Enzymatic and chemoenzymatic approaches to polymer synthesis and modification.
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Biopolymers↗
Biomedical subjects
J S Dordick
Publications and source records attributed to J S Dordick.
How do organic solvents affect peroxidase structure and function?
The effect of organic solvents on horseradish peroxidase structure and function has been studied. Some, but not complete, enzyme denaturation occurs even in low volumes of water-miscible organic solvents (e.g., greater than 30% v/v dioxane, greater than 50% v/v methanol, and greater than 20% v/v acetonitrile) as determined by the decreased difference between the fluorescence of peroxidase's sole tryptophan residue and free L-tryptophan in solution. Absorbance and electron paramagnetic resonance spectroscopies indicate exposure of peroxidase's active site to the organic solvent. This reduces the local polarity in the enzyme's active site and results in stronger hydrogen bonding of phenolic substrates to the enzyme. In extreme cases (e.g., 95% v/v dioxane, 90% v/v acetonitrile, and ethyl and butyl acetate containing 2 and 1% v/v aqueous buffer, respectively), the transition state of the enzymic reaction is sufficiently perturbed so as to alter the magnitude of the Hammett rho value. This is most likely the result of the increased strength of hydrogen bonding between electron-donating alkoxyphenols (negative sigma values) and an electrophilic group in the enzyme's active site, thereby reducing catalytic efficiencies for such substrates relative to alkyl- and chlorophenols. Perhaps the most important effect of the organic solvent, however, is the significant ground-state stabilization of phenolic substrates in organic media as opposed to aqueous buffer. This stabilization can account for nearly 4 orders of magnitude in reduction of catalytic efficiency and is manifested in increased Km's. This study indicates that enzymes can maintain much of their native active-site structure in organic media and that the effect of solvent on substrate thermodynamics must be considered.
Acetonitriles↗
Enzymatic catalysis and dynamics in low-water environments.
Enzymes suspended in organic solvents represent a versatile system for studying the involvement of water in enzyme structure and function. Addition of less than 1% (vol/vol) water to tetrahydrofuran containing 1 M 1-propanol leads to a substantial increase in the transesterification activity of subtilisin Carlsberg (from Bacillus licheniformis) that correlates with a sharp increase in the active-site polarity and a 90% decrease in the rotational correlation time (i.e., increase in mobility) of a nitroxide spin label within the active site. Water in excess of 1% has little additional effect on active-site polarity and coincides with a further increase in spin-label mobility, yet the transesterification activity decreases dramatically. Thus, transesterification activity increases and then decreases with increasing enzyme hydration and flexibility (which are presumably coupled through dielectric screening), suggesting that the conformation of partially hydrated subtilisin is different from that of the nearly dry enzyme--i.e., enzyme containing less than 9% (wt/wt) water.
Bacillus↗
Enzymatic and chemoenzymatic approaches to polymer synthesis.
The function of many specialized polymers calls for properties such as chirality and biodegradability. The stereo, -positional-and chemo-selectivities characteristic of enzymatic catalysis are highly desirable attributes for incorporation into strategies for synthesizing such polymers. Enzymes alone, or in combination with chemical synthesis (i.e. chemoenzymatic methodologies), are finding increased use in the synthesis of novel materials. Potential applications include water-absorbents, hydrogels, biodegradable materials, chiral adsorbents, liquid crystals and permselective membranes.
Acrylic Resins↗
Enzymology in monophasic organic media.
Over the past year, an important area of research has been directed towards the fundamental aspects of enzymes and new applications of enzymology in monophasic organic media. Much of this research has focused on the factors that influence enzymatic catalysis in monophasic organic solvents, including the importance of enzyme-associated water, and the effect of organic solvents on enzyme structure and thermodynamic features. From an applications perspective, new advances in the use of enzymes in organic and polymer syntheses and optical resolutions have been made.
Biotechnology↗
Thiol and Mn(2+)-mediated oxidation of veratryl alcohol by horseradish peroxidase.
Horseradish peroxidase has been shown to catalyze the oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) and benzyl alcohol to the respective aldehydes in the presence of reduced glutathione, MnCl2, and an organic acid metal chelator such as lactate. The oxidation is most likely the result of hydrogen abstraction from the benzylic carbon of the substrate alcohol leading to eventual disproportionation to the aldehyde product. An aromatic cation radical intermediate, as would be formed during the oxidation of veratryl alcohol in the lignin peroxidase-H2O2 system, is not formed during the horseradish peroxidase-catalyzed reaction. In addition to glutathione, dithiothreitol, L-cysteine, and beta-mercaptoethanol are capable of promoting veratryl alcohol oxidation. Non-thiol reductants, such as ascorbate or dihydroxyfumarate (known substrates of horseradish peroxidase), do not support oxidation of veratryl alcohol. Spectral evidence indicates that horseradish peroxidase compound II is formed during the oxidation reaction. Furthermore, electron spin resonance studies indicate that glutathione is oxidized to the thiyl radical. However, in the absence of Mn2+, the thiyl radical is unable to promote the oxidation of veratryl alcohol. In addition, Mn3+ is unable to promote the oxidation of veratryl alcohol in the absence of glutathione. These results suggest that the ultimate oxidant of veratryl alcohol is a Mn(3+)-GSH or Mn(2+)-GS. complex (where GS. is the glutathiyl radical).
Benzyl Alcohols↗
Affinity-based separations and purifications. Patents and literature.
The separation and purification of biologically functional molecules (e.g., proteins, antibodies, peptides, hormones, low molecular weight biologicals) is of fundamental importance to biotechnology. Affinity separations have become a particularly attractive method for bioseparations due to their high degree of selectivity. Numerous affinity ligands have been prepared in recent years including lectins, nucleic acids, inhibitors, and immunoresponse agents. Furthermore, a variety of novel supports have been synthesized to aid in the development of commercially useful affinity separation systems. Recent US patents and scientific literature on affinity separations and purifications are surveyed. Patent abstracts are summarized individually and a list of literature references are given.
Chemistry Techniques, Analytical↗
Non-aqueous enzymology.
Compelling evidence has been obtained during the past year that enzymes retain their native active-site structure in organic solvents, and yet the properties of the solvent significantly affect enzyme kinetics. Fundamental advances in enzymatic catalysis in monophasic organic media are discussed and selected applications in the areas of asymmetric, polymer and chemoenzymatic syntheses are highlighted.
Binding Sites↗
Protein engineering and site-directed mutagenesis. Patents and literature.
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Mutagenesis, Site-Directed↗
Selective biotransformations. Patents and literature.
Stereo- and regioselective biotransformations is an area of intense research interest. The ability to take advantage of microorganisms and isolated enzyme systems to perform selective organic syntheses is well known. In recent years, several new methodologies have caused a rapid growth in the area of selective biotransformations. These novel techniques include genetic and protein engineering, biocatalysis in organic media, improved screening procedures, as well as an increased awareness of the value of biotechnology to solve important problems in organic synthesis. Recent US patents and scientific literature on stereo- and regioselective biotransformations are surveyed. Patent abstracts are summarized individually and a list of literature references are given.
Bibliographies as Topic↗
Biocatalysis in nonaqueous media. Patents and literature.
Biocatalysis in nonaqueous media is being used in increasing regularity both in academic and industrial research. A variety of biocatalysts have been used in organic media including enzymes, multi-enzyme systems, and whole cells. In addition, the nonaqueous media has encompassed both monophasic and biphasic solvent systems, enzymes and whole cells in reversed micelles, enzymes and cells in nearly anhydrous (no added water) solvents, and enzymes catalytically active in supercritical fluids and the gas phase. Recent US and overseas patents and scientific literature on biocatalysis in nonaqueous media are surveyed. Patent abstracts are summarized individually, and literature references are divided into major subheadings.
Bibliographies as Topic↗
Monoclonal antibodies for clinical applications. Patents and literature.
The use of monoclonal antibodies for clinical applications has increased dramatically in the past few years. Monoclonal antibodies are used for the detection of bacterial and viral infections, determination of the presence and stage of cancer, and as diagnostics for the detection and quantitation of low molecular weight compounds of clinical and biochemical interest. The latter application has also taken place through the development of monoclonal antibody-based sensors. The ease of monoclonal antibody synthesis and their high degree of antigen specificity has made monoclonal antibodies ideal for the aforementioned clinical applications. Recent US patents and scientific literature on clinical applications of monoclonal antibodies are surveyed. Patent abstracts are summarized individually and a list of literature references are given.
Antibodies, Monoclonal↗
Purification of glycoproteins by selective transport using concanavalin-mediated reverse micellar extraction.
A novel methodology for coupling liquid-liquid extraction with affinity interaction has been developed to selectively and efficiently purify and separate glycoproteins. The basis for the separation is the selective extraction of glycoproteins from an aqueous solution into a reverse micellar organic phase by using concanavalin A (a sugar-binding lectin) as a facilitative carrier. Specifically, horseradish peroxidase (a common glycoprotein) can be bound to concanavalin A in an aqueous phase and then extracted into an AOT-isooctane organic phase with negligible loss in enzyme activity. Virtually no extraction of peroxidase occurs in the absence of concanavalin A. Electron spin resonance studies have shown that the large lectin-glycoprotein complex (96,000 daltons) resides in a nonaqueous environment within the reverse micelle, perhaps at the surfactant, water-pool interface; hence, extraction of the large complex is feasible. The facilitative extraction has been extended to selective transport of peroxidase from a mixture of peroxidase and alkaline phosphatase (a nonglycosylated protein). This results in an efficient separation strategy with a separation factor of 16.
Chemistry Techniques, Analytical↗
Designing enzymes for use in organic solvents.
Enzymes are routinely used in organic solvents where numerous reactions of interest to synthetic and polymer chemists can be performed with high selectivity. Recently, it has become apparent that the catalytic properties of an enzyme can be tailored to a specific catalytic requirement by the use of solvent and protein engineering. The former involves altering the polarity, hydrophobicity, water content, etc., of the organic milieu, while the later applies site-directed mutagenesis to alter the physicochemical properties of the biocatalyst. The dominant effects of organic solvents on enzyme structure and function, and the potential of solvent and protein engineering to design enzymes to function optimally in organic media, are the major foci of this review.
Binding Sites↗