55 Matching Annotations
  1. Jul 2019
    1. directed evolution
    2. downstream processing

      Refers to the process of separating desired products from biosynthetic pathways

    3. functional-group protection and/or manipulation

      Functional group protection and deprotection increases the number of steps and auxiliary agents during synthesis and is not considered green. The methodology described in this research obeys one of the principles of green chemistry: "Unnecessary derivatization (use of blocking groups, protection/deprotection, temporary modification of physical/chemical processes) should be minimized or avoided if possible, because such steps require additional reagents and can generate waste."

    4. free alcohols and primary amines

      Free alcohols and primary amines are very reactive functional groups that are inert during this carbene transfer reaction.

    5. Relative to the wild-type protein, the evolved triple mutant catalyzes the reaction more than seven times faster, with turnover frequency (TOF) of 46 min–1 (Fig. 1E).

      Via site saturation mutagenesis, V75 and M103 positions along the protein sequence were identified as likely beneficial mutations and were randomized, i.e., the amino acids at these positions are replaced by random ones. A large number of random variants, which together constitute a library, are produced and then screened in an attempt to discover a highly active variant among them. The evolved triple mutant fits the bill.

    6. a 12-fold improvement over the wild-type protein (Fig. 1D).

      Recombinant Protein is a protein encoded by a gene that has been cloned in a system that supports expression of the gene (in this case, it is M100). Modification of the gene by recombinant DNA technology can lead to expression of a mutant protein. In this study, M100D mutation is more highly activating than the wild-type protein (as it occurs in nature).

    7. site-saturation mutagenesis

      M100 is the specific amino acid residue within the protein sequence that has been identified to be critical for the protein’s function. it is very important to determine the ideal amino acid residue for this position. Site saturation mutagenesis is a form of random mutagenesis, allowing the substitution of specific sites against all 20 possible amino acids at once. In this study, this technique is employed to generate a series of enzymes with enhanced activity and enantiospecificity.

    8. Synthetic methodologies such as carbene insertion into silanes can be rendered enantioselective using chiral transition metal complexes based on rhodium (11, 12), iridium (13), and copper (14, 15).

      Certain iridium catalytic systems show 97% ee while copper catalysts show 35% ee and rhodium has been shown to exhibit 77% ee.

    9. chiral reagents

      Any reagent that exhibits chirality (or asymmetry) in its molecular structure

    10. carbene insertion

      carbene is a neutral reactive intermediate; a carbene insertion reaction is the insertion of carbene in a carbon-hydrogen bond.

    11. “Active site” structure of wild-type Rma cyt c showing a covalently bound heme cofactor ligated by axial ligands H49 and M100. Amino acid residues M100, V75, and M103 residing close to the heme iron were subjected to site-saturation mutagenesis.

      Proposed theory for the binding mode for the iron-carbene complex is one where the carbene complex forms such that it takes the place of the axial methionine. The silane may approach from the more exposed side in the wild-type protein. This further explains the observed stereochemistry of the organosilicon product. The V75T, M100D, and M103E mutations may improve reactivity by providing better access of the substrate to the iron center.

      A complete carbene transfer to the protein may be the reason for the catalyst to be inactivated. The activity and lifetime of Rma cyt c may be improved with further mutagenesis.

    12. Carbon–silicon bond forming rates over four generations of Rma cyt c.

      Turnover frequency for each variant relative to wild type protein: WT: 1 M100D 2.8 +/- 0.2 V75T M100D 4.6 +/- 0.3 V75T M100D M103E 7.1 +/- 0.4

      From this experimental data, it is clear that directed evolution has resulted from changing the enzyme from unselective wild type into a highly enantioselective variant.

    13. directed evolution

      Is a method of engineering proteins towards a defined goal or purpose. Directed evolution mimics 'real' evolution and is accelerated in the laboratory by focusing on individual genes expressed in fast‐growing microorganisms such as E.Coli. Enzyme chosen (known as wild type) must show at least a minimal desired reactivity. Mutations are introduced at strategic locations in the wild type protein. Then, the library of mutant proteins is screened for the mutated enzymes with enhanced reactivity. The improved enzymes are used as parents for the next round of mutation and screening. Additional beneficial mutations are introduced if needed. This can continue for several cycles until a desired and beneficial evolution of the enzyme is attained.

    14. physiological

      conditions that occur in nature for an organism in contrast to laboratory conditions

    15. In addition, diazo compounds other than Me-EDA could be used for carbon–silicon bond formation

      Additional diazo compounds that were successful were are R3 = -CH3, -CH2CH3, -Ph.

    16. Fig. 2 Scope of Rma cyt c V75T M100D M103E-catalyzed carbon–silicon bond formation.
      1. Rma cyt c V75T M100D M103E shows excellent enantioselectivity and turnover over a wide range of substrates. Silicon substrates with weakly electron donating or activating methyl substituents (4), strongly electron donating -OMe (5), weakly deactivating -Cl (6), strongly deactivating -CF3 (7), and moderately deactivating COOMe and CONMe (9 and 10 respectively) show moderate to excellent turnover and high selectivity. No direct relationship exists between turnover number and substituents effects from this study. Enantioselectivity is excellent in all substrates. All products were identified using GC-MS and no traditional organic chemistry techniques were used.
    17. We thus chose Rmacyt c as the platform for evolving a carbon–silicon bond-forming enzyme.

