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.

Edwardsiella tarda Sip2: A Serum-Induced Protein That Is Essential to Serum Survival, Acid Resistance, Intracellular Replication, and Host Infection

“…Hydrogenases are an important mechanism of bacterial adaption to acidic pH ( Ogata et al, 2015 ). Currently, hydrogenases are grouped into three classes based on the metal cofactor present at the active site, namely [Fe-Fe], [Ni-Fe], and [Fe] hydrogenases ( Mangayil et al, 2016 ), all which possess the iron active site and are involved in adapting to acidic environment ( Ogata et al, 2015 ). In E. coli , S. enterica , and Shigella flexneri , hydrogenase mutants display impaired acid resistance ( Hayes et al, 2006 ; Zbell et al, 2008 ; Noguchi et al, 2010 ; McNorton and Maier, 2012 ; Sui et al, 2017 ).…”

A Review of Enhancement of Biohydrogen Productions by Chemical Addition Using a Supervised Machine Learning Method

“…From a holistic point of view, all the steps involved in BioH 2 generation metabolism could be targeted as steps to enhance BioH 2 generation (HY and HER). Two major metabolic pathways, namely, butyrate and acetate, are mainly associated with the activities of hydrogenase and the generation of H 2 during dark fermentation [93][94][95]. From a stoichiometric perspective, the metabolic route towards acetate generates two times that of butyrate pathways [96,97].…”