Department of Chemistry, Middle East Technical University, 06531 Ankara, Turkey.
J Biotechnol. 2011 Apr 10;152(4):176-83. doi: 10.1016/j.jbiotec.2011.03.002. Epub 2011 Mar 15.
Rapid immobilization with the one-pot purification of galactitol dehydrogenase (GatDH) and formate dehydrogenase (FDH) is achieved by using iminodiacetic acid (IDA) with chelated Co(2+) modified magnetic nanoparticles as a carrier. Lactate dehydrogenase (LDH) from recombinant Escherichia coli and FDH commencing Candida methylica were used as an auxiliary enzyme for the regeneration of NADH/NAD(+) with a representative synthesis of (S)-1,2-propanediol and l-tagatose starting from hydroxyacetone and galactitol. The affinity magnetic nanoparticles were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), while the purity of GatDH and FDH was assayed by SDS-PAGE analysis. The immobilized two-enzyme system, reflecting the pH dependence of its constituent enzymes, showed optimal activity at pH 7 and 8 for (S)-1,2-propanediol and l-tagatose production, respectively. The immobilized enzyme system retained up to 70% of its activity after one week of repeated use. The use of affinity magnetic nanoparticles offers the advantage of a one-pot purification of His(6)-tagged GatDH and FDH followed by the production of rare sugar and chiral diol.
通过使用亚氨基二乙酸（IDA）与螯合 Co(2+)修饰的磁性纳米颗粒作为载体，实现了半乳糖醇脱氢酶（GatDH）和甲酸脱氢酶（FDH）的一锅法快速固定化和纯化。重组大肠杆菌的乳酸脱氢酶（LDH）和起始于 Candida methylica 的 FDH 被用作辅助酶，用于以羟基丙酮和半乳糖醇为起始原料，再生 NADH/NAD(+)，以合成（S）-1,2-丙二醇和 l-塔格糖。亲和磁性纳米颗粒通过扫描电子显微镜（SEM）和傅里叶变换红外光谱（FTIR）进行了表征，而 GatDH 和 FDH 的纯度则通过 SDS-PAGE 分析进行了测定。固定化的双酶系统反映了其组成酶的 pH 依赖性，在（S）-1,2-丙二醇和 l-塔格糖生产中分别在 pH 7 和 8 下表现出最佳活性。固定化酶系统在重复使用一周后仍保留了 70%的活性。亲和磁性纳米颗粒的使用具有以下优点：可以一锅法纯化 His(6)-标记的 GatDH 和 FDH，然后生产稀有糖和手性二醇。
