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dc.contributor.authorWang, Yiranen_US
dc.contributor.authorZhang, Y-H Percivalen_US
dc.date.accessioned2012-08-24T12:03:11Z
dc.date.available2012-08-24T12:03:11Z
dc.date.issued2009-06-04
dc.identifier.citationMicrobial Cell Factories. 2009 Jun 04;8(1):30en_US
dc.identifier.urihttp://hdl.handle.net/10919/18896
dc.description.abstractBackground 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_US
dc.format.mimetypeapplication/pdf
dc.language.isoen_US
dc.rightsCreative Commons Attribution 4.0 International*
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/*
dc.titleOverexpression and simple purification of the Thermotoga maritima 6-phosphogluconate dehydrogenase in Escherichia coli and its application for NADPH regenerationen_US
dc.typeArticle - Refereed
dc.date.updated2012-08-24T12:03:11Z
dc.description.versionPeer Reviewed
dc.rights.holderYiran Wang et al.; licensee BioMed Central Ltd.en_US
dc.title.serialMicrobial Cell Factories
dc.identifier.doihttps://doi.org/10.1186/1475-2859-8-30
dc.type.dcmitypeText


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