Introduction:

Fructooligosaccharides (FOS) are soluble fibers with prebiotic effects in humans and animals. Inulin-type FOS [fructosyl-fructose β (2→1) linkages] are currently in the market, while levan-type FOS [fructosyl-fructose β 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) β-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.

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