Overexpression and simple purification of the Thermotoga maritima 6-phosphogluconate dehydrogenase in Escherichia coli and its application for NADPH regeneration
| dc.contributor.author | Wang, Yiran | en |
| dc.contributor.author | Zhang, Y. H. Percival | en |
| dc.contributor.department | Biological Systems Engineering | en |
| dc.contributor.department | Institute for Critical Technology and Applied Science | en |
| dc.date.accessioned | 2012-08-24T12:03:11Z | en |
| dc.date.available | 2012-08-24T12:03:11Z | en |
| dc.date.issued | 2009-06-04 | en |
| dc.date.updated | 2012-08-24T12:03:11Z | en |
| dc.description.abstract | Background Thermostable enzymes from thermophilic microorganisms are playing more and more important roles in molecular biology R&D and industrial applications. However, over-production of recombinant soluble proteins from thermophilic microorganisms in mesophilic hosts (e.g. E. coli) remains challenging sometimes. Results An open reading frame TM0438 from a hyperthermophilic bacterium Thermotoga maritima putatively encoding 6-phosphogluconate dehydrogenase (6PGDH) was cloned and expressed in E. coli. The purified protein was confirmed to have 6PGDH activity with a molecular mass of 53 kDa. The kcat of this enzyme was 325 s-1 and the Km values for 6-phosphogluconate, NADP+, and NAD+ were 11, 10 and 380 μM, respectively, at 80°C. This enzyme had half-life times of 48 and 140 h at 90 and 80°C, respectively. Through numerous approaches including expression vectors, hosts, cultivation conditions, inducers, and codon-optimization of the 6pgdh gene, the soluble 6PGDH expression levels were enhanced to ~250 mg per liter of culture by more than 500-fold. The recombinant 6PGDH accounted for >30% of total E. coli cellular proteins when lactose was used as a low-cost inducer. In addition, this enzyme coupled with glucose-6-phosphate dehydrogenase for the first time was demonstrated to generate two moles of NADPH per mole of glucose-6-phosphate. Conclusion We have achieved a more than 500-fold improvement in the expression of soluble T. maritima 6PGDH in E. coli, characterized its basic biochemical properties, and demonstrated its applicability for NADPH regeneration by a new enzyme cocktail. The methodology for over-expression and simple purification of this thermostable protein would be useful for the production of other thermostable proteins in E. coli. | en |
| dc.description.version | Published version | en |
| dc.format.mimetype | application/pdf | en |
| dc.identifier.citation | Microbial Cell Factories. 2009 Jun 04;8(1):30 | en |
| dc.identifier.doi | https://doi.org/10.1186/1475-2859-8-30 | en |
| dc.identifier.uri | http://hdl.handle.net/10919/18896 | en |
| dc.language.iso | en | en |
| dc.rights | Creative Commons Attribution 4.0 International | en |
| dc.rights.holder | Yiran Wang et al.; licensee BioMed Central Ltd. | en |
| dc.rights.uri | http://creativecommons.org/licenses/by/4.0/ | en |
| dc.title | Overexpression and simple purification of the Thermotoga maritima 6-phosphogluconate dehydrogenase in Escherichia coli and its application for NADPH regeneration | en |
| dc.title.serial | Microbial Cell Factories | en |
| dc.type | Article - Refereed | en |
| dc.type.dcmitype | Text | en |