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At least 73 records · Page 4Linked to original sources
PubMed2000

Modeling solid-to-solid biocatalysis: integration of six consecutive steps.

A quantitative model for the conversion of a solid-substrate salt to a solid-product salt in a batch bioreactor seeded with product crystals is presented. The overall process consists of six serial steps (with dissolution and crystallization each in themselves complex multistep processes): solid-salt dissolution, salt dissociation into an ionic substrate and a counter-ion, bioconversion accompanied by biocatalyst inactivation, complexation of the ionic product with the counter-ion, and salt crystal growth. In the model, the consecutive steps are integrated, including biocatalyst inactivation and assuming that salt dissociation and complexation of ions are at equilibrium. Model parameters were determined previously in separate independent experiments. To validate the model, either dissolved or solid Ca-maleate was converted to solid Ca-D-malate by permeabilized Pseudomonas pseudoalcaligenes in a batch bioreactor seeded with Ca-D-malate crystals. The model very well predicted the concentrations of all components in the liquid phase (Ca-maleate, Ca(2+), maleate(2-), D-malate(2-), and Ca-D-malate) and the amounts of the solid phases (Ca-maleate. H(2)O and Ca-D-malate. 3H(2)O), especially when high initial amounts of Ca-maleate. H(2)O and Ca-D-malate. 3H(2)O were present.

Bioreactors↗
PubMed2000

Phenylalanine ammonia-lyase: the use of its broad substrate specificity for mechanistic investigations and biocatalysis--synthesis of L-arylalanines.

Several fluoro- and chlorophenylalanines were found to be good substrates of phenylalanine ammonia-lyase (PAL/EC 4.3.1.5) from parsley. The enantiomerically pure L-amino acids were obtained in good yields by reaction of the corresponding cinnamic acids with 5M ammonia solution (buffered to pH 10) in the presence of PAL. The kinetic constants for nine different fluoro- and chlorophenylalanines do not provide a rigorous proof for but are consistent with the previously proposed mechanism comprising an electrophilic attack of the methylidene-imidazolone cofactor of PAL at the aromatic nucleus as a first chemical step. In the resulting Friedel-Crafts-type sigma complex the beta-protons are activated for abstraction and consequently the pro-S is abstracted by an enzymic base. Results from semi-empirical calculations combined with a proposed partial active site model showed a correlation between the experimental kinetic constants and the change in polarization of the pro-S Cbeta-H bond and heat of formation of the sigma complexes, thus making the electrophilic attack at the neutral aromatic ring plausible. Furthermore, while 5-pyrimidinylalanine was found to be a moderately good substrate of PAL, 2-pyrimidinylalanine was an inhibitor.

Apiaceae↗
PubMed2007

A versatile esterase from Bacillus subtilis: cloning, expression, characterization, and its application in biocatalysis.

An esterase from Bacillus subtilis DSM402 (BS2) was cloned and functionally expressed in E. coli. The enzyme is active up to 50 degrees C, and the V(max) (1449 mM/min) and K(M) values (119 mM) were determined using p-nitrophenyl acetate as substrate. BS2 belongs to the few hydrolases that can act on tertiary alcohols and was therefore used to resolve racemic acetates of selected tertiary alcohols, but also to selectively remove the tert-butyl ester protecting group from peptides. In addition, the enzyme shows promiscuous amidase activity.

Alcohols↗
PubMed2003

A combinatorial biocatalysis approach to an array of cholic acid derivatives.

A 39-member library of bile acid derivatives was prepared starting from 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid methyl ester using a combinatorial biocatalytic approach. A regioselective oxidation step, catalyzed by hydroxysteroid dehydrogenases, followed by an acylation step with a series of different acyl donors catalyzed by Candida antarctica lipase B, led to the modification of the bile acid scaffold. Each member of the library was obtained in high purity and good yield.

Acylation↗
PubMed2003

Use of the two-liquid phase concept to exploit kinetically controlled multistep biocatalysis.

