DNA from uncultured organisms as a source of 2,5-diketo-D-gluconic acid reductases
DNA from uncultured organisms as a source of 2,5-diketo-D-gluconic acid reductases
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DOI：
10.1128/aem.67.9.4206-4214.2001
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发表时间：
2001-09-01
影响因子：
4.4
通讯作者：
Donnelly, MI
中科院分区：
文献类型：
--
作者：
Eschenfeldt, WH;Stols, L;Donnelly, MI
从土壤样品中提取未培养生物群体的总DNA，通过PCR方法，回收了两种不同的2，5-二酮-D-葡萄糖酸还原酶（DKGRs）基因。基于已发表的序列信息的简并PCR引物得到与已知DKGR同源的内部基因片段。将对内部片段具有特异性的巢式引物与随机引物组合以从环境DNA扩增侧翼基因片段，并且从组合序列预测两个假设的全长基因。基于这些预测，使用特异性引物在单个PCR中扩增两个完整基因。这些基因被克隆并在大肠杆菌中表达。纯化的基因产物催化2，5-二酮基-D-葡萄糖酸还原为2-酮基-L-古洛糖酸。与先前描述的从棒状杆菌属分离的DKGR相比，这些环境还原酶具有一些有价值的性质。两者都表现出大于20倍高的k（猫）/K-m值比先前确定的，主要是由于更好的结合底物。两种新还原酶的Km值分别为57和67 μ M，而棒状杆菌的Km值分别为2和13 mM。两种环境DKGR都接受NADH和NADPH作为共底物;其他DKGR和大多数相关的醛酮还原酶仅使用NADPH。此外，一种新的还原酶比已知的DKGR更耐热。
Total DNA of a population of uncultured organisms was extracted from soil samples, and by using PCR methods, the genes encoding two different 2,5-diketo-D-gluconic acid reductases (DKGRs) were recovered. Degenerate PCR primers based on published sequence information gave internal gene fragments homologous to known DKGRs. Nested primers specific for the internal fragments were combined with random primers to amplify flanking gene fragments from the environmental DNA, and two hypothetical full-length genes were predicted from the combined sequences. Based on these predictions, specific primers were used to amplify the two complete genes in single PCRs. These genes were cloned and expressed in Escherichia coli. The purified gene products catalyzed the reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid. Compared to previously described DKGRs isolated from Corynebacterium spp., these environmental reductases possessed some valuable properties. Both exhibited greater than 20-fold-higher k(cat)/K-m values than those previously determined, primarily as a result of better binding of substrate. The K-m values for the two new reductases were 57 and 67 muM, versus 2 and 13 mM for the Corynebacterium enzymes. Both environmental DKGRs accepted NADH as well as NADPH as a cosubstrate; other DKGRs and most related aldo-keto reductases use only NADPH. In addition, one of the new reductases was more thermostable than known DKGRs.