Engineered enzymes that retain and regenerate their cofactors enable continuous-flow biocatalysis

Hartley, Carol J.; Williams, Charlotte C.; Scoble, Judith A.; Churches, Quentin I.; North, Andrea J.; French, Nigel G.; Nebl, Thomas; Coia, Gregory; Warden, Andrew C.; Simpson, Greg; Frazer, Andrew R.; Jensen, C.N.; Turner, Nicholas J.; Scott, Colin

doi:10.1038/s41929-019-0353-0

Cited by 114 publications

(101 citation statements)

References 34 publications

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“…The proof of principle was demon-strated using a nicotinamide adenine dinucleotide phosphate [(NADP(H)]-dependent alcohol dehydrogenase (ADH, Scheme 1.A). Three different conceptual approaches were developed in the past, [6] which will be briefly explained below (Scheme 1.B-D).…”

Section: Introductionmentioning

confidence: 99%

Efficient Nicotinamide Adenine Dinucleotide Phosphate [NADP(H)] Recycling in Closed‐Loop Continuous Flow Biocatalysis

et al. 2020

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Biocatalytic redox reactions regularly depend on expensive cofactors that require recycling. For continuous conversions in flow chemistry, this is often an obstacle since the cofactor is washed away. Here, we present a quasi-stationary recycling system for nicotinamide adenine dinucleotide phosphate utilizing an immobilized alcohol dehydrogenase. Four model substrates were reduced with high enantioselectivity as a proof of concept. The two-phase system enables continuous production as well as quick substrate changes. This setup may serve as a general cofactor regeneration module for continuous biocatalytic devices employing (co-)substrates being miscible in organic solvent. The system resulted in space-time yields up to 117 g L À 1 h À 1 and total turnover numbers for nicotinamide adenine dinucleotide phosphate higher than 12,000 mol/mol are possible.

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Section: Introductionmentioning

confidence: 99%

Efficient Nicotinamide Adenine Dinucleotide Phosphate [NADP(H)] Recycling in Closed‐Loop Continuous Flow Biocatalysis

et al. 2020

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“…The performance of the cascade can potentially be improved by implementing enzymes in a continuous flow setup using column reactors and tethered cofactors (Hartley et al, 2019). A continuous flow setup would circumvent inhibition of enzymes by cascade intermediates and help to alleviate substrate inhibition by high mannose concentrations.…”

Section: Resultsmentioning

confidence: 99%

Cell-Free Enzymatic Conversion of Spent Coffee Grounds Into the Platform Chemical Lactic Acid

Kopp

Willows

Sunna

2019

Front. Bioeng. Biotechnol.

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The coffee industry produces over 10 billion kg beans per year and generates high amounts of different waste products. Spent coffee grounds (SCG) are an industrially underutilized waste resource, which is rich in the polysaccharide galactomannan, a polysaccharide consisting of a mannose backbone with galactose side groups. Here, we present a cell-free reaction cascade for the conversion of mannose, the most abundant sugar in SCG, into L-lactic acid. The enzymatic conversion is based on a so far unknown oxidative mannose metabolism from Thermoplasma acidophilum and uses a previously characterized mannonate dehydratase to convert mannose into lactic acid via 4 enzymatic reactions. In comparison to known in vivo metabolisms the bioconversion is free of phosphorylated intermediates and cofactors. Assessment of enzymes, adjustment of enzyme loadings, substrate and cofactor concentrations, and buffer ionic strength allowed the identification of crucial reaction parameters and bottlenecks. Moreover, reactions with isotope labeled mannose enabled the monitoring of pathway intermediates and revealed a reverse flux in the conversion process. Finally, 4.4 ± 0.1 mM lactic acid was produced from 14.57 ± 0.7 mM SCG-derived mannose. While the conversion efficiency of the process can be further improved by enzyme engineering, the reaction demonstrates the first multi-enzyme cascade for the bioconversion of SCG.

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“…88 A recent publication correspondingly dealing with the cofactor regeneration in continuously operated biocatalytic reactions was published by Hartley et al in late 2019. 89 The authors cover the topics of cofactor supply and cofactor regeneration in continuous mode and enzyme immobilization without loss of activity. A complex multistep continuous flow reactor with three PBRs was designed for the cofactor-dependent continuous-flow biocatalysis of the antidiabetic drug D-fagomine ( Fig.…”

Section: Selected Examples Of Continuously Operated Meso-scale Reactomentioning

confidence: 99%

The rise of continuous flow biocatalysis – fundamentals, very recent developments and future perspectives

2020

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Engineered enzymes that retain and regenerate their cofactors enable continuous-flow biocatalysis

Cited by 114 publications

References 34 publications

Efficient Nicotinamide Adenine Dinucleotide Phosphate [NADP(H)] Recycling in Closed‐Loop Continuous Flow Biocatalysis

Efficient Nicotinamide Adenine Dinucleotide Phosphate [NADP(H)] Recycling in Closed‐Loop Continuous Flow Biocatalysis

Cell-Free Enzymatic Conversion of Spent Coffee Grounds Into the Platform Chemical Lactic Acid

The rise of continuous flow biocatalysis – fundamentals, very recent developments and future perspectives

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