Charaterization Of Hyperthermophilic Mannitol Dehydrogenases And Their Contribution In D-mannitol Production

Polyalcohols(sugar alcohol)are a group of polyols which reduces sugar’s carbonyl(aldehyde or ketone)to the corresponding primary or secondary hydroxyl group.D-mannitol is a rare monosaccharide sugar alcohol containing six carbons atoms.Dmannitol is a common and safe chemical compound used in the food,pharmaceutical industries,and chemical production.Mannitol dehydrogenase(Mt DH;EC 1.1.1.67)is the key enzyme which catalyzes the conversion of D-fructose to D-mannitol.Our study was focused on the enzymatic synthesis of D-mannitol by highly thermostable mannitol dehydrogenase(Mt DH)which was produced from hyperthermostable strains.Due to the unique specificity of target enzyme;it can be utilized for direct synthesis of D-mannitol from D-fructose without byproduct formation.Selecting thermostable Mt DH was the target of our study because it is generally considered as a vital enzyme for the enzymatic production of D-mannitol due to their unique thermostability under high temperature.A new mannitol dehydrogenase gene was cloned from highly thermostable Caldicellulosiruptor hydrothermalis 108 and Thermotoga neapolitana DSM 4359 and then was successfully expressed in Escherichia coli BL21.The aim of this study was to purify and characterize a thermophlilc mannitol dehydrogenase from our selected strains.The enzyme from both strains was purified by nickel affinity chromatography.The molecular mass of the purified enzyme form C.hydrothermalisand T.neapolitana was determined as~38 and 36 k Da through sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),and liquid chromatography/mass spectrometry results were recorded as 76.7 and 135 k Da,respectively.The results of the native molecular mass suggested that C.hydrothermalis Mt DH was functioned as a homodimer and T.neapolitana Mt DH was a tetramer protein.Optimum activity was determined at 60 °C and 90 °C and p H 7.0 and 6.5 for C.hydrothermalis Mt DH and T.neapolitana Mt DH,respectively.C.hydrothermalis Mt DH was retained 80,75,60 and 10 % of its initial activity at 55,60,65 and 70 oC within 4 h intervals,respectively.The conversion yield of D-mannitol from both enzymes was in range of 40-41% from 1% D-frucyose(W/V).The Conversion ratio of D-fructose to D-mannitol reached more than 80% in T.neapolitana MtDH.Nevertheless,T.neapolitana Mt DH was quite stable at 75 oC and had half of initial activity after 1 h of incubation at 90 oC.The Km,kcat and kcat/Km values of C.hydrothermalis Mt DH for D-fructose direction were 6 m M,37.25 s1 and 6.2 m M-1 s-1,respectively.Km,Vmax,Kcat and kcat/Km values for T.neapolitana were recorded to be 20 m M,200 U mg-1,180s-1 and 9 m M-1 s-1,respectively.Whole cell biotransformation considered the most accurate method for converting D-sugars structures to the corresponding sugars.A thermostable mannitol dehydrogenase(Mt DH)from C.hydrothermalis and formate dehydrogenase(FDH)from Ogataea parapolymorpha were co-expressed to design the multi-enzyme coupling system for Dmannitol bio-synthesis.The cofactor recycling system was constructed using formate dehydrogenase gene from O.parapolymorpha for continuous supplying of NADH.The recombinant Escherichia coli was produced a maximum yield of D-mannitol about 41.0 mg/m L from 0.5% fructose.The results showed that the D-mannitol yield was inversely proportional when high concentration of 1% and 2% of D-fructose was used.Optimum conditions for the reaction optimization were recorded at p H 7.0 and optimum temperature 60 oC.E.coli are efficient biocatalyst to produce D-mannitol with no other sugar formation at high temperature and staple p H and it resulted in a significant conversation of D-mannitol.The protein sequence of C.hydrothermalis Mt DH and T.neapolitana Mt DH were utilized for homology modeling through the Swiss model.The 3D structure was showed D-mannitol docking using Auto Dock Vina software version 5.6.The results of homology modeling and docking exposed that the conserved residues of Mt DH which directly involved in the chemistry reaction were Asp205,Arg203,Glu187,Lys188,Asp189 and Glu190 for C.hydrothermalis Mt DH.For T.neapolitana Mt DH,Asn86,Leu109 and Asp82 were identified as catalytic residues that in direct contact with D-mannitol through hydrogen bonding.D-mannitol recorded a great interaction with C.hydrothermalis Mt DH presenting a grid score of-0.27,and the grid score for T.neapolitana Mt DHwas-0.29 which is considered a good score for chemical intraction binding.