Cloning And Characterization Of A H₂O-forming NADH Oxidase And A Galactitol Dehydrogenase

NADH oxidase can catalyze the oxidation of NADH to NAD+ in the presence of O2. It can be coupled with any NAD+-dependent dehydrogenase to regenerate cofactors by improving catalytic efficiency of redox reaction. However, although there are many reports about NADH oxidase, very feware high catalytic efficiency and no H2O2 production. Therefore, the preparation of a new NADH oxidase with high efficiency will have practical significance.In this work, a water-forming NADH oxidase from S. mutans ATCC 25175 was analyzed and cloned based on the gene sequence（NOX, Genbank:EMP63766.1）. And the expression and characterization of the NOX enzyme were also investigated.Firstly, two assumed NOX gene encoding NADH oxidases were screened from Pubmed, and biological information were obtained as follows.The Sm NOX gene sequence was 1374 bp length, it encoded 457 amino acids with molecular weight of 49.93 k Da. Sequence comparison of the protein and others in database showed that the NOX encoding product had 1 cysteine, which was considered as the catalytic center. There were also two FAD-binding domains（FAD-binding1:ANHAGT, FAD-binding2: TSIPDVYAIGDCA） and a NAD-binding domain（GAGYIGVELAE）.Seccondly, the gene Sm NOX was cloned and expressed.After the extraction of the genomic DNA, PCR was runned with the degenerate primers which were designed by their gene sequences. The amplified fragments were ligated to plasmid p GEM-T vector and transformed into E. coli DH5α. Gene sequencing indicated that a mutation was happened in Sm NOX gene. We cloned the NOX gene into expression vector p ET-28a（+） with Bam HI and Xho I restriction sites and then transformed into strain E. coli BL21（DE3）. Whereafter, inducing and expression of the recombinant protein was performed. The expression product was verified by SDS-PAGE electrophoresis.Thirdly, purification and characterization of Sm NOX were carried out. Ni-NTA was used to purify the intracellular soluble protein. Then the reaction conditions and enzymatic properties of the NADH oxidase were carried out. The results showed thethe purified NADH oxidase（Sm NOX） had highest specific activity（202.8 U/mg） at optimal p H, FAD concentration and temperature of 7.0, 30 m M and 35 oC, with a Km of 247.9 μM and a Vmax of 281.7 U/mg. Thermal stability revealed that Sm NOX showed good stability at room temperature, no decrease of activity was observed in 3 h.In the chapter Three, a short-chain dehydrogenase from Rhodobacter sphaeroides 2.4.1 ATCC BAA-808; GDH, NCBI:YP₃52081.1) was cloned. The gene sequence revealed the product galactitol dehydrogenase was 780 bp length, encoding 259 amino acids with molecular weight of 26.614 k Da. By sequence blast, a NAD-binding domain（TGGTQGAG）,which is to combined cofactor NAD+ was observed and the residues Asn, Ser, Tyr, Lys constitutes the catalytic active sites, which are all conserved with those reported galactitol dehydrogenase. Therefore, the short-chain dehydrogenase was presumed as galactitol dehydrogenase（Rs GDH）. Then the Rs GDH gene was cloned and expressed as the previous route of NADH oxidase and a mutation was found in the cloning product. The expression product was verified by SDS-PAGE electrophoresis.Finally, characterization of the recombinant Rs GDH was carried out. The Rs GDH product has broad substrate specificity especially taking galactitol as substrate. Comparing with NADP+, Rs GDH was more NAD+ dependent and taking Ca2+ as cofactor. Then the reaction conditions and enzymatic properties of the galactitol dehydrogenase were observed. The results showed that the optimal reaction conditions of Rs GDH were 25 m M NAD+ concentration, p H8.0, and 75 oC. The maximum reaction rate Vmax was 23.5 μmol/L/min with Km value of 30.7 m M. The thermal stability of the enzyme showed that it was a thermophilic galactitol dehydrogenase. More than 60 % of residual activity was still obtained after 1 h incubation at 75 oC. So the galactitol dehydrogenase（Rs GDH） will have good applications in the redox reactions under high temperature conditions.