Identification Of L-fucose Isomerase And D-arabinose Isomerase And Their Applications In Rare Sugar Production

L-Fuculose and D-ribulose are potentially valuable rare sugars used in food,agriculture,and medicine industries.These are pentoses and organized into aldo pentoses and ketopentoses,the two main groups.There are 8 aldo-and 4 ketopentoses,and only few are natural,while others are rare sugars found in a very small amount in nature.These sugars have great commercial applications,especially in many kinds of drugs in the medicine industry.The synthesis of these sugars is very expensive and difficult by chemical methods,so the enzymatic way of converting L-fucose into L-fuculose and D-arabinose into D-ribulose by isomerization would be an effective method with great industrial applications.L-Fucose isomerase(L-FI;E.C.5.3.1.25)and D-arabinose isomerase(D-AI;EC 5.3.1.3)belongs to the same family of enzymes with broad substrate specificities,having a similar function.Both enzymes have similar kinds of isomerizing mechanism,and each enzyme can catalyze both L-fucose and D-arabinose.Furthermore,the isomerization of L-fucose and D-arabinose by L-FI and D-AI from bacterial sources for the production of L-fuculose and D-ribulose have not yet become industrial due to the shortage of biocatalysts,resulting in poor yield and high cost of production.The pentose izumoring strategy offers a complete enzymatic tactic to link all kinds of pentoses by using different enzymes.In general,the enzymatic production of L-fuculose and D-ribulose through L-FI and D-AI is the inexpensive and uncomplicated method up till now.Firstly,L-FI,which is an aldose-ketose isomerase,plays a significant role in producing rare sugars.A recommended L-FI gene was cloned from Caldanaerobiuspolysaccharolyticus and purified with a single band of 65 kDa using nickel-affinity chromatography,with a specific activity of 108.23 U mg-1.The native molecular mass existed with 214 kDa was a trimer.The purified enzyme showed a maximum activity in 1 mM of Mn2+at 55℃ and pH 6.5 with a melting temperature(Tm)of 80.3℃ in the presence of one molecule per monomer.L-FI from C.polysaccharolyticus(Capo-Lflase)exhibited the highest activity of L-fucose with Michaelis-Menten constant(Km),the turnover number(kcat)and catalytic efficiency(kcat/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℃.The 9 putative active site residues of the L-fucose substrate were described using a homology model,and the results showed that Met185,Tyr440,Trp499,and Asn527 are the candidates of metal-binding residues,while Glu302,Glu337,Asp361,Ser393,and His528 would be involved in substrate binding.The conversion rate of L-fuculose from L-fucose was almost 28.2%,with 80 g L-1 of L-fucose,and no by-product was found.To the best of our knowledge,Capo-LfIase produces a high yield of L-fuculose from L-fucose by enzymatic methods.Secondly,a thermostable D-ribulose-and L-fuculose producing D-AI from the bacterium Thermanaeromonas toyohensis was characterized.The recombinant D-AI from T.toyohensis(Thto-DaIase)was purified with a single band at 66 kDa using His-trap affinity chromatography.The native enzyme existed as a tetramer with a molecular weight of 310 kDa,and the specific activities for both D-arabinose and L-fucose were observed to be 98.08 and 85.52 U mg-1,respectively.The thermostable recombinant Thto-Dalase was activated when 1 mM Mn2+was added to the reactions at an optimum pH of 9.0 at 75℃ and showed approximately 50%activity for both D-arabinose and L-fucose at 75℃ after 10 h.The Km,kcat,and kcat/Km for D-arabinose/L-fucose were 111/81.24 mM,18466/10688 min-1,and 166/132 mM-1 min-1,respectively.When the reaction reached to equilibrium,the conversion rates of D-ribulose from D-arabinose and L-fuculose from L-fucose were almost 27%(21 g L-1)and 24.9%(19.9 g L-1)from 80 g L-1 of D-arabinose and L-fucose,respectively.Thirdly,the production of L-fuculose was done from cheap and natural sources(fucoidan)and commercial source(Sigma-Aldrich)by a recombinant enzyme L-FI from Paenibacillus rhizosphaerae(Pa-LFI).Fucose containing polysaccharide(FPs)called fucoidan was extracted,hydrolyzed,and characterized by Undaria pinnatifida for enzymatic production of L-fuculose.The FPs provide 35.9%of fucose along with few other monosaccharides.Pa-LFI was characterized and purified with a single band at 65 kDa.It showed an activity of 104.5 U mg-1 and exhibited as a hexamer with native molecular mass 396 kDa.The maximum activity for recombinant Pa-LFI was detected at pH 6.5 and 50℃ in 1 mM of Mn2+.The Tm was observed at 75℃ and half-life at 50℃ was 12.6 h.The isomerizing activity of Pa-LFI with aldose substrate(L-fucose)was higher,exposing Km,kcat,and kcat/Km 86.2 mM,32831 min-1,and 335 min-1 mM-1 respectively.The conversion ratio of L-fuculose from 100 g L-1 of FPs and commercial fucose after the equilibrium state was about 6%(5.6 g L-1)and 30%(30.2 g L-1),respectively.Pa-LFI catalyzed the reaction to convert L-fucose into L-fuculose.The enzyme will be helpful in the production of L-fuculose with an efficient and simple method without producing by-products.