Investigation Of The Structures And Catalytic Properties Of Three NADH Oxidases

H₂O-forming NADH oxidases are oxidoreductases that can directly consume dissolved oxygen to catalyze the oxidation of NADH to NAD⁺.Various organisms contain these enzymes which play important role in regulating cellular redox and osmotic pressure balance to maintain normal cell growth and development.H₂O-forming NADH oxidases have been used in NAD?P?⁺regeneration because these enzymes can catalyze the oxidation of NADH to NAD⁺.They also have potential in industrial application for the advantages of high-efficiency catalysis and no by-products in aqueous solution.In this work,two NADH oxidases?FAD or FMN-dependent NADH oxidases?were selected,rational designed and investigated to establish highly efficient NAD⁺regeneration for L-tagatose production.In the second chapter,the amino acid residues on the surface of H₂O-forming NADH oxidase?LrNox?derived from L.rhamnosus were rationally selected according to their position and physicochemical properties and were mutated to Cys residue respectively.All mutants were expressed,purified and characterized.The results showed that the catalytic activity of surface Cys mutants is highly dependent on the polarity and charge of the selected amino acid residues.When the polar charged amino acid residues on the surface of LrNox were mutated to Cys,the catalytic activity of the mutants significantly reduced?the specific activity of K380C is only 25.7%of the wild type?.When the selected amino acid residue was a polar uncharged residue or Ala,the Cys mutant exhibits higher catalytic activity?the catalytic activity of the T96C mutant is 2.7 times of the wild type?.CD spectrum results showed that the T96C mutant affects the secondary structure of the protein,but the molecular docking results showed that the number of hydrogen bonds between the position 96 and the local solvent increased obviously,which may have certain effect on the conformation of the active center substrate and nearby amino acid residues,thereby improving the catalytic activity of LrNox.In the third chapter,an FMN-dependent NADH oxidase?LrFOR?from L.rhamnosus was cloned,expressed and biochemical characterized.The optimal LrFOR FMN concentration?15?M?,temperature?30 ^oC?,pH value?7.5?,and half-life at 45^oC(t_1/2=279 min)were all determined.Molecular docking was used to find the amino acid residues which have important roles in the catalytic function of LrFOR.When Thr29 was mutated to Ala with small side chain,the catalytic activity of the T29G mutant towards NADH increased by 2.7 times.The position 29 mutation results suggested that when the Thr29 was mutated to Gly with smaller side chain or Asp with negative side chain,the activities of LrFOR were improved 3.6 and 3.8 times respectively.However,when an amino acid residue with a larger side chain such as Tyr was introduced into this site,the catalytic activity of the obtained mutant decreased to only 50%of the wild type.The CD results showed that there were less changes in the secondary structure of the all single-point mutants.However,the molecular simulation results showed that the introduction of amino acid residues with larger side chains at position 29 could hinder the binding of the substrate NADH to the binding pocket.And,mutating Thr29 to a smaller amino acid residue will facilitate the entry and binding of NADH,thereby improving the catalytic activity of the enzyme.In the fourth chapter,several amino acid residues in the substrate-specific region of the LrNox were selected based on the results of sequence comparison,the polarity and the location.4 single-point mutants and 3 multi-point mutants was obtained by site-directed mutagenesis of these sites.The results showed that the substrate specificity of these mutants changed significantly.Among them,the catalytic activity of the L179S mutant on NADPH was 47.7 times higher than that of wild type,while its catalytic activity towards NADH was dropped to 51%of wild type.In addition,the changes on the optimal pH of these mutants were also observed.For example,with NADPH as the substrate,the optimal pH of the L179S mutant increased from 5.5 to 6.0.Therefore,this mutant was more suitable for the regeneration of the depleted coenzyme NADP⁺in industrial application.The CD spectrum results showed that the single point mutation has no significant effect on the secondary structure of the protein but the molecular simulation results showed that when the Leu 179 of LrNox was mutated to Ser,the hydroxyl group of Ser form a new hydrogen bond with the phosphate group of NADPH.In addition,K185 also formed more hydrogen bonds with the substrate NADPH.These newly formed or shortened hydrogen bonds can increase the affinity of the substrate and enzyme,thereby improving the enzyme catalytic activity.In the fifth chapter,a galactitol dehydrogenase?GatDH?derived from Rhodobacter sphaeroides D was coupled with NADH oxidase derived from Streptococcus mutans to construct a NAD⁺regeneration system.The characterization results showed that D-galactitol is the optimal substrate,and the optimal pH and temperature were 30 ^oC and9.0.The catalytic conditions including the SmNox/GatDH ratio,substrate concentration and reaction time were then optimized with the D-galactitol as the substrate.The results showed that when the catalytic reaction time reached 12 h,the ratio of SmNox to GatDH was 2/3 and the substrate concentration was 100 mM,NAD⁺was efficiently regenerated.The highest conversion rate of 100 mM D-galactitol?88%?and the highest space-time yield of L-tagatose(1.33 g·L^-1·h^-1)were achieved by only adding 3 mM of NAD⁺.In this thesis,the structures of two H₂O-forming NADH oxidases were analyzed,and site-directed mutagenesis was then used to mutate residues on surface,active pocket,and substrate-specific domain of the enzyme.Mutants with higher catalytic activity for NADH or NADPH were both obtained.In addition,a NAD⁺regeneration method contains a H₂O-forming NADH oxidase and galactitol dehydrogenase was used to achieve the highly efficient production of L-tagatose by adding a low concentration of NAD⁺.This thesis provides new ideas for the further rational design and structural analysis of NADH oxidase as well as for recycling the NAD⁺using H₂O-forming NADH oxidase.