Characterization Of A Novel D-lyxose Isomerase And Its Application On Enzymatic Production Of D-mannose

D-mannose,a kind of aldohexose,has promising physiological benefits such as anti-inflammation,prevention of the urinary tract infections,good prebiotic properties,and it can be used as the material for the synthesis of drugs and some functional substances,which has drawn much attention of the researchers.D-lyxose isomerase can not only calalyze the isomerization between D-lyxose and D-xylulose,but also the isomerization between D-fructose and D-mannose.This study was mainly focused on the characterization of recombinant Thermosedminibacter oceani D-lyxose isomerase and the production of D-mannose.And we also tried to construct a co-expression system to produce D-mannose with the cheaper material D-glucose.The gene encoding D-lyxose isomerase from T.oceani(protein ID: ADL08607.1)consisted of 546 bp nucleotides encoding 181 residues.The recombinant plasmid was constructed with pET-22b(+)as the expression vector and then transformed into E.coli BL21(DE3)strain for overexpression with IPTG as the inducer.The target enzyme was purified by Ni2+ affinity chromatography and the protein molecular weight on the SDS-PAGE displayed 22 kDa,which was in consistent with the calculation.And the result from gel filtration chromatography analysis was approximiately 44 kDa,indicating the recombinant enzyme a homodimer.It displayed maximal activity in presence of 1 mM Mn2+ at pH 6.5 and 65oC,and was determined to be highly thermostable.The specific activity,Km,kcat and catalytic efficiency(kcat/Km)for D-fructose were 5.3 ± 0.1U/mg,27.3 ± 1.3 mM,1,030 ± 26 min-1,38 ± 1 mM-1 min-1,respectively.Under optimal condition,26.8% D-mannose was produced from 100 g/L D-fructose by a 4 U/mLD-lyxose isomerases fromT.oceani after reaction for 2 h.The gene encoding D-glucose isomerase from Acidothermus cellulolyticus 11B(protein ID: ABK53836.1)consisted of 1224 bp nucleotides encoding 407 residues.A co-expression system including the D-lyxose isomerase gene and D-glucose isomerase gene was constructed using pETDuet-1 as the expression vector and then the plasmid was transformed into E.coli BL21(DE3)strain for overexpression with IPTG as the inducer.The protein molecular weight on the SDS-PAGE displayed 22 kDa and 42 kDa,which agreed with the calculation.The maximal activity of the co-expression system was observed at pH 6.5 and 65oC,and Co2+ can improve the enzyme activity most.Under optimal condition,16.4% D-mannose was produced from 100 g/L D-glucose by 100 g/L wet cells after reaction for 2 h.