Regeneration of enzymatic cofactors is the most limiting technical challenge of employing biochemical synthetic reactions for industrial chemical manufacturing. Consequently, requirement and the high cost of these cofactors has limited, isolated biocatalysts for production of fine chemicals. We strived to engineer a drop-in, cofactor regeneration technology, circumventing limited use of isolated biocatalyst for consumer chemicals. Presented here is a biologically engineered artificial organelle that allows for proficient and environmental affable reduction of nicotinamide coenzymes; using light and sourcing electrons from water, generating oxygen as the sole by-product. This platform relies on the elegant nexus between two complex membrane
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To successfully co-reconstitute both enzymes into a single liposome it was necessary to first conduct detergent screening experiments for the reconstitution of each enzyme independently. To establish the most suitable detergent and detergent concentration, the magnitude of PMF generation along with enzymatic activity was measured for each reconstituted enzyme. Of the detergents tested, the zwitterionic surfactant CHAPS was found most suitable for co-reconstitution. To confirm CMI was reconstituted into the liposomes both NADH:decylubiquinone (DQ) oxidoreductase activity AND proton translocation were measured by monitoring NADH oxidation with the corresponding quenching of the pH sensitive fluorophore 9-amino-6-chloro-2-methoxyacridine (ACMA), respectively.
CMI proteoliposome preparations would routinely result in a ~80% decrease in ACMA fluorescence with an NADH:DQ oxidoreductase specific activity of 3,200 nmol NADH min-1 mg CMI-1 (Figure S 1.A). The decrease in ACMA signal was confirmed to be the result of CMI activity by incubation with the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) as minimal change in ACMA fluorescence was observed (Figure S 1.B). Additionally, the NADH:DQ oxidoreductase specific activity of CMI proteoliposomes was inhibited >95% when incubated with 50 M Piercidin A(Figure S 1.A); a potent CMI …show more content…
3B. To determine if this phenomenon is due to disruption to the integrity of the system, light-dark cycle photoreduction experiments were performed (Fig. 4). During the first two light-dark cycles of 20 mins the maximum rates increased from 161.76 44.57 to 286.78 1.45 nmol min-1 mg CMI-1, respectively. This suggests the function of the construct remains, therefore NADH must have an inhibitory effect on the RET for CMI. Nonetheless, little work has been completed on the RET for E. coli CMI, nonetheless it has been reported for the ATP-driven, succinate-supported RET in SMPs(20). Given the highly conservative nature of CMI between species, it would be logical that NADH would surely have an inhibitory effect on RET in this
To successfully co-reconstitute both enzymes into a single liposome it was necessary to first conduct detergent screening experiments for the reconstitution of each enzyme independently. To establish the most suitable detergent and detergent concentration, the magnitude of PMF generation along with enzymatic activity was measured for each reconstituted enzyme. Of the detergents tested, the zwitterionic surfactant CHAPS was found most suitable for co-reconstitution. To confirm CMI was reconstituted into the liposomes both NADH:decylubiquinone (DQ) oxidoreductase activity AND proton translocation were measured by monitoring NADH oxidation with the corresponding quenching of the pH sensitive fluorophore 9-amino-6-chloro-2-methoxyacridine (ACMA), respectively.
CMI proteoliposome preparations would routinely result in a ~80% decrease in ACMA fluorescence with an NADH:DQ oxidoreductase specific activity of 3,200 nmol NADH min-1 mg CMI-1 (Figure S 1.A). The decrease in ACMA signal was confirmed to be the result of CMI activity by incubation with the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) as minimal change in ACMA fluorescence was observed (Figure S 1.B). Additionally, the NADH:DQ oxidoreductase specific activity of CMI proteoliposomes was inhibited >95% when incubated with 50 M Piercidin A(Figure S 1.A); a potent CMI …show more content…
3B. To determine if this phenomenon is due to disruption to the integrity of the system, light-dark cycle photoreduction experiments were performed (Fig. 4). During the first two light-dark cycles of 20 mins the maximum rates increased from 161.76 44.57 to 286.78 1.45 nmol min-1 mg CMI-1, respectively. This suggests the function of the construct remains, therefore NADH must have an inhibitory effect on the RET for CMI. Nonetheless, little work has been completed on the RET for E. coli CMI, nonetheless it has been reported for the ATP-driven, succinate-supported RET in SMPs(20). Given the highly conservative nature of CMI between species, it would be logical that NADH would surely have an inhibitory effect on RET in this