strains and represent a first step towards the development of a FOS production industrial process using recombinant microorganism carrying the ftase gene from A. oryzae N74.

Recent trends in fructooligosaccharides production.

Prebiotics are food ingredients that promote host health beneficially due to their effect over the growth and activity of probiotic bacterial species. Prebiotic properties have been demonstrated for inulin-type fructans, galactoolicosaccharides and lactulose. Fructooligosaccharides (FOS), considered as inulin-type fructans, represent an important source of prebiotic compounds that are widely used as an ingredient in functional foods. FOS are produced by the action of fructosyltransferase from many plants, fungi and bacteria, and they are mainly composed of 1-kestose, nystose, and 1-beta-fructofuranosyl nystose. Among them, 1-kestose has better therapeutic properties than those with a high polymeric degree (GF(n > 4)). FOS exhibited properties than those with a high polymeric degree (GF(n>4)). FOS exhibited properties such as low caloric values, non-cariogenic properties, decrease levels of lipids and cholesterol, help gut absorption of ions, and stimulate the bifidobacteria growth in the human colon. This review presents a summary of the patents related with FOS production by industrial sucrose biotransformation or the use of recombinant fructosyltransferase enzymes. Also, a brief description of recent FOS applications will be discussed.