Exceptionally High Rates of Biological Hydrogen Production by Biomimetic In Vitro Synthetic Enzymatic Pathways

Abstract

Hydrogen production by water splitting energized by biomass sugars is one of the most promising technologies for distributed green H production. Direct H generation from NADPH, catalysed by an NADPH-dependent, soluble [NiFe]-hydrogenase (SH1) is thermodynamically unfavourable, resulting in slow volumetric productivity. We designed the biomimetic electron transport chain from NADPH to H by the introduction of an oxygen-insensitive electron mediator benzyl viologen (BV) and an enzyme (NADPH rubredoxin oxidoreductase, NROR), catalysing electron transport between NADPH and BV. The H generation rates using this biomimetic chain increased by approximately five-fold compared to those catalysed only by SH1. The peak volumetric H productivity via the in vitro enzymatic pathway comprised of hyperthermophilic glucose 6-phosphate dehydrogenase, 6-phosphogluconolactonase, and 6-phosphogluconate dehydrogenase, NROR, and SH1 was 310 mmol H /L h , the highest rate yet reported. The concept of biomimetic electron transport chains could be applied to both in vitro and in vivo H production biosystems and artificial photosynthesis.

Building a Thermostable Metabolon for Facilitating Coenzyme Transport and In Vitro Hydrogen Production at Elevated Temperature

“…Plasmid pET20b-cbm3-scaf encoding miniscaffoldin [7a] and pET20b-g6pdh encoding glucose 6-phosphate dehydrogenase (G6PDH) from Thermotoga maritima [22] were constructed as described previously.T he NAD + -preferring mutant G6PDH (mG6PDH S33E/R65M/ T66S) was obtained by site-directed mutagenesis.…”

“…The increase in the absorbance at 340 nm was measured over 5min. [22] The reduced BV that was generated by the reactions with sodium dithionite (Na 2 S 2 O 4 ,2 0mm)i nd egassed HEPES buffer (100 mm, pH 7.5) contained SHI, SHI-CMmCtDoc, or SHI-CMmCtDoc in the final assembled structure. [25] Specific ac- ChemSusChem 2018, 11,3120 -3130 www.chemsuschem.org tivities of DI, DI-CcDoc, and DI-CcDoc in the final assembled structure were determined at 70 8Cb ym easuring the absorbance change at 578 nm over 5min.…”

“…The millimolar extinction coefficient of NADH (e 340 )w as 6.2 mm À1 cm À1 .T he activity was expressed as units (U) per micromole mG6PDH, mG6PDH-RfDoc and mG6PDH-RfDoc in the final assembled structure, where 1U was defined as 1 mmol NADH generated per minute. [22] Activityassays of the metabolon and enzyme cocktail The enzyme reactions were carried out in an anaerobic screw-cap IR quartz cuvette (Reflex Analytical Co.,R idgewood, NJ, USA) with degassed HEPES buffer (100 mm, pH 7.5) containing MgCl 2 (5 mm), MnCl 2 (0.5 mm), and NADH (2 mm).…”

“…The millimolar extinction coefficient e 578 for reduced BV was 8.65 mm À1 cm À1 .D Ia ctivity was expressed as units (U) per micromole DI, DI-CcDoc, and DI-CcDoc in the final assembled structure, where 1U was defined as 1 mmol reduced BV generated per minute. [22] Specific activities of SHI, SHI-CMmCtDoc and SHI-CMmCtDoc in the final assembled structure in H 2 generation were determined under anaerobic conditions as described previously. [22] The reduced BV that was generated by the reactions with sodium dithionite (Na 2 S 2 O 4 ,2 0mm)i nd egassed HEPES buffer (100 mm, pH 7.5) contained SHI, SHI-CMmCtDoc, or SHI-CMmCtDoc in the final assembled structure.…”

“…[22] Specific activities of SHI, SHI-CMmCtDoc and SHI-CMmCtDoc in the final assembled structure in H 2 generation were determined under anaerobic conditions as described previously. [22] The reduced BV that was generated by the reactions with sodium dithionite (Na 2 S 2 O 4 ,2 0mm)i nd egassed HEPES buffer (100 mm, pH 7.5) contained SHI, SHI-CMmCtDoc, or SHI-CMmCtDoc in the final assembled structure. The reaction was performed at 70 8C, and 0.2 mL of headspace gas was drawn out with ag as-tight syringe (Hamilton Co.,R eno, NV) to measure the H 2 concentration.…”