Development of NADH regeneration system by thermostable phosphite dehydrogenase

亚磷酸脱氢酶(PtxD)催化亚磷酸盐几乎不可逆地氧化成磷酸盐，同时将NAD+还原为NADH，在工业生物催化剂中具有巨大的再生NADH的潜力。为了获得一株耐热的PtxD菌株，我们分离到一株Ralstonia sp.菌株4506能在45℃下生长在以亚磷酸盐为唯一磷来源的最低限度的培养基上。利用该菌株PtxD的NADH再生系统(RsPtxD)成功地应用于制药行业重要的手性构建块L-叔亮氨酸的生产。然后对RsPtxD进行定点突变，以提高对NADP的底物专一性。合成的酶RsPtxD-DM作为NADPH再生系统成功地用于莽草酸的生产。

Phosphite dehydrogenase (PtxD), which catalyzes the nearly irreversible oxidation of phosphite to phosphate with the concomitant reduction of NAD+to NADH, has great potential for NADH regeneration in industrial biocatalysts. In order to obtain a heat-stable PtxD, we isolated a Ralstonia sp. strain 4506 that can grow at 45 oC on a minimal medium containing phosphite as the sole source of phosphorus. NADH regeneration system using PtxD of this strain (RsPtxD) was successfully applied to the production of L-tert-leucine, an important chiral building block used in the pharmaceutical industry. Then RsPtxD was modified by site-specific mutation to increase the substrate specificity for NADP. The resultant enzyme RsPtxD-DM was successfully used for shikimic acid production as a NADPH regeneration system.