Catalizadores enzimáticos novedosos para la producción de fructooligosacáridos a partir del azúcar de caña

Introduction: Fructooligosaccharides (FOS) are soluble fibers with prebiotic effects in humans and animals. Inulin-type FOS [fructosyl-fructose · (2·1) linkages] are currently in the market, while levan-type FOS [fructosyl-fructose · 2·6) linkages] are not commercially available. The work was attempted to produce modified enzymes capable to yield short-chain FOS with different types of linkages. Methods: The genes encoding the enzymes 1) ·-fructosidase (BfrA, EC 3.2.1.26) from the bacterium Thermotoga maritima and 2) sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from the plant Schedonorus arundinaceus were modified by directed mutagenesis and expressed constitutively in the yeast Pichia pastoris. Results. Three BfrA mutants (W14Y, W14Y-N16S and W14Y-W256Y) enhanced 4 fold the ratio of fructosylation/hydrolysis activities. The reaction with sucrose (1,75 M) yielded 37 % (w/w) FOS with a predominant composition of 6-kestose and neokestose. On the other hand, 1-SST synthesized 1-kestose and nystose in ratio 9:1, with their sum representing 55%-60 % (w/w) of total carbohydrates. The culture supernatants from the recombinant P. pastoris clones expressing either BfrA or 1-SST were submitted to ultrafiltration (concentration, dialysis) and lyophilization. The resultant water-soluble powders displayed high specific activity (> 8 000 U/g), high protein purity (> 50 %) and remained stable after 1-year storage at 4 oC. The enzymatic catalysts BfrA and 1-SST provide attractive alternatives for cane sugar conversion into short-chain FOS of the levan- and inulin-type, respectively.