Biochemical characterization of a novel α-L-fucosidase from Pedobacter sp. and its application in synthesis of 3′-fucosyllactose and 2′-fucosyllactose

Fucosyllactoses have gained much attention owing to their multiple functions, including prebiotic, immune, gut, and cognition benefits. In this study, human milk oligosaccharide (HMO) 2 '-fucosyllactose (alpha-L-Fuc-(1,2)-D-Gal beta-1,4-Glu, 2 ' FL) and its isomer 3 '-fucosyllactose (alpha-L-Fuc-(1,3)-D-Gal beta-1,4-Glu, 3 ' FL) with potential prebiotic effect were synthesized efficiently by a novel recombinant alpha-L-fucosidase. An alpha-L-fucosidase gene (PbFuc) from Pedobacter sp. CAU209 was successfully cloned and expressed in Escherichia coli (E. coli). The deduced amino acid sequence shared the highest identity of 36.8% with the amino sequences of other reported alpha-L-fucosidases. The purified alpha-L-fucosidase (PbFuc) had a molecular mass of 50 kDa. The enzyme exhibited specific activity (26.3 U/mg) towards 4-nitrophenyl-alpha-L-fucopyranoside (pNP-FUC), 3 ' FL (8.9 U/mg), and 2 ' FL (3.4 U/mg). It showed the highest activity at pH 5.0 and 35 degrees C, respectively. PbFuc catalyzed the synthesis of 3 ' FL and 2 ' FL through a transglycosylation reaction using pNP-FUC as donor and lactose as acceptor, and total conversion ratio was up to 85% at the optimized reaction conditions. The synthesized mixture of 2 ' FL and 3 ' FL promoted the growth of Lactobacillus delbrueckii subsp. bulgaricus NRRL B-548, L. casei subsp. casei NRRL B-1922, L. casei subsp. casei AS 1.2435, and Bifidobacterium longum NRRL B-41409. However, the growths of E. coli ATCC 11775, S. enterica AS 1.1552, L. monocytogenes CICC 21635, and S. aureus AS 1.1861 were not stimulated by the mixture of 2 ' FL and 3 ' FL. Overall, our findings suggest that PbFuc possesses a great potential for the specific synthesis of fucosylated compounds.