Biochemical characterization of recombinant L-fucose isomerase from Caldanaerobius polysaccharolyticus for L-fuculose production

L-fuculose is a rare sugar that is useful for the agriculture and medicine industries. L-fucose isomerase (E.C.5.3.1.25), which is an aldose-ketose isomerase, plays a significant role in producing rare sugars. A recommended L-fucose isomerase gene was cloned from Caldanaerobius polysaccharolyticus and purified with a single band of 65 kDa using nickel-affinity chromatography, with a specific activity of 10823 U mg-1. The native molecular mass existed with 214 kDa was a trimer. The purified enzyme showed a maximum activity in 1 mM Mn2+ at 55 C and pH 6.5 with a melting temperature (T,) of 80.3 C in the presence of one molecule per monomer. L-fucose isomerase from C polysaccharolyticus (Capo-Lflase) exhibited the highest activity of L-fucose with Km, kca, and Kat/km values of 94.2 mM, 23854 min -1 and 253.3 min -1 mM-1, respectively. Capo-Lflase showed more than 50% thermostability after 20 h of incubation at 45, 55, 65, 75 and 85 C. The 9 putative active site residues of the L-fucose substrate were described using a homology model, and the results showed that Tyr440, Met185, Trp499 and Asn527 are the candidates of metal -binding residues, while Ser393, G1u337, Glu302, His528 and Asp361 would be involved in substrate binding. The conversion rate of L-fuculose from L-fucose was almost 28.2%, with 80 g L-1 L-fucose, and no byproduct was found. To the best of our knowledge, CapoLflase produces high yield of L-fuculose from L-fucose by enzymatic methods. (C) 2019 Elsevier B.V. All rights reserved.