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One-step production of α-ketoglutaric acid from glutamic acid with an engineered L-amino acid deaminase from Proteus mirabilis.
MedLine Citation:
PMID:  23333917     Owner:  NLM     Status:  Publisher    
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 MgCl(2), 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.
Long Liu; Gazi Sakir Hossain; Hyun-Dong Shin; Jianghua Li; Guocheng Du; Jian Chen
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-1-17
Journal Detail:
Title:  Journal of biotechnology     Volume:  -     ISSN:  1873-4863     ISO Abbreviation:  J. Biotechnol.     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-1-21     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8411927     Medline TA:  J Biotechnol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2013. Published by Elsevier B.V.
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
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