Directed Evolution And Immobilization Of Phosphite Dehydrogenase

Phosphite dehydrogenase (EC 1.20.1.1), it catalyzed oxidation of phosphite to phosphate coupled with reduction of NAD+ to generate NADH. Phosphite dehydrogenase not only involved in the metabolism of phosphite, also can generate the NADH. These properties bring about higher using value and broad application prospect in coenzyme regeneration process of industrial biocatalysis and research field of transgenic plants.In this dissertation, we start from sequences of phosphite dehydrogenase from Ralstonia sp. strain 4506 and obtain this ptxD gene by artificial optimization synthesis. The ptxD (1011bp) encoding phodphite dehydrogenase of 336 amino acids was cloned and expressed in Escherichia coli BL21(DE3).The recombinant PTDH display its maximum activity at 45℃ and pH7.0. The activity is steady after 1 OOmin of incubation at 40℃ and 45℃, but it is inactive after 20min of incubation at 50℃、55℃ and 60℃。 In addition, wide-pH-range sTable. was observed in PTDH with the retention of 80% initial activity at different buffer pH4.5 to pH 9.5.The Km and kcat values for phosphite were 9.35±0.37 mM and 157.48±1.99min-1, respectively. The catalytic efficiency kcat/Km was 16.85 mM-1·min-1. The Km and kcat values for NAD+were 0.53±0.02 mM and 190.24±2.09min-1,respectively. The catalytic efficiency towards NAD+kcat/Km was 353.02 mM-1·min-1.PTDH with improved catalytic efficiency were obtained using error-prone PCR. One variant PTDH-M20 was screened from the mutant library containing 10000 clonies. Compared with wide-type, PTDH-M20’s sequences have one site changed (tyrosine to tryptophan). The variant PTDH-M20 display its maximum activity at 45℃ and pH7.0. Compared with wide type, PTDH-M20 showed a 30% decrease in Km (6.52±0.34mM) for phosphite and a 3 folds increase in catalytic efficiency(kcat/Km=70.46 mM-1. min-1). PTDH-M20 showed a 83% increase in Km (0.97±0.04mM) for NAD+ and a 45.8% increase in catalytic efficiency(kcat/Km=514.71mM-1. min-1). In order to study the role of the mutation site, the other 18 amino acids were respectively introduced into the site by site-saturation mutagenesis. The result showed, when tyrosine was replaced by phenylalanine、glutamine、methionine、leucine and arginine, the mutants have catalytic activity. When mutated to phenylalanine and glutamine, the catalytic efficiency (kcat/Km) for phosphite were exhibited a 4.19-fold and 4.83-fold of increase respectively. The catalytic efficiency (kcat/Km) for NAD+ were exhibited a 1.74-fold and 1.89-fold of increase respectively. This study provides new information and experience data for structure-function relationship of phosphite dehydrogenase, and could lay the basis for the development and application of industry and transgenic crops.Phosphite dehydrogenase is co-precipitated along with Fe3O4 magnetic nanoparticle and cross-linked by using glutaraldehyde cross linker. Immobilized enzyme Fe3O4-PTDH display its maximum activity at 50℃ and pH6.5. It retained approximately 50%、 25% and 25% of its initial activity after 100min of incubation at 50℃,55℃ and 60℃.The thermostability was increased compared with free enzyme. Immobilized enzyme Fe3O4-PTDH kept 57% activity after 10 times usage. This study of immobilized phosphite dehydrogenase provide new application value for industrial production. This method is energy-saving economic and environmentally friendly.