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At least 163 records · Page 9Linked to original sources
PubMed1997

A direct electrode-driven P450 cycle for biocatalysis.

The large potential of redox enzymes to carry out formation of high value organic compounds motivates the search for innovative strategies to regenerate the cofactors needed by their biocatalytic cycles. Here, we describe a bioreactor where the reducing power to the cycle is supplied directly to purified cytochrome CYP101 (P450cam; EC 1.14.15.1) through its natural redox partner (putidaredoxin) using an antimony-doped tin oxide working electrode. Required oxygen was produced at a Pt counter electrode by water electrolysis. A continuous catalytic cycle was sustained for more than 5 h and 2,600 enzyme turnovers. The maximum product formation rate was 36 nmol of 5-exo-hydroxycamphor/nmol of CYP101 per min.

Bioreactors↗
PubMed2000

Changing the donor cofactor of bovine alpha 1, 3-galactosyltransferase by fusion with UDP-galactose 4-epimerase. More efficient biocatalysis for synthesis of alpha-Gal epitopes.

Two fusion enzymes consisting of uridine diphosphogalactose 4-epimerase (UDP-galactose 4-epimerase, EC ) and alpha1, 3-galactosyltransferase (EC ) with an N-terminal His(6) tag and an intervening three-glycine linker were constructed by in-frame fusion of the Escherichia coli galE gene either to the 3' terminus (f1) or to the 5' terminus (f2) of a truncated bovine alpha1, 3-galactosyltransferase gene, respectively. Both fusion proteins were expressed in cell lysate as active, soluble forms as well as in inclusion bodies as improperly folded proteins. Both f1 and f2 were determined to be homodimers, based on a single band observed at about 67 kDa in SDS-polyacrylamide gel electrophoresis and on a single peak with a molecular mass around 140 kDa determined by gel filtration chromatography for each of the enzymes. Without altering the acceptor specificity of the transferase, the fusion with the epimerase changed the donor requirement of alpha1, 3-galactosyltransferase from UDP-galactose to UDP-glucose and decreased the cost for the synthesis of biomedically important Galalpha1,3Gal-terminated oligosaccharides by more than 40-fold. For enzymatic synthesis of Galalpha1,3Galbeta1,4Glc from UDP-glucose and lactose, the genetically fused enzymes f1 and f2 exhibited kinetic advantages with overall reaction rates that were 300 and 50%, respectively, higher than that of the system containing equal amounts of epimerase and galactosyltransferase. These results indicated that the active sites of the epimerase and the transferase in fusion enzymes were in proximity. The kinetic parameters suggested a random mechanism for the substrate binding of the alpha1, 3-galactosyltransferase. This work demonstrated a general approach that fusion of a glycosyltransferase with an epimerase can change the required but expensive sugar nucleotide to a less expensive one.

Animals↗
PubMed1991

Denaturation capacity: a new quantitative criterion for selection of organic solvents as reaction media in biocatalysis.

The process of reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was investigated using both our own and literature data, based on the results of kinetic and spectroscopic measurements. In all systems studied, the denaturation proceeded in a threshold manner, i.e. an abrupt change in catalytic and/or spectroscopic properties of dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of the reversible protein denaturation by organic solvents was developed, based on the widely accepted notion that an undisturbed water shell around the protein globule is a prerequisite for the retention of the native state of the protein. The quantitative treatment led to the equation relating the threshold concentration of the organic solvent with its physicochemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation described well the experimental data for all proteins tested. Based on the thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents, called the denaturation capacity (DC), was suggested. Different organic solvents, arranged according to their DC values, form the DC scale of organic solvents which permits theoretical prediction of the threshold concentration of any organic solvent for a given protein. The validity of the DC scale for this kind of prediction was verified for all proteins tested and a large number of organic solvents. The experimental data for a few organic solvents, such as formamide and N-methylformamide, did not comply with equations describing the denaturation model. Such solvents form the group of so-called 'bad' solvents; reasons for the occurrence of 'bad' solvents are not yet clear. The DC scale was further extended to include also highly nonpolar solvents, in order to explain the well-known ability of enzymes to retain catalytic activity and stability in biphasic systems of the type water/water-immiscible organic solvent. It was quantitatively demonstrated that this ability is accounted for by the simple fact that nonpolar solvents are not sufficiently soluble in water to reach the inactivation threshold concentration.

Catalysis↗
PubMed2002

Microbial carboxyl esterases: classification, properties and application in biocatalysis.

Esterases (EC 3.1.1.x) represent a diverse group of hydrolases catalyzing the cleavage and formation of ester bonds and are widely distributed in animals, plants and microorganisms. Beside lipases, a considerable number of microbial carboxyl esterases have also been discovered and overexpressed. This review summarizes their properties and classification. Special emphasis is given on their application in organic synthesis for the resolution of racemates and prostereogenic compounds. In addition, recent results for altering their properties by directed evolution are presented.

Bacteria↗
PubMed2006

Application of biocatalysis to drug metabolism: preparation of mammalian metabolites of a biaryl-bis-sulfonamide AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor potentiator using Actinoplanes missouriensis.

LY451395 (2-propanesulfonamide, N-[(2R)-2-[4'-[2-[methylsulfonyl)amino]ethyl][1,1'-biphenyl]-4-yl]propyl]-) is a potent and highly selective potentiator of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. It is a biaryl-bis-sulfonamide and is known to be highly metabolized in preclinical species. In those metabolism studies, the metabolite structures were proposed exclusively by the analysis of mass spectrometric data. Although mass spectrometry is clearly a technique of choice for rapid identification of drug metabolites, occasionally, nuclear magnetic resonance spectroscopy is required to unambiguously assign and characterize, particularly, the regio- and stereochemistry of metabolic changes. Nuclear magnetic resonance spectroscopy, in general, is less sensitive than other detection methods and demands several micrograms of material for the analysis. To support full structure characterization of metabolites by NMR, in this study we demonstrated the application of a microbial-based surrogate biocatalytic system to produce sufficient amounts of the mammalian metabolites of LY451395. The results revealed that incubation of LY451395 with Actinoplanes missouriensis NRRL B3342 generated several metabolites that were previously detected in the in vivo metabolism studies of the preclinical species. Subsequent large-scale bioconversion resulted in the isolation of seven mammalian metabolites in milligram quantities for structural characterization by nuclear magnetic resonance spectroscopy. Furthermore, a selected group of metabolites generated from the microbial conversion served as analytical standards to monitor and quantify drug metabolites during clinical investigations.

Biotechnology↗
PubMed1996

New designs of macroporous polymers and supports: from separation to biocatalysis.

Reactive polymers play many roles, from supports for solid-phase synthesis or catalysis to media for separations. Although macroporous polymer beads that provide high reactive capacities and excellent solvent tolerance are well established, approaches to monosized beads with optimized pore structures or multiple chemistries segregated within pores of different sizes have expanded their realm of application. Polymer monoliths containing intricate pore networks can be obtained in any desired shape by a simple molding process and provide unique advantages such as fast kinetics, high reactivity, and high throughput. Applications ranging from immobilized enzyme reactors to fast media for the separation of synthetic or biopolymers are presented.

Catalysis↗