      The wildtype protein chosen must exhibit some degree of activity for the desired reaction. When compared to hemin, hemin with bovine serum, a range of protein engineered cytochrome P450 and myoglobin variants, it was found that Rmacyt c showed 97% ee. This was chosen as the enzyme that would form the basis for directed evolution.

    18. 22

      In this study, Professor Arnold's group used protein engineered variants of cytochrome P450BM3 to bring about highly diastereoselective and enantioselective cyclopropanation reaction of styrenes from diazoester. Variant BM3-CIS was identified as a competent cyclopropanation catalyst. It exhibits a strong preference for the cis product and forms both diasteromers over 90% ee and is as stable as the wild type enzyme. P450-BM3 works on a wide range of substrates with both electron donating and electron withdrawing substituents in styrene.

    19. Evolution, natural or in the laboratory, can use these promiscuous functions to generate catalytic novelty
    20. Such enzymes would expand the catalytic repertoire of biology
    21. Chemoselectivity

      With each mutation, the chemoselectivity of the enzyme is greatly enhanced. V75T M100D M103E favors the carbon-silicon bond 29 times more than the wild type. This is attributed to the improved binding and orientation of the silicon donor in the enzyme's active site.

    22. the triple mutant catalyzed the formation of 20 silicon-containing products,

      A generally applicable protocol, as a result of directed evolution, over a variety of substrates illustrates the synthetic utility of the mutant.

    23. Carbon–silicon bond formation catalyzed by heme and purified heme proteins.

      Heme proteins that were readily available were screened to identify the one that gave the most enantioselectivity. This served as starting point for directed evolution. Purified heme protein, silane, diazoester, thiosulfate, methyl cyanide and M9-N buffer as the medium for microbial growth were stirred at room temperature in anaerobic conditions. Reactions were performed in triplicate. Unreacted starting materials was obtained in all cases and no further purification was carried out.

    24. bioorthogonal chemistry

      A new approach of conducting chemical reactions where reactants must react rapidly and selectively with each other under physiological conditions. Two key and relevant features of bioorthogonal reactions is high selectivity and compatibility with naturally occurring functional groups.

    25. metabolic engineering

      Metabolic engineering is the production of specific target chemicals in high yield and stereoselectivity by altering the metabolic pathways. Metabolic pathway is changed via recombinant DNA technology.

    26. heteroarenes

      aromatic compounds where one or more ring carbon atoms are replaced by a heteroatom such as nitrogen, sulfur or oxygen.

    27. naphthalenes

      compounds that contain two fused benzene rings; also referred to as polycyclic aromatic hydrocarbon

    28. amides

      organic compounds that contain -CONH2 structural feature

    29. esters

      organic compounds that contain -COOR functional group

    30. alkyl halides

      organic compounds that contain a halogen connected to an alkyl group such as methyl ethyl etc.

    31. aryl halides

      organic compounds that contain a halogen connected to a benzene ring

    32. ethers

      organic compounds that contains C-O-C structural feature

    33. anaerobic

      oxygen free conditions

    34. recombinantly expressed in Escherichia coli.

      At the theoretical level, the steps needed for obtaining a recombinant protein are straightforward. Take your gene of interest, clone it, transform it into the host of choice (here it is E.Coli), induce and then, the protein is ready for purification and characterization.

    35. adventitious “active site”

      an active site created by chance rather than by design

    36. Met

      The amnio acid, methionine

    37. distal

      located at a further distance

    38. His

      The amino acid, histadine

    39. proximal

      located at a closer distance

    40. hydrophobic

      repels or has no affinity towards water

    41. eukaryotic

      cells with membrane bound organelles

    42. functionally conserved

      relatively unchanged when one goes back in genealogical time

    43. thermohalophilic

      An organism that thrives in extreme high temperature and high salt concentrations

    44. enantioinduction

      Enantioinduction is also popularly known as asymmetric induction. This process is the preferential formation of one enantiomer over the other as a result of the influence of a chiral feature present in reactants or the catalyst.

    45. molecular biology

      branch of biology that deals with structure and function of nucleic acids and proteins

    46. genetically encoded

      Is the order of nucleotides that make up the genetic codes that is translated into proteins

    47. isostere

      elements that have the same number of electrons in the outermost shell (also known as valence shell) and have similar electronic properties

    48. biocompatible

      not harmful to living cells

    49. organosilicon

      Compounds that contain carbon-silicon bonds

    50. enantiopure

      A compound available in one enantiomeric form

    51. turnover

      The number of moles of substrata that a catalyst can convert into the desired product before becoming inactive

    52. chemo- and enantioselectivity

      Chemoselectivity is the preferential reaction of a reagent with a specific functional group over others. Enantioselectivity is the extent to which one enantiomer is formed over the other in a chemical reaction.

    53. heme proteins

      is a type of metalloprotein and contains a heme group which is required for the functionality of the protein

    54. catalyze

      speed up a reaction with the use of external agent, typically a chemical compound

    55. streamlined alternative to transition-metal catalysis

      Although transition metal catalysis offer several advantages, the metals typically used are toxic. Removal of these metals after the synthetic process is time consuming and expensive. The process described in the research ensures sustainability and reduces cost.