Characterization Of D-Lyxose Isomerase And Its Application In The Monosaccharide Biotransformation

D-Mannose is the epimer of D-glucose at the C-2 position,as well as the aldose isomer of D-fructose.It has prebiotic effect and has a role in T cell activation.D-Mannose can also be used as the substrate to produce D-mannitol.L-Ribose,the enantiomer of D-ribose,is a kind of rare sugar.It is a key intermediate for the synthesis of L-nucleoside-based antiviral agents against human immunodeficiency virus,cytomegalovirus and hepatitis virus.Thus,the industrial demand for D-mannose and L-ribose is gradually increasing.D-Lyxose isomerase?D-LI,EC 5.3.1.15?is an important aldose-ketose isomerase,which efficiently catalyzes the reverse isomerization reaction between D-xylulose and D-lyxose,D-fructose and D-mannose,as well as L-ribulose and L-ribose.In this study,a thermostable D-lyxose isomerase was characterized from Peptococcus bacterium 1109,and the recombinant E.coli co-expressing D-LI and D-glucose isomerase genes was constructed to produce D-mannose from D-glucose,and the recombinant E.coli coexpressing D-LI and L-arabinose isomerase genes was developed to produce L-ribose from L-arabinose.The gene encoding the D-lyxose isomerase gene was derived from P.bacterium 1109?GenBank accession No:KLU40859.1?and consisted of 543 bp.The gene fragment was synthesized and inserted into the vector pET-22b?+?to construct the recombinant plasmid.The recombinant plasmid was transformed into E.coli BL21?DE3?strain for overexpression.The target protein was purified by using a nickel affinity column and analyzed by SDS-PAGE.The molecular weight of the protein was determined to be 21 kDa,which was consistent with the theoretical molecular weight results.The total molecular weight of the protein was measured to be 42 kDa,indicating that the recombinant enzyme was a homodimeric protein.The recombinant enzyme exhibited maximum catalytic activity at pH 7.5 and 70? with Co²⁺supplementation.The enzyme was determined to be highly thermostable between 70? and 75?.It showed the highest specific activity towards D-lyxose and displayed catalytic activity for D-fructose,D-mannose and L-ribose.The specific activity for D-fructose was 2.5±0.2 U/mg,and 96.0 g/L D-mannose was produced from 500 g/L D-fructose?19.2%conversion?after 8 h.The coding genes of D-glucose isomerase from Acidothermus cellulolyticus and D-LI from P.bacterium 1109 were inserted into plasmid pCDFDuet-1,and then transformed into E.coli BL21?DE3?cells for co-expression.The co-expression system exhibited maximum activity at pH 6.0 and 70? with Co²⁺supplement,and displayed relative good thermostability under 60?.Approximately 185.0 g/L D-fructose and 62.6 g/L D-mannose were produced from 500 g/L D-glucose.The co-expression plasmid encoding L-arabinose isomerase from Alicyclobacillus hesperidum and D-LI from P.bacterium 1109 was constructed.The co-expression system exhibited maximum activity at pH 6.5 and 65? with Co²⁺supplement,and showed relative good thermostability at 50?.In the long term reaction,approximately 85.6 g/L L-ribulose and 45.9 g/L L-ribose were produced from 500 g/L L-arabinose.