Document Detail

Protein engineering of hydrogenase 3 to enhance hydrogen production.
MedLine Citation:
PMID:  18335216     Owner:  NLM     Status:  MEDLINE    
The large subunit (HycE, 569 amino acids) of Escherichia coli hydrogenase 3 produces hydrogen from formate via its Ni-Fe-binding site. In this paper, we engineered HycE for enhanced hydrogen production by an error-prone polymerase chain reaction (epPCR) using a host that lacked hydrogenase activity via the hyaB hybC hycE mutations. Seven enhanced HycE variants were obtained with a novel chemochromic membrane screen that directly detected hydrogen from individual colonies. The best epPCR variant contained eight mutations (S2T, Y50F, I171T, A291V, T366S, V433L, M444I, and L523Q) and had 17-fold higher hydrogen-producing activity than wild-type HycE. In addition, this variant had eightfold higher hydrogen yield from formate compared to wild-type HycE. Deoxyribonucleic acid shuffling using the three most-active HycE variants created a variant that has 23-fold higher hydrogen production and ninefold higher yield on formate due to a 74-amino acid carboxy-terminal truncation. Saturation mutagenesis at T366 of HycE also led to increased hydrogen production via a truncation at this position; hence, 204 amino acids at the carboxy terminus may be deleted to increase hydrogen production by 30-fold. This is the first random protein engineering of a hydrogenase.
Toshinari Maeda; Viviana Sanchez-Torres; Thomas K Wood
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2008-03-12
Journal Detail:
Title:  Applied microbiology and biotechnology     Volume:  79     ISSN:  0175-7598     ISO Abbreviation:  Appl. Microbiol. Biotechnol.     Publication Date:  2008 May 
Date Detail:
Created Date:  2008-04-14     Completed Date:  2008-07-28     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  8406612     Medline TA:  Appl Microbiol Biotechnol     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  77-86     Citation Subset:  IM    
Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, TX 77843-3122, USA.
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MeSH Terms
Amino Acid Sequence
DNA Shuffling
Directed Molecular Evolution
Escherichia coli / enzymology*
Escherichia coli Proteins / metabolism*
Formates / metabolism
Hydrogen / metabolism*
Hydrogenase / metabolism*
Models, Molecular
Molecular Sequence Data
Polymerase Chain Reaction
Protein Engineering*
Reg. No./Substance:
0/Escherichia coli Proteins; 0/Formates; 1333-74-0/Hydrogen; EC 1.12.-/hycE protein, E coli; EC

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