High‐Throughput Screening of Catalytic H2 Production

Hydrogenases, ferredoxins, and ferredoxin‐NADP⁺ reductases (FNR) are redox proteins that mediate electron metabolism in vivo, and are also potential components for biological H₂ production technologies. A high‐throughput H₂ production assay device (H₂PAD) is presented that enables simultaneous evaluation of 96 individual H₂ production reactions to identify components that improve performance. Using a CCD camera and image analysis software, H₂PAD senses the chemo‐optical response of Pd/WO₃ thin films to the H₂ produced. H₂PAD‐enabled discovery of hydrogenase and FNR mutants that enhance biological H₂ production is reported. From a library of 10 080 randomly mutated Clostridium pasteurianum [FeFe] hydrogenases, we found a mutant with nearly 3‐fold higher H₂ production specific activity. From a library of 400 semi‐randomly mutated Oryza sativa FNR, the top hit enabled a 60 % increase in NADPH‐driven H₂ production rates. H₂PAD can also facilitate elucidation of fundamental biochemical mechanisms within these systems.