The two-liquid phase concept was used to develop a whole cell biocatalytic system for the efficient multistep oxidation of pseudocumene to 3,4-dimethylbenzaldehyde. Recombinant Escherichia coli cells were employed to express the Pseudomonas putida genes encoding xylene monooxygenase, which catalyzes the multistep oxygenation of one methyl group of toluene and xylenes to corresponding alcohols, aldehydes, and acids. A fed-batch based two-liquid phase bioconversion was established with bis(2-ethylhexyl)- phthalate as organic carrier solvent and a phase ratio of 0.5; the product formation pattern, the impact of the nutrient feeding strategy, and the partitioning behavior of the reactants were studied. On the basis of the favorable conditions provided by the two-liquid phase system, engineering of the initial pseudocumene concentration allowed exploiting the complex kinetics of the multistep reaction for the exclusive production of 3,4-dimethyl- benzaldehyde. Further oxidation of the product to 3,4-dimethylbenzoic acid could be inhibited by suitable concentrations of pseudocumene or 3,4-dimethylbenzyl alcohol. The optimized biotransformation setup includes a completely defined medium with high iron content and a nutrient feeding strategy that avoids severe glucose limitation as well as high inhibitory glucose levels. Using such a system on a 2-liter scale, we were able to produce, within 14.5 h, 30 g of 3,4-dimethylbenzaldehyde as predominant reactant in the organic phase and reached a maximal productivity of 1.6 g per liter liquid volume per hour. The present study implicates that the two-liquid phase concept is an efficient tool to exploit the kinetics of multistep biotransformations in general.

Benzaldehydes↗
PubMed2001

Enzyme stabilization by covalent binding in nanoporous sol-gel glass for nonaqueous biocatalysis.

A unique nanoporous sol-gel glass possessing a highly ordered porous structure (with a pore size of 153 A in diameter) was examined for use as a support material for enzyme immobilization. A model enzyme, alpha-chymotrypsin, was efficiently bound onto the glass via a bifunctional ligand, trimethoxysilylpropanal, with an active enzyme loading of 0.54 wt%. The glass-bound chymotrypsin exhibited greatly enhanced stability both in aqueous solution and organic solvents. The half-life of the glass-bound alpha-chymotrypsin was >1000-fold higher than that of the native enzyme, as measured either in aqueous buffer or anhydrous methanol. The enhanced stability in methanol, which excludes the possibility of enzyme autolysis, particularly reflected that the covalent binding provides effective protection against enzyme inactivation caused by structural denaturation. In addition, the activity of the immobilized alpha-chymotrypsin was also much higher than that of the native enzyme in various organic solvents. From these results, it appears that the glass-enzyme complex developed in the present work can be used as a high-performance biocatalyst for various chemical processing applications, particularly in organic media. Published by John Wiley & Sons

Chymotrypsin↗
PubMed2001

Biocatalysis of isomerization of 1,2-benzisoxazole by bovine serum albumin in an organic solvent.

The catalytic properties of bovine serum albumin (BSA) have been studied rather extensively. The protein has shown catalytic activities toward a variety of reactions in aqueous media. However, there appears to be no reports of reactions catalyzed exclusively in organic media. In this study, we report the catalytic property of BSA on the isomerization of 1,2-benzisoxazole in an organic solvent. Kinetics were performed using a continuous flow system in a spectrophotometer at 330 nm. The catalysis showed a Michaelis-Menten behavior with an estimated catalytic efficiency (k(cat)/K(M)) of 8.77 M(-1)S(-1). The influence of water content and inhibitors was also studied. The activity of BSA in acetonitrile is approximately 20% of that in water under similar conditions. Inhibition studies show that the active site may be the pyridoxal binding site involving Lys-220.

Acetonitriles↗
PubMed2004

Enabling multienzyme biocatalysis using nanoporous materials.

Multistep reactions catalyzed by a covalently immobilized enzyme-cofactor-enzyme system were achieved. Lactate dehydrogenase (LDH), glucose dehydrogenase (GDH), and cofactor NADH were incorporated into two porous silica glass supports. One of the glass supports had pores of 30 nm in diameter, while the other was of 100-nm pore size. Effective shuttling of the covalently bound NADH between LDH and GDH was achieved, such that regeneration cycles of NADH/NAD(+) were observed. The glass of 30-nm pore size afforded enzyme activities that were about twice those observed for the glass of 100-nm pore size, indicating the former provided better enzyme-cofactor integration. The effect of the size of spacers was also examined. The use of longer spacers increased the reaction rates by approximately 18 times as compared to those achieved with glutaraldehyde linkage. It appeared that the concave configuration of the nanopores played an important role in enabling the multistep reactions. The same multienzyme system immobilized on nonporous polystyrene particles of 500-nm diameter was only approximately 2% active as the glass-supported system. It is believed that the nanoporous structure of the glass supports enhances the molecular interactions among the immobilized enzymes and cofactor, thus improving the catalytic efficiency of the system.

Catalysis↗
PubMed2005