Engineered thermostable β–fructosidase from Thermotoga maritima with enhanced fructooligosaccharides synthesis

Abstract The thermostable β–fructosidase (BfrA) from the bacterium Thermotoga maritima converts sucrose into glucose, fructose, and low levels of short–chain fructooligosaccharides (FOS) at high substrate concentration (1.75 M) and elevated temperatures (60–70 °C). In this research, FOS produced by BfrA were characterized by HPAE–PAD analysis as a mixture of 1–kestotriose, 6 G –kestotriose (neokestose), and to a major extent 6–kestotriose. In order to increase the FOS yield, three BfrA mutants (W14Y, W14Y–N16S and W14Y–W256Y), designed from sequence divergence between hydrolases and transferases, were constructed and constitutively expressed in the non–saccharolytic yeast Pichia pastoris . The secreted recombinant glycoproteins were purified and characterized. The three mutants synthesized 6–kestotriose as the major component of a FOS mixture that includes minor amounts of tetra– and pentasaccharides. In all cases, sucrose hydrolysis was the predominant reaction. All mutants reached a similar overall FOS yield, with the average value 37.6% (w/w) being 3–fold higher than that of the wild–type enzyme (12.6%, w/w). None of the mutations altered the enzyme thermophilicity and thermostability. The single mutant W14Y, with specific activity of 841 U mg −1 , represents an attractive candidate for the continuous production of FOS–containing invert syrup at pasteurization temperatures.