Confining Enzyme Clusters in Bacteriophage P22 Enhances Cofactor Recycling and Stereoselectivity for Chiral Alcohol Synthesis

Abstract

Cofactor recycling is important in the synthesis of chiral alcohols via bioreduction of ketones. Herein, a carbonyl reductase from Scheffersomyces stipitis (SsCR) and a glucose dehydrogenase from Bacillus megaterium (BmGDH) were confined in bacteriophage P22 nanoparticles. The recycling efficiency of nicotinamide adenine dinucleotide phosphate (NADPH) in these nanoparticles was enhanced by a factor of from 3 to 45 compared with the free enzyme system, which is attributed to the higher local concentrations of NADPH resulting from the confinement of the enzymes in the P22 nanoparticles. The consumption of NADPH can be reduced by an order of magnitude in scale-up synthesis compared with a free enzyme system.

Enzyme-photo-coupled catalysis in gas-sprayed microdroplets

“…GCN, graphite carbon-nitride; NAD, nicotinamide adenine dinucleotide; SsCR, Sporobolomyces salmonicolor carbonyl reductase; TEOA, triethanolamine. Note that according to the sequence rule of chiral compounds, there are differences in the stereo configuration of 2a-2f, which is in consistent with previous report 51,52. (c) Variation of product concentration and outlet flow rate with reflux ratio b.…”

“…Note that according to the sequence rule of chiral compounds, there are differences in the stereo configuration of 2a-2f, which is in consistent with previous report. 51,52 A different concentration distribution with faster reaction rates was observed in microdroplets. The concentration of NADH in microdroplets gradually increased as the reaction proceeded, whereas the bulk solution system concentration remained constant aer equilibrium was reached.…”

“…A typical EPC catalysis system is composed of a photosensitizer, an electron transfer intermediate, and a redox enzyme. We used GCN, [46][47][48] M, 49,50 and SsCR 51,52 to construct an EPC catalysis system. TEOA was used as sacricial electron donor, while GCN was used to absorb light energy, and the excited electrons subsequently reduced NAD + into NADH with the assistance of M. NADH was eventually utilized for stereoselective reduction of ketones to chiral alcohols, catalyzed by SsCR.…”

Rapid design and prototyping of biocatalytic virus-like particle nanoreactors

“…Encapsulation of enzymes by heterologous protein cages such as VLPs can improve resistance to proteases, chaotropes, and thermal denaturation [5][6][7][8]. These protective properties, combined with the flexibility to encapsulate multiple enzymes [9][10][11], have increased interest in VLP engineering as nanocompartments for applied in vitro [12,13] and in vivo [7,8,14,15] biocatalysis in manufacturing and health.…”

“…While not reproducing the complex environment of a cell, VLPs provide a tool enabling very high ‘cell-like’ protein concentrations of encapsulated proteins [24,25], allowing the study of enzyme behaviour in crowded environments. Using VLPs to concentrate and confine enzymes has led to intriguing effects on protein stability [26] and catalytic properties [11]. A generalizable yet not fully understood impact is one of increased thermal stability.…”