Cloning And Characterization Of A New Ribitol Dehydrogenase And Its Application In Allitol Synthesis

Sugar alcohols are organic compounds derived from sugars and contain class of polyol.They are commonly added to foods as they have a lower calorie content than that of sugars.Allitol is a rare alcohol monosaccharide containing six carbons atoms.It is a major product of the D-psicose reduction pathway that exerts various physiological effects,for example laxative effects for the treatment of constipation.Allitol can be also used as an anti-crystallization agent.In addition,allitol cross-links D-and L-hexoses,thus contributing to production of L-psicose.Whole cell biotransformation and enzymatic reactions are very suitable for converting Dsugars to various sugars.Ribitol dehydrogenase(ribitol: NAD+ 2-oxidoreductase;RDH;EC 1.1.1.56)catalyses the conversion of ribitol to D-ribulose and D-psicose into allitol.A new ribitol dehydrogenase gene was cloned from Providencia alcalifaciens RIMD 1656011 and expressed in Escherichia coli BL21.This study aimed to purify and characterize the ribitol dehydrogenase from P.alcalifaciens RIMD 1656011 and investigate its substrate specificity for potential use as an industrial enzyme.The protein was purified by nickel affinity chromatography.The molecular mass of the purified enzyme was determined as ?25 000 and 26 650 Da through sodium dodecyl sulfate polyacrylamide gel electrophoresis and liquid chromatography/mass spectrometry respectively.The result for native molecular mass(104 kDa)suggested that the enzyme functions as a tetramer.Optimum activity of the enzyme was determined at pH 10.0 and a temperature of 35 °C.Regarding its thermal stability,the enzyme retained 72,72,48 and 0% of its initial activity after 4 h at 25,30,40 and 50 °C respectively.The Km,kcat and kcat /Km values of the enzyme for the substrate ribitol were determined as 13.9 mmol L-1,10.0 s-1 and 0.71 mmol L-1 s-1 respectively.The Km of NAD+ was 0.042 mmol L-1.In addition,the substrate specificity indicated that the ribitol dehydrogenase from P.alcalifaciens RIMD 1656011 can be used for direct production of allitol from D-fructose without any byproduct formation.Allitol was synthesized from D-psicose by a combination of new ribitol dehydrogenase(RDH)from P.alcalifaciens and formate dehydrogenase(FDH)under optimized production conditions.RDH and FDH genes were cloned and introduced into pET-22b(+)vectors for expression in Escherichia coli to produce the corresponding enzymes.The effects of temperature,pH,shaking velocity(75,100,125,and 150 rpm),and shaking type(horizontal and vortex)were optimized to maximize the production yield of allitol.The final product was purified and subjected to nuclear magnetic resonance(NMR)spectroscopy,infrared(IR)spectrometry,and liquid chromatography-mass spectrometry(LC-MS)to confirm its structure.The optimal pH and temperature for the reaction were 7.5 and 40 °C,respectively.The results revealed that allitol yield significantly increased with increase in reaction shaking velocity and reached a maximum yield of 95.60 ± 0.54% at 150 rpm shaking velocity after 6 h of reaction.When the reaction was run under horizontal shaking,allitol yield increased from 100 ± 6.05(without shaking)to 124.20 ± 9.70%.Twenty milligrams of D-psicose were successfully reduced to allitol under optimum conditions with a high production yield of 16.7 ± 0.62 mg after 6 h.No by-products were formed during or after the reaction.The separated final produced allitol had a purity of 95%,and its structure was confirmed by HPLC,IR,LC-MS,and NMR spectral analyses.In addition,thermal properties of allitol measured by Differential Scanning Calorimetry(DSC).Using D-psicose as a substrate,allitol was successfully produced by the combination of novel RDH and FDH.The production of allitol by ribitol dehydrogenase(RDH)was investigated,its action of the enzyme through homology and molecular docking studies.We have investigated ribitol dehydrogenase(RDH)from providencia alcalifaciens RIMD 1656011.The protein sequence of RDH was performed for homology modeling through the Swiss model.The 3D structure revealed was docked with NAD+ and D-psicose using AutoDock Vina software version 5.6.The results of homology modeling and docking studies showed that the conserved residues of RDH were Tyr 153,Tyr 92,Ser 17 and Lys157 with NAD+,while conserved residues with D-psicose were Gln67 and Asp61.The NAD+ has good interaction with RDH showing grid score of-49.84,which is a good score for binding.Cofactor regeneration is the hot research topic in recent studies.It has been developed by using various methods.The enzymatic reaction is the common procedure employed in industrial cofactor regeneration.Formate dehydrogenase(FDH)is an enzyme that catalysis the oxidation of formate into carbon dioxide(CO2).It is NAD+ dependant enzyme and is responsible for regeneration of NADH from NAD+.We investigated the regeneration of NADH from NAD+ by formate dehydrogenase.Its action of the enzyme through homology and molecular docking,exploring the active site interaction.We have investigated formate dehydrogenase from Ogataea parapolymorpha DL-1.The protein sequence of FDH was processed for homology modeling using the Swiss model.The 3D structure revealed was docked with NAD+ using AutoDock Vina software.The results of presented homology modeling and docking studies revealed that the conserved residues of FDH interact with NAD+ were Pro 68,Arg 258,Asn 119,Asn 228 and His 97.The cofactor NAD+ has a good interaction with FDH showing grid score of-60.23,which is the appropriate score for binding.An engineered strain for reduction of D-psicose into allitol was designed by constructing allitol synthesis pathway and cofactor recycling system in Escherichia coli.Ribitol dehydrogenase from providencia alcalifaciens was coexpressed to form the pathway for allitol production.The cofactor recycling system was constructed using the formate dehydrogenase gene from Ogataea parapolymorpha for continuous NADH supply.The recombinant strain produced 96% allitol after 48 hours.The optimum conversion condition temperature,pH,Dpsicose concentration,and biomass concentration for whole cell biotransformation were 30°C,7.0,1% and OD600 = 40,respectively.Based on these results,this constructed E.coli shows enormous potential for application in the production of allitol.