title: One-step production of α-ketoglutaric acid from glutamic acid with an engineered L-amino acid deaminase from Proteus mirabilis.
abstract: Currently, α-ketoglutaric acid (α-KG) is industrially produced by multi-step chemical synthesis, which can cause heavy environmental pollution. Here we reported a simple one-step approach for the production of α-KG by transforming l-glutamic acid with an engineered l-amino acid deaminase (l-AAD) from Proteus mirabilis. First, to facilitate the purification of membrane-bound l-AAD, one N-terminal transmembrane region (from 21 to 87th nucleotide) was removed from l-AAD to block the binding of l-AAD with membrane, and the relatively low-usage codons were replaced by high-usage codons in Escherichia coli to improve the expression level. However, inclusion bodies formed when expressing the ΔN-LAAD in E. coli BL 21, and then the soluble and active ΔN-LAAD was obtained by the solubilization and renaturation of ΔN-LAAD. Furthermore, the biochemical properties of the refolded ΔN-LAAD were characterized and compared with those of full-length l-AAD. Finally, the ΔN-LAAD was used to synthesize α-KG and the maximal formation rate of α-KG reached 12.6% (w/w) at 6h under the following conditions: 12g/L l-glutamic acid, 0.1g/L ΔN-LAAD, 5mM MgCl2, temperature 45°C and pH 8.0. Compared with the multi-step chemical synthesis, the transformation approach has less environmental pollution and has a great potential for α-KG production.
authors: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
authors: Long
authors: L
authors: Liu
authors: Gazi Sakir
authors: GS
authors: Hossain
authors: Hyun-dong
authors: HD
authors: Shin
authors: Jianghua
authors: J
authors: Li
authors: Guocheng
authors: G
authors: Du
authors: Jian
authors: J
authors: Chen
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid / Q000032:analysis / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007656:Ketoglutaric Acids / Q000032:analysis / Q000378:metabolism*; D019216:Metals, Heavy; D015202:Protein Engineering / Q000379:methods; D058849:Protein Refolding; D011513:Proteus mirabilis / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; D019216:Metals, Heavy; D011994:Recombinant Proteins; D018698:Glutamic Acid; D000581:Amidohydrolases