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PubMed2000

Molecular modeling and biocatalysis: explanations, predictions, limitations, and opportunities.

Rapid advances in structural biology have revealed the three-dimensional structures of many biocatalysts. Molecular modeling is the tool that links these structures with experimental observations. As a qualitative tool, current modeling methods are extremely useful. They can explain, on a molecular level, unusual features of reactions. They can predict how to increase the selectivity either by substrate modification or by site-directed mutagenesis. Quantitative predictions, for example the degree of enantioselectivity, are still not reliable, however. Modeling is limited also by the availability of three-dimensional structures. Most current modeling involves hydrolases, especially proteases and lipases, but structures for other types of enzymes are starting to appear.

Camphor 5-Monooxygenase↗
PubMed2003

Solid/gas biocatalysis: an appropriate tool to study the influence of organic components on kinetics of lipase-catalyzed alcoholysis.

The influence of the addition of an extra component in a gaseous reaction medium, on the kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by immobilized lipase B from Candida antarctica was studied in a continuous solid/gas reactor. In this reactor, the solid phase is composed of a packed enzymatic sample, which is percolated by gaseous nitrogen, simultaneously carrying gaseous substrates and additional components to the enzyme while removing reaction products. The system permits to set thermodynamic activity of all gaseous components (substrates or not) independently at the desired values. This allows in particular to study the influence of an extra added component at a constant thermodynamic activity value, contrary to classical solid/liquid system, which involves large variations of thermodynamic activity of added solvent, when performing full kinetic studies. Alcohol inhibition constant (K(I)) and methyl propionate and propanol dissociation constants (K(MP) and K(P)) have been determined in the solid/gas reactor in the presence of 2-methyl-2-butanol, and compared with values previously obtained in the absence of added component and in the presence of water. Complementary experiments were carried out in the presence of an apolar compound (hexane) and led to the conclusion that the effect of added organic component on lipase-catalyzed alcoholysis is related to their competitive inhibitory character towards first substrate methyl propionate. The comparison of data obtained in liquid or with gaseous 2-methyl-2-butanol shows that lower K(MP) and K(I) are found in gaseous medium, which would correspond on the one hand to a lower acylation rate k(2), and on the other hand to a higher binding rate k(1) between substrate and free enzyme in gaseous medium.

1-Propanol↗
PubMed2004

Magnetite-containing spherical silica nanoparticles for biocatalysis and bioseparations.

The simultaneous entrapment of biological macromolecules and nanostructured silica-coated magnetite in sol-gel materials using a reverse-micelle technique leads to a bioactive, mechanically stable, nanometer-sized, and magnetically separable particles. These spherical particles have a typical diameter of 53 +/- 4 nm, a large surface area of 330 m(2)/g, an average pore diameter of 1.5 nm, a total pore volume of 1.427 cm(3)/g and a saturated magnetization (M(S)) of 3.2 emu/g. Peroxidase entrapped in these particles shows Michaelis-Mentan kinetics and high activity. The catalytic reaction will take place immediately after adding these particles to the reaction solution. These enzyme entrapping particles catalysts can be easily separated from the reaction mixture by simply using an external magnetic field. Experiments have proved that these catalysts have a long-term stability toward temperature and pH change, as compared to free enzyme molecules. To further prove the application of this novel magnetic biomaterial in analytical chemistry, a magnetic-separation immunoassay system was also developed for the quantitative determination of gentamicin. The calibration for gentamicin has a working range of 200-4000 ng/mL, with a detection limit of 160 ng/mL, which is close to that of the fluorescent polarization immunoassay (FPIA) using the same reactants.

Adsorption↗
PubMedDate not supplied

High temperature biocatalysis by chemically modified cytochrome C.

Chemically modified cytochrome c with poly(ethylene glycol) (PEG) showed activity at temperatures higher than 100 degrees C and to be highly thermostable. The molecular size of PEG moieties and the coupling site affected the thermal stabilization. An optimal PEG/protein mass ratio of 2.8 was found, producing a fully thermostable biocatalyst at 80 degrees C. Site-directed mutagenesis on yeast cytochrome c showed an increased thermostabilization when lysine 79 residue, localized at the edge of the active site, was replaced by a nonreactive residue. Tertiary, secondary, and active-site structures were analyzed by fluorescence, CD, and UV/visible spectroscopies. Besides its disordered structure, the pegylated protein showed a lower unfolding rate at the active-site than the unmodified ones. A shell-like structure seems to protect the heme environment, in which PEG is coiled on the protein surface with a primary shield of rigid water molecules solvating the hydrophilic region of bound-PEG, and the PEG hydrophobic regions interacting with the hydrophobic clusters on protein surface.

Animals↗
PubMedDate not supplied

Improving the cytochrome P450 enzyme system for electrode-driven biocatalysis of styrene epoxidation.

Cytochrome P450 enzymes catalyze a vast array of oxidative and reductive biotransformations that are potentially useful for industrial and pharmaceutical syntheses. Factors such as cofactor utilization and slow reaction rates for nonnatural substrates limit their large-scale usefulness. This paper reports several improvements that make the cytochrome P450cam enzyme system more practical for the epoxidation of styrene. NADH coupling was increased from 14 to 54 mol %, and product turnover rate was increased from 8 to 70 min(-1) by introducing the Y96F mutation to P450cam. Styrene and styrene oxide mass balance determinations showed different product profiles at low and high styrene conversion levels. For styrene conversion less than about 25 mol %, the stoichiometry between styrene consumption and styrene oxide formation was 1:1. At high styrene conversion, a second doubly oxidized product, alpha-hydroxyacetophenone, was formed. This was also the exclusive product when Y96F P450cam acted on racemic, commercially available styrene oxide. The alpha-hydroxyacetophenone product was suppressed in reactions where styrene was present at saturating concentrations. Finally, styrene epoxidation was carried out in an electroenzymatic reactor. In this scheme, the costly NADH cofactor and one of the three proteins (putidaredoxin reductase) are eliminated from the Y96F P450cam enzyme system.

Camphor 5-Monooxygenase↗
PubMedDate not supplied

Solvent selection strategies for extractive biocatalysis.

This report follows the development of systematic solvent screening strategies for the identification of superior pure solvents and introduces techniques for the identification of effective coextractants. Specifically, methods to predict the biocompatibility and extractant capability of solvents are discussed. Biocompatibility is predicted by using heuristic data or the correlations between bioactivity and the logarithm of the partition coefficient of the solvent or the concentration of solvent in the cell membrane. A computer program, known as the extractant screening program or ESP, has been developed to effectively predict the behavior of virtually any product in any solvent/aqueous system. It is demonstrated that a biocompatible yet poor solvent can be mixed with a toxic solvent that has better extractant properties to yield a mixture with improved solvent characteristics that is still biocompatible. The fact that solvents do not mix in an ideal manner is exploited by using ESP to identify solvent mixtures that are still biocompatible at relatively high concentrations of toxic solvent.

Animals↗
PubMedDate not supplied

Asymmetric synthesis with immobilized yeast in organic solvents: equilibrium conversion and effect of reactant partitioning on whole cell biocatalysis.

A newly isolated strain of the yeast Saccharomyces cerevisiae is investigated for the biocatalytic reduction of ketones and the oxidation of alcohols in organic solvents. The yeast cells are immobilized by entrapment within calcium alginate beads and are found to possess the ability to stereoselectively reduce prochiral ketones and oxidize chiral alcohols to equilibrium conversions. The effect of reactant partitioning on the initial rate of the reactions is also investigated. The observed initial rates are found to vary inversely with reactant partitioning between the organic solvent and the biocatalyst beads. A kinetic model is developed to describe the initial reaction rate of hexanone reduction as a function of substrate concentration within the alginate beads.

Alcohols↗
PubMed2002

Molecular imprinting of enzymes with water-insoluble ligands for nonaqueous biocatalysis.

Attaining higher levels of catalytic activity of enzymes in organic solvents is one of the major challenges in nonaqueous enzymology. One of the most successful strategies for enhancing enzyme activity in organic solvents involves tuning the enzyme active site by molecular imprinting with substrates or their analogues. Unfortunately, numerous imprinters of potential importance are poorly soluble in water, which significantly limits the utility of this method. In the present study, we have developed strategies that overcome this limitation of the molecular-imprinting technique and that thus expand its applicability beyond water-soluble ligands. The solubility problem can be addressed either by converting the ligands into a water-soluble form or by adding relatively high concentrations of organic cosolvents, such as tert-butyl alcohol and 1,4-dioxane, to increase their solubility in the lyophilization medium. We have succeeded in applying both of these strategies to produce imprinted thermolysin, subtilisin, and lipase TL possessing up to 26-fold higher catalytic activity in the acylation of paclitaxel and 17beta-estradiol compared to nonimprinted enzymes. Furthermore, we have demonstrated for the first time that molecular imprinting and salt activation, applied in combination, produce a strong additive activation effect (up to 110-fold), suggesting different mechanisms of action involved in these enzyme activation techniques.

Catalysis↗
PubMed2002

An enzyme library approach to biocatalysis: development of nitrilases for enantioselective production of carboxylic acid derivatives.

The discovery, from Nature, of a large and diverse set of nitrilases is reported. The utility of this nitrilase library for identifying enzymes that catalyze efficient production of valuable hydroxy carboxylic acid derivatives is demonstrated. Unprecedented enantioselectivity and substrate scope are highlighted for three newly discovered and distinct nitrilases. For example, a wide array of (R)-mandelic acid derivatives and analogues were produced with high rates, yields, and enantiomeric excesses (95-99% ee). We also have found nitrilases that provide direct access to (S)-phenyllactic acid and other aryllactic acid derivatives, again with high yields and enantioselectivities. Finally, different nitrilases have been discovered that catalyze enantiotopic hydrolysis of 3-hydroxyglutaronitrile to afford either enantiomer of 4-cyano-3-hydroxybutyric acid with high enantiomeric excesses (>95% ee). The first enzymes are reported that effect this transformation to furnish the (R)-4-cyano-3-hydroxybutyric acid which is a precursor to the blockbuster drug Lipitor.

Aminohydrolases↗
PubMed2002

Smart nanotubes for bioseparations and biocatalysis.

Tube-shaped nanostructures (nanotubes) have a number of attributes that make them potentially useful for biomedical applications such as drug delivery/detoxification and enzyme immobilization. Template synthesis provides a route for preparing monodisperse nanotubes of nearly any size and composed of nearly any material. We show here that template-synthesized silica nanotubes can be biochemically functionalized such that they act as biocatalysts and highly selective nano-phase extraction agents for bioseparations. For example, nanotubes containing an enantioselective antibody selectively extract the enantiomer of a drug molecule that binds to the antibody, relative to the enantiomer that has no specific interaction with the antibody. Nanotubes containing the enzyme glucose oxidase function as nanophase bioreactors to catalyze the oxidation of glucose.

Antibodies↗
PubMed2006

Electrodes modified with the phase transition polymer and heme peptide: biocatalysis and biosensing with tunable activity and dynamic range.

An electrode was modified with a phase transition polymer, poly(N-isopropylacrylamide), and the polymer was further modified with a peroxidase model compound, heme peptide (HP). As the polymer layer shrank at temperatures above 30-40 degrees C, the catalytic activity of the HP molecules for H(2)O(2) reduction improved, and simultaneously, the number of HP molecules that can communicate electrochemically with the electrode increased. As a result, the catalytic current for H(2)O(2) reduction in the shrunken state was 4 times larger than that in the swollen state. This reversible change was exploited for tuning the sensitivity and dynamic range of the HP electrode in H(2)O(2) biosensing. The dynamic range in inhibition-based biosensing of imidazole derivatives was also tunable.

Acrylic Resins↗
PubMed2003

Applying slow-release biocatalysis to the asymmetric reduction of ethyl 4-chloroacetoacetate.

Amberlite XAD 2 resin enhanced the asymmetric reduction of ethyl 4-chloroacetoacetate (ECA) to S-4-chloro-3-hydroxybutyric acid ethyl ester as catalyzed by Saccharomyces cerevisiae. The absorbed ECA was released slowly to the solution during the reaction so that the substrate inhibition and the spontaneous chemical hydrolysis of ECA were considerably lessened. With 75 g resin l(-1) and ECA at 74 mM, the reaction yield and the product's optical purity increased from 75% to 84% and from 88% to 93%, respectively.

Absorption↗
PubMed2003

Biocatalysis of nitro substituted styrene oxides by non-conventional yeasts.

Yeast strains (410) from more than 45 different genera were screened for the enantioselective hydrolysis of nitro substituted styrene oxides. These strains included 262 yeasts with known epoxides hydrolase activity for various other epoxides. Epoxide hydrolase activity for p-nitrostyrene oxide (pNSO) (177 strains) and m-nitrostyrene oxide (mNSO) (148 strains) was widespread in the yeasts, while activity for o-nitrostyrene oxide (oNSO) was less ubiquitous (22 strains). The strains that displayed enantioselectivity in the hydrolysis of one or more of the nitro substituted styrene oxides (35 strains) were also screened against styrene oxide (SO). Rhodosporidium toruloides UOFS Y-0471 displayed the highest enantioselectivity for pNSO (ee 55%, yield 35%) while Rhodotorula glutinis UOFS Y-0653 displayed the highest enantioselectivity for mNSO (ee > 98%, yield 29%), oNSO (ee 39%, yield 19%) and SO (ee > 98%, yield 19%). (R)-Styrene oxide was preferentially hydrolysed to the corresponding (R)-diol with retention of configuration at the stereogenic centre. In the case of the nitro substituted styrene oxides the absolute configurations of the remaining epoxides and the formed diols were not established.

Catalysis↗
PubMed2004

Toward the synthesis of pyroglutamate lauroyl ester: biocatalysis versus chemical catalysis.

The synthesis of dodecyl pyroglutamate (or pyroglutamate lauroyl ester) was achieved in a two-step process involving a pyroglutamic acid alkyl ester intermediate. The reaction was carried out either by lipase or by chemical catalysis using ion exchange resin. Among the various tested lipases, the one from Candida antarctica B gave the best results allowing 73% formation of the desired ester after 6 h. Comparing the efficiency of this latter lipase with the one of Amberlyst IR120H resin in catalyzing this reaction, the biocatalyst gave a molar yield of pyroglutamate lauroyl ester of 79% compared to 69% when using the ion exchange resin starting with 1.04 mmol substrate in each case.

Catalysis↗