Studies On Highly Efficient Cofactor Recycling Enabled By Coupled Reactions Catalyzed By Self-assembled Dehydrogenase Tagged With SsDNA

As an oxidoreductase, dehydrogenase belongs to the first calss in the classification of six enzyme classes, and has been widely used in the fields of biosensor, coenzyme regeneration and multienzyme catalysis. In order to establish a dehydrogenase-coupled catalytic system to achieve efficient coenzyme regeneration, we conducted a series of studies from characterization of fundamental properties to application level. Our studies covered the characterization of a dehydrogenase, establishing dehydrogenase-ssDNA crosslinking reaction method, and demonstration of ssDNA-mediated dehydrogenase coupled reaction system to achieve coenzyme regeneration. The main work consists of three parts:1. Dehydrogenase encoded by orf382from E.coli MG1655was identified and characterized regarding its enzymatic properties and structure. Studies on substrate spectrum and michaelis constant for different substrates （Km is222.5μM for ethanol, Km is11.29μM for L-threonine） were carried out, and a mutant strain with the disrupted orf382was constructed and its phenotype was analyzed. These studies confirmed it as an L-Threonine Dehydrogenase （LTDH） and could play roles in threonine catabolism. Subsequent studies were conducted with L-threonine as substrate. The enzyme exhibited best catalytic performance at39℃and pH9.8with NAD+as the preferential cofactor, and the isoelectronic point was pH5.4. The LTDH was an ion containing protein （Fe, Zn） based on inductively coupled plasma-atomic emission spectrometry （ICP-AES）. Circular dichroism analysis was taken for characterization of its secondary structure in different contexts.2. Protein A*（proA*. protein ID:NP₀40704） from bacteriophage ΦX174was used to construct fusion protein with dehydrogenase as a fusion tag in this study. An Efficient protein-ssDNA crosslinking reaction method was established by taking advantage of the properties of protein A*, that it could recognize, cleave and catalyze specific ssDNA to form a phosphodiester bond between the5’-phosphoryl group of the cleavage site and the tyrosine residue of the protein A*moiety. Protein-ssDNA conjugates were prepared in the crosslinking reaction buffer in37℃for lOmin.3. Through the established protein-ssDNA cross-linking reaction method, the cross-linking of glycerol dehydrogenase （GlyDH）-protein A*and glutamate dehydrogenase （GluDH）-protein A*were carried out under mild conditions to prepare protein-ssDNA conjugates. Complementary ssDNA was designed to guide the spatial arrangement of dehydrogenase-ssDNA conjugates through base-pairing to assemble dehydrogenase complex and construct programmable self-assembled multi-enzyme synergistic catalytic system. Results showed that the fusion with protein A*could improve the specific activity of the enzymes to some extent （GlyDH-A*:GlyDH=116.0%; GluDH-A*:GluDH=105.2%）. Additionally, in the coupled reaction system with glycerol and a-Ketoglutaric acid as substrates, regarding the production of glutamic acid based on HPLC analysis, the efficiency was enhanced by31.2to53.9folds, indicating the existence of substrate channeling mechanism in the spatially assembled reaction system due to the proximity effects. This study exemplified a novel strategy of cofactor recycling for enhanced performance of coupled oxidoreductive reactions.