title: Recombinant antibodies for specific detection of clostridial [Fe-Fe] hydrogenases.
abstract: Biological hydrogen production is based on activity of specific enzymes called hydrogenases. Hydrogenases are oxygen sensitive metalloenzymes containing Ni and/or Fe atoms at the active site, catalyzing reversible reduction of protons. Generally, [Fe-Fe] hydrogenases prefer proton reduction to molecular hydrogen, a potential energy carrier molecule that can be produced by bioprocesses in sustainable manner. Thus, monitoring tools have been developed to study the relationship between [Fe-Fe] hydrogenases and biohydrogen production in bioreactors at DNA and RNA levels. In the present study, novel molecular tools are introduced for quantitative monitoring of clostridial [Fe-Fe] hydrogenases at the protein level. Aerobic and anaerobic biopanning (for inactive and active [Fe-Fe] hydrogenase, respectively) of phage displayed single-chain variable fragment (scFv) antibody libraries aided in isolating nine potential scFvs. The enriched antibodies demonstrated high specificity towards Clostridium spp. [Fe-Fe] hydrogenases allowing detection from pure and mixed cultures. Additionally, the antibodies showed different binding characteristics towards hydrogenase catalytic states, providing a possible means for functional detection of clostridial [Fe-Fe] hydrogenases. From hydrogenase-antibody interaction studies we observed that though antibody binding reduced the enzyme catalytic activity, it facilitated to retain hydrogen evolution from oxygen exposed hydrogenases.
authors: Tampere University of Technology, Department of Chemistry and Bioengineering, Tampere, 33720, Finland.
authors: Rahul
authors: R
authors: Mangayil
authors: Tampere University of Technology, Department of Chemistry and Bioengineering, Tampere, 33720, Finland.
authors: Matti
authors: M
authors: Karp
authors: University of Turku, Department of Biotechnology, Turku, 20014, Finland.
authors: Urpo
authors: U
authors: Lamminmäki
authors: Tampere University of Technology, Department of Chemistry and Bioengineering, Tampere, 33720, Finland.
authors: Ville
authors: V
authors: Santala
mesh_terms: D000907:Antibodies, Bacterial / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D003013:Clostridium / Q000201:enzymology*; D006864:Hydrogenase / Q000032:analysis* / Q000276:immunology; D019151:Peptide Library; D011485:Protein Binding; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D000907:Antibodies, Bacterial; D019151:Peptide Library; D057127:Single-Chain Antibodies; D006864:Hydrogenase