Biochemical Characteristics And Application Of L-arabinose/D-galactose 1-dehydrogenase Of Thermotoga Marina

D-Galactonic acid is a rare aldonic acid obtained from the oxidation of D-galactose.It is a valuable raw material in the cosmetics,pharmaceutical,food and construction industries.Enzymatic synthesis of D-galactonic acid has the advantages of easy control of the reaction process,fast reaction speed,mild conditions,few by-products,short production cycle and environmental friendliness.L-Arabinose/D-Galactose 1-dehydroge-nase is a member of the Gfo/Idh/Moc A family of oxidoreductases,which can convert D-galactose to synthesize D-galactonic acid.In this study,L-arabinose/D-galactose1-dehydrogenase was screened for the first time from Thermotoga maritima,and then L-arabinose/D-galactose 1-dehydrogenase was cloned,expressed and purified;the enzymatic properties of recombinant L-arabinose/D-galactose 1-dehydrogenase were studied,and the recombinant enzyme was used to convert lactose to synthesize D-galactonic acid,providing an effective way for the high-value utilization of cheap lactose.The specific research results are as follows:1.Cloning and expression and activity identification of L-arabinose/D-galactose1-dehydrogenase from Thermotoga maritimaThrough bioinformatics,four pseudo-L-arabinose/D-galactose 1-dehydrogenase genes Tm0441,Tm0297,Tm0312 and Tm0325 were screened and cloned from T.maritima.The gene fragments obtained were expressed in the expression cell E.coli BL 21(DE 3)by means of genetic engineering,and the expression products were separated and purified by Ni-NTA affinity chromatography column to obtain high-purity recombinant enzymes.The bands obtained by SDS-PAGE inspection were in general agreement with the theoretical values of 27.89 KDa,36.54 KDa and 26.64 KDa of recombinant enzymes Tm0297,Tm0312 and Tm0325.The recombinant enzyme obtained was reacted with 20 kinds of substrates,and the results of enzyme activity measurement showed that the best substrate of recombinant enzyme Tm0297 was D-arabinitol,followed by D-xylitol;the recombinant enzyme Tm0312 has more preference for L-arabinose and D-galactose,followed by D-fucose.The products obtained from the conversion of D-galactose by the recombinant enzyme Tm0312 were further identified and analyzed by LC-MS and¹H-NMR spectrum to determine the presence of D-galactonic acid and its intermediate D-galactose-1,4-lactone and NADH.2.Enzymatic characterization of recombinant L-arabinose/D-galactose1-dehydrogenaseThe multiple sequence alignment analysis of the amino acid sequence of Tm0312shows that the amino acid conservation relationship between Tm0312 and the oxidoreductase of the Gfo/Idh/Moc A family and the sequence of L-arabinose/D-galactose1-dehydrogenase from other strains,respectively.The catalytically active amino acids of Tm0312 are Lys 101,Tyr 240,Asp 186,Arg 174 and Gly 13,and the conserved domain is Asp 172-Lys 176.Secondly,a phylogenetic tree of Tm0312 was constructed,and the result showed that Tm0312 is relatively distantly related to the bacterial L-arabinose/D-galactose1-dehydrogenase family,which also proves that Tm0312 is a novel L-arabinose/D-galacto-se 1-dehydrogenase that can act on both L-arabinose and D-galactose substrates.The enzymatic properties of the purified recombinant enzyme Tm0312 were studied,and the results showed that:The optimum p H of recombinant enzyme Tm0312 was 8.0 and the optimum temperature was 75°C;the enzyme has good heat resistance,the residual activity of the enzyme is 63.69%and 47.41%after keeping it at 60 and 65?for 24 hours;and it has good alkali resistance under the alkaline condition of p H 7.0-9.0,and the relative activity is above 60%;Zn²⁺,Ca²⁺and EDTA can enhance the enzyme activity of recombinant enzyme Tm0312.When NAD⁺is used as a cofactor,the K_mof recombinant enzyme Tm0312 for L-arabinose,D-galactose and D-fucose are 1.74,1.65 and 2.60 m M,respectively;and the k_cat/K_mare 80.70,76.89 and 34.82 min^-1m M^-1,respectively;when NADP⁺is used as a cofactor,the K_mof recombinant enzyme Tm0312 for L-arabinose,D-galactose and D-fucose are 1.57,1.71 and 4.80 m M,respectively;and the k_cat/K_mare92.91,75.66 and 39.04 min^-1m M^-1,respectively.When L-arabinose was used as the substrate,the K_mvalues of recombinant enzyme Tm0312 for NAD⁺and NADP⁺were 0.22and 0.09 m M,respectively,and the k_cat/K_mvalues were 921.96 and 2647.83 min^-1m M^-1,respectively;when D-galactose was used as the substrate,the K_mvalues of recombinant enzyme Tm0312 for NAD⁺and NADP⁺were 0.22 and 0.089 m M,and the k_cat/K_mvalues were 845.87 and 2474.65 min^-1m M^-1,respectively.By means of homology modeling and molecular docking,the structure of the recombinase Tm0312 was predicted,the substrate NAD⁺,D-galactose/L-arabinose and the key amino acids of Tm0312 and their interaction forces were analyzed.Tm0312 consists of11?-helices and 15?-folds;84.7%of the amino acids are in the most reasonable region and 12.5%are in the more reasonable region,indicating that the predicted three-dimensional structure of Tm0312 is reliable.The docking results showed that the binding free energy of Tm0312 with NAD⁺,D-galactose and L-arabinose were-6.74Kcal/mol,-4.29 Kcal/mol and-4.05 Kcal/mol,respectively,which are consistent with the results of kinetic parameters K_m;and the interaction between Tm0312 and the substrate is mostly through hydrogen bonds.3.Synthesis of D-galactonic acid by recombinant L-arabinose/D-galactose1-dehydrogenaseIn this study,the optimum conditions for the conversion of D-galactose to D-galactonic acid by recombinant L-arabinose/D-galactose 1-dehydrogenase Tm0312 were optimized:enzyme addition of 0.16 mg/m L,p H 8.5,and temperature 70°C.Under these optimum conditions,the yield of D-galactonic acid was 8.97 m M at 4 h and 13.93 m M at24 h of reaction.The recombinant enzyme Tm0312 transformed 5 m M D-galactose and D-galactose was completely converted at 12 h of reaction;when the recombinant enzyme Tm0312 transformed D-galactose with different substrate concentrations,the conversion rate of the system was slow after 12 h and the yield decreased relatively with the increase of substrate concentration.Using cheap lactose as a substrate,combined with the recombinant enzyme Tm0312 and?-galactosidase from T.maritima to convert and produce D-galactonic acid,and lactose was completely converted at 20 h,while D-galactonic acid almost stopped growing after 16 h of reaction,at which time 2.80 m M of D-galactonic acid was produced,and the yield reached 56%.