Postdoctoral fellow, The university of Rochester
Enzymes and proteins are increasingly used in organic transformations due to excellent chemo-, regio- and stereo- selectivity, environmental sustainability, milder reaction conditions, improved productivity, simplified work-streams and greater economical saving potential. Use of engineered and artificial biocatalysis is an effective way to tap chemical transformations that lie beyond the realm of natural enzymes. Fasan group at the University of Rochester discovered that engineered variants of myoglobin (Mb), would support the cyclopropanation of variety of aryl-substituted olefins with excellent trans diastereoselectivity (97 to 99.9%) and enantioselectivity (96 to 99.9%, trans-1S,2S isomer) while undergoing high catalytic turnovers (up to 46,800 TON). The ability to access both enantiomeric forms of a target cyclopropane pharmacophore is critical in the context of the synthesis of bioactive molecules. We have used a systemic active-site mutagenesis approach combined with structure-reactivity-relationship (SRR) guided design for developing trans-(1R,2R)-selective Mb catalysts for 1-carboxy-2-aryl-cyclopropane synthesis. SRR strategy yielded Mb(L29T,F43W,H64V,V68F) variant that catalyzes cyclopropanation between styrene and ethyl diazoacetate (EDA) with excellent diastereoselectivity (>99%, trans) and enantioselectivity (95%, trans-1R,2R). In order to expand the scope of Mb catalyzed organic transformations, we have investigated the effect of altering the nature of the metal center and proximal ligand on the carbene transfer activity of these metalloproteins. Using this strategy, Mb catalysts capable of promoting cyclopropanation and carbene Y-H insertion reactions (Y= N, S) under aerobic conditions were identified. Aerobic enzyme reactions would further simplify the biocatalytic process. Performing hemoprotein biocatalysis under aerobic conditions is a challenge due to its greater affinity towards molecular oxygen. Replacing the porphyrin ring of the metal cofactor with other naturally occurring or artificial porphyrinoids could improve the aerobic myoglobin biocatalyst activity. To this end, we have used chlorin e6 (ce6, has 20 π electron containing tetrapyrrole ring system and a chlorophyll analog) as a porphyrin replacement in Mb heme prosthetic group and prepared various Mb variants containing Fe ce6 prosthetic group either through recombinant expression or reconstitution.
Abstract: Chemistry plays a crucial role in creating synthetic analogues of biomacromolecular structures. Of particular scientific and technological interest are biomimetic vesicles that are inspired by natural membrane compartments and organelles but avoid their drawbacks, such as membrane instability and limited control over cargo transport across the boundaries. In this study, completely synthetic vesicles were developed from stable polymeric walls and easy-to-engineer membrane DNA nanopores. The hybrid nanocontainers feature selective permeability and permit the transport of organic molecules of 1.5 nm size. Larger enzymes (ca. 5 nm) can be encapsulated and retained within the vesicles yet remain catalytically active. The hybrid structures constitute a new type of enzymatic nanoreactor. The high tunability of the polymeric vesicles and DNA pores will be key in tailoring the nanocontainers for applications in drug delivery, bioimaging, biocatalysis, and cell mimicry.
Pub.: 25 Aug '16, Pinned: 04 Jul '17
Abstract: We herein describe the engineering of E. coli strains that display orthogonal tags for immobilization on their surface and overexpress a functional heterologous “protein content” in their cytosol at the same time. Using the outer membrane protein Lpp-ompA, cell-surface display of the streptavidin-binding peptide, the SpyTag/SpyCatcher system, or a HaloTag variant allowed us to generate bacterial strains that can selectively bind to solid substrates, as demonstrated with magnetic microbeads. The simultaneous cytosolic expression of functional content was demonstrated for fluorescent proteins or stereoselective ketoreductase enzymes. The latter strains gave high selectivities for specific immobilization onto complementary surfaces and also in the whole-cell stereospecific transformation of a prochiral CS-symmetric nitrodiketone.
Pub.: 20 Jan '17, Pinned: 04 Jul '17
Abstract: Nature applies enzymatic assembly lines to synthesize bioactive compounds. Inspired by such capabilities, we have developed a facile method for spatially segregating attached enzymes in a continuous-flow, vortex fluidic device (VFD). Fused Hisn -tags at the protein termini allow rapid bioconjugation and consequent purification through complexation with immobilized metal affinity chromatography (IMAC) resin. Six proteins were purified from complex cell lysates to average homogeneities of 76 %. The most challenging to purify, tobacco epi-aristolochene synthase, was purified in only ten minutes from cell lysate to near homogeneity (>90 %). Furthermore, this "reaction-ready" system demonstrated excellent stability during five days of continuous-flow processing. Towards multi-step transformations in continuous flow, proteins were arrayed as ordered zones on the reactor surface allowing segregation of catalysts. Ordering enzymes into zones opens up new opportunities for continuous-flow biosynthesis.
Pub.: 31 Jan '17, Pinned: 04 Jul '17
Abstract: Recent developments of biocatalytic and chemocata-lytic methods are discussed. The review provides a guide to the use of biocatalytic methods in the area of chemical synthesis with focused attention on retrosynthetic considerations and analysis. The methods presented are organized according to bond disconnections and attendant synthetic methods. The review is expected to lead to better understanding of the charac-teristics and distinctions of the two complementary approaches. It depicts for researchers in bio- and chemocatalysis a road map of challenges and opportunities for the evolution (and at times revolution) in chemical synthesis.
Pub.: 14 Apr '17, Pinned: 04 Jul '17
Abstract: For the first time, CO2-expanded bio-based liquids were reported as novel and sustainable solvents for biocatalysis. Herein, it was found that by expansion with CO2, 2-methyltetrahydrofuran (MeTHF), and other bio-based liquids, which were not favorable solvents for immobilized Candida antarctica lipase B (Novozym 435) catalyzed transesterification, were tuned into excellent reaction media. Especially, for the kinetic resolution of challenging bulky secondary substrates such as rac-1-adamantylethanol, the lipase displayed very high activity with excellent enantioselectivity (E value > 200) in CO2-expanded MeTHF (MeTHF concentration 10% v/v, 6 MPa), whereas there was almost no activity observed in conventional organic solvents.
Pub.: 12 Apr '17, Pinned: 04 Jul '17
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