Department of Biotechnology, Faculty of Engineering (Biotechnology Research Center), Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
J Biosci Bioeng. 2010 Mar;109(3):218-23. doi: 10.1016/j.jbiosc.2009.09.040.
Dihydrolipoamide dehydrogenase (LPD), a useful biocatalyst for regenerating NAD(+), was purified from Microbacterium luteolum JCM 9174, and the gene encoding LPD was cloned from the genomic DNA. The gene contained an opening reading frame consisting of 1395 nucleotides encoding 465 amino acid residues with a predicted molecular weight of 49912.1 Da, which displayed 36-78% homology to known LPDs. Moreover, the FAD- and NAD(+)-binding sites and the two catalytic residues in the LPDs were conserved. The enzyme was expressed in recombinant Escherichia coli cells and purified to homogeneity by column chromatography. LPD of M. luteolum (MluLPD) accepted not only lipoamide but also some artificial electron acceptors such as dichlorophenolindophenol (DCIP) and nitrotetrazolium blue (NTB), that is, it functions as a diaphorase. NAD(+) demonstrated a strong activating effect on MluLPD, and the activity was 5.2 times higher than that without NAD(+). The enzyme was suitable for regenerating NAD(+) in biocatalytic reactions because of its high affinity for NADH (6.1 microM). An NAD(+)-regenerating system with MluLPD and laccase using 2,5-dimethoxy-1,4-benzoquinone as a hydrogen acceptor was demonstrated.
二氢硫辛酰胺脱氢酶（LPD）是一种可用于再生 NAD（+）的有用生物催化剂，它从黄色微杆菌 JCM 9174 中被分离出来，并从基因组 DNA 中克隆了编码 LPD 的基因。该基因包含一个开放阅读框，由 1395 个核苷酸组成，编码 465 个氨基酸残基，预测分子量为 49912.1 Da，与已知的 LPD 具有 36-78%的同源性。此外，LPD 中的 FAD 和 NAD（+）结合位点以及两个催化残基是保守的。该酶在重组大肠杆菌细胞中表达，并通过柱层析法纯化至均一性。黄色微杆菌的 LPD（MluLPD）不仅接受硫辛酰胺，还接受一些人工电子受体，如二氯酚靛酚（DCIP）和硝基四氮唑蓝（NTB），也就是说，它具有二氢醌还原酶的功能。NAD（+）对 MluLPD 具有很强的激活作用，其活性比没有 NAD（+）时高 5.2 倍。由于其对 NADH 的高亲和力（6.1 μM），该酶非常适合用于生物催化反应中 NAD（+）的再生。使用 2,5-二甲氧基-1,4-苯醌作为氢受体，展示了一个含有 MluLPD 和漆酶的 NAD（+）再生系统。
