High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP(+) to NAD(+)
文献类型：期刊论文
| 作者 | Huang, Rui1; Chen, Hui1; Zhong, Chao1; Kim, Jae Eung1; Zhang, Yi-Heng Percival1,2 |
| 刊名 | SCIENTIFIC REPORTS
 |
| 出版日期 | 2016-09-02 |
| 卷号 | 6 |
| 英文摘要 | Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP+ to NAD+. Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD(+), and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP+ to NAD+. This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P) H reacted with the redox dye TNBT. |
| WOS标题词 | Science & Technology |
| 类目[WOS] | Multidisciplinary Sciences |
| 研究领域[WOS] | Science & Technology - Other Topics |
| 关键词[WOS] | 2,5-DIKETO-D-GLUCONIC ACID REDUCTASE ; COFACTOR-BINDING POCKET ; ESCHERICHIA-COLI ; SACCHAROMYCES-CEREVISIAE ; PHOSPHITE DEHYDROGENASE ; ALCOHOL-DEHYDROGENASE ; THEORETICAL YIELD ; RATIONAL DESIGN ; SPECIFICITY ; NADH |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS记录号 | WOS:000382476200001 |
| 源URL | [http://124.16.173.210/handle/834782/2921]  |
| 专题 | 天津工业生物技术研究所_体外合成生物学研究组 张以恒_期刊论文 |
| 作者单位 | 1.Virginia Tech, Dept Biol Syst Engn, 304 Seitz Hall, Blacksburg, VA 24061 USA 2.Chinese Acad Sci, Tianjin Inst Ind Biotechnol, Tianjin Airport Econ Area, 32 West 7th Ave, Tianjin 300308, Peoples R China |
| 推荐引用方式 GB/T 7714 | Huang, Rui,Chen, Hui,Zhong, Chao,et al. High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP(+) to NAD(+)[J]. SCIENTIFIC REPORTS,2016,6. |
| APA | Huang, Rui,Chen, Hui,Zhong, Chao,Kim, Jae Eung,&Zhang, Yi-Heng Percival.(2016).High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP(+) to NAD(+).SCIENTIFIC REPORTS,6. |
| MLA | Huang, Rui,et al."High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP(+) to NAD(+)".SCIENTIFIC REPORTS 6(2016). |
入库方式： OAI收割
来源：天津工业生物技术研究所
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