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 H-2 production technologies. A high-throughput H-2 production assay device (H(2)PAD) is presented that enables simultaneous evaluation of 96 individual H-2 production reactions to identify components that improve performance. Using a CCD camera and image analysis software, H(2)PAD senses the chemo-optical response of Pd/WO3 thin films to the H-2 produced. H(2)PAD-enabled discovery of hydrogenase and FNR mutants that enhance biological H-2 production is reported. From a library of 10 080 randomly mutated Clostridium pasteurianum [FeFe] hydrogenases, we found a mutant with nearly 3-fold higher H-2 production specific activity. From a library of 400 semi-randomly mutated Oryza sativa FNR, the top hit enabled a 60% increase in NADPH-driven H-2 production rates. H(2)PAD can also facilitate elucidation of fundamental biochemical mechanisms within these systems.