中文摘要：

辅酶NAD（H）相比NADP（H）有稳定性好、价格低廉及更广的辅酶循环方法等优势,因此在实际应用中常需将NADP（H）依赖型的脱氢酶改造成为NAD（H）依赖型的。来源于嗜热共生杆菌Symbiobacterium thermophilum的NADP（H）依赖型内消旋-2,6-二氨基庚二酸脱氢酶（meso-2,6-diaminopimelate dehydrogenase,St DAPDH）及其突变体酶是催化还原氨化合成D-氨基酸的优良催化剂,本研究试图改变其辅酶偏好性,增强其应用优势。对其晶体结构分析可知,氨基酸残基Y76距离腺嘌呤较近,R35及R36和辅酶上磷酸基团有直接相互作用。依氨基酸侧链基团性质对Y76进行了定点突变,发现不同突变子对两种辅酶的偏好性都发生了变化;对与磷酸基团直接作用的R35、R36进行的双突变R35S/R36V,导致酶对NADP^＋的催化活力降低;将R35S/R36V和部分Y76突变进行了组合,发现三突变组合以NAD^＋为辅酶时的活力均大于以NADP^＋为辅酶的活力,实现了辅酶偏好性转变。这些研究工作为进一步实现St DAPDH的辅酶偏好性完全转变提供依据。

英文摘要：

In industrial application of NAD（P）H-dependent dehydrogenases, NAD（H） has the advantages over NADP（H） in higher stability, lower price and wider recycling system. Recently, a meso-2,6-diaminopimelate dehydrogenase from Symbiobacterium thermophilum（St DAPDH） has been found to be a useful biocatalyst for the production of D-amino acids, but it requires NADP（H） as co-enzyme. To switch the co-enzyme specificity from NADP（H） to NAD（H）, we studied the effect of Y76 on the co-enzyme specificity of St DAPDH, because the crystal structural analysis indicated that residue Y76 is near the adenine ring. The mutation of Y76 exerted significant effect on the co-enzyme specificity. Furthermore, the double mutant R35S/R36 V significantly lowered the specific activity toward NADP^＋, and the combination of R35S/R36 V with some of the Y76 mutants resulted in mutant enzymes favorable NAD^＋ over NADP^＋. This study should provide useful guidance for the further development of highly active NAD^＋-dependent St DAPDH by enzyme engineering.