10 research outputs found
Biocatalytic reductive amination by native Amine Dehydrogenases to access short chiral alkyl amines and amino alcohols
Small optically active molecules, and more particularly short-chain chiral amines, are key 20 compounds in the chemical industry and precursors of various pharmaceuticals. Their chemo-21 biocatalytic production on a commercial scale is already established, mainly through lipase-22 catalyzed resolutions leading to ChiProsTM products among others. Nevertheless, their 23 biocatalytic synthesis still remains challenging for very short-chain C4 to C5 amines due to low 24 enantiomeric excess. To complement the possibilities recently offered by transaminases, this 25 work describes alternative biocatalytic access using amine dehydrogenases (AmDHs). Without 26 any protein engineering, some of the already described wild-type AmDHs (CfusAmDH, 27 MsmeAmDH, MicroAmDH and MATOUAmDH2) were shown to be efficient for the synthesis 28 of hydroxylated or unfunctionalized small 2-aminoalkanes. Conversions up to 97.1% were 29 reached at 50 mM, and moderate to high enantioselectivities were obtained, especially for (S)-30 1-methoxypropan-2-amine (98.1%), (S)-3-aminobutan-1-ol (99.5%), (3S)-3-aminobutan-2-ol 31 (99.4%) and the small (S)-butan-2-amine (93.6%) with MsmeAmDH. Semi-preparative scale 32 up experiments were successfully performed at 150 mM substrate concentrations for the 33 synthesis of (S)-butan-2-amine and (S)-1-methoxypropan-2-amine, the latter known as “(S)-34 MOIPA”. Modelling studies provided some preliminary results explaining the basis for the 35 challenging discrimination between similarly sized substituents in the active sites of these 36 enzymes
Chiral amine production with cofactor regeneration in self-aseembled protein-inorganic supraparticles
Protein-inorganic supraparticles are utilized as an enzyme immobilization support. Using leucine zipper affinity domains, supraparticles can specifically immobilize enzymes fused to the complementary domain. Dimensional analysis, image processing, and slow-motion video are used to determine the effects of composition and fluid mechanics on supraparticle assembly. Successful fusion constructs of amine dehydrogenase and formate dehydrogenase with leucine zippers are synthesized via a library of linkers, and preliminary design heuristics for leucine zipper addition to enzymes are delineated. The enzymes are co-immobilized onto the supraparticles and demonstrate comparable conversion of substrate to product to the unaltered enzymes. Further, supraparticles demonstrate many desirable traits for an immobilized biocatalyst, including controllable loading ratios of multiple proteins, recoverability, reloadability, and stability in organic solvent. Recommendations for continued work are discussed. This work demonstrates the utility delocalizing binding and catalysis of immobilized enzymes via fusion protein design, emphasizing the importance of linker engineering to retain activity.Ph.D
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Polynorbornene-based bioconjugates by aqueous grafting-from ring-opening metathesis polymerization reduce protein immunogenicity
Protein-polymer conjugates (PPCs) improve therapeutic efficacy of proteins and have been widely used for the treatment of various diseases such as cancer, diabetes, and hepatitis. PEGylation is considered as the "gold standard" in bioconjugation, although in practice its clinical applications are becoming limited because of extensive evidence of immunogenicity induced by pre-existing anti-PEG antibodies in patients. Here, optimized reaction conditions for living aqueous grafting-from ring-opening metathesis polymerization (ROMP) are utilized to synthesize water-soluble polynorbornene (PNB)-based PPCs of lysozyme (Lyz-PPCs) and bacteriophage Qβ (Qβ-PPCs) as PEG alternatives. Lyz-PPCs retain nearly 100% bioactivity and Qβ-PPCs exhibit up to 35% decrease in protein immunogenicity. Qβ-PPCs derived from NB-PEG show no reduction in recognition by anti-PEG antibodies while Qβ-PPCs derived from NB-Zwit show >95% reduction as compared with Qβ-PEG. This work demonstrates a new method for PPC synthesis and the utility of grafting from PPCs to evade immune recognition
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Inter-coat protein loading of active ingredients into Tobacco mild green mosaic virus through partial dissociation and reassembly of the virion
Chemical pesticide delivery is a fundamental aspect of agriculture. However, the extensive use of pesticides severely endangers the ecosystem because they accumulate on crops, in soil, as well as in drinking and groundwater. New frontiers in nano-engineering have opened the door for precision agriculture. We introduced Tobacco mild green mosaic virus (TMGMV) as a viable delivery platform with a high aspect ratio and favorable soil mobility. In this work, we assess the use of TMGMV as a chemical nanocarrier for agriculturally relevant cargo. While plant viruses are usually portrayed as rigid/solid structures, these are "dynamic materials," and they "breathe" in solution in response to careful adjustment of pH or bathing media [e.g., addition of solvent such as dimethyl sulfoxide (DMSO)]. Through this process, coat proteins (CPs) partially dissociate leading to swelling of the nucleoprotein complexes-allowing for the infusion of active ingredients (AI), such as pesticides [e.g., fluopyram (FLP), clothianidin (CTD), rifampicin (RIF), and ivermectin (IVM)] into the macromolecular structure. We developed a "breathing" method that facilitates inter-coat protein cargo loading, resulting in up to ~ 1000 AIs per virion. This is of significance since in the agricultural setting, there is a need to develop nanoparticle delivery strategies where the AI is not chemically altered, consequently avoiding the need for regulatory and registration processes of new compounds. This work highlights the potential of TMGMV as a pesticide nanocarrier in precision farming applications; the developed methods likely would be applicable to other protein-based nanoparticle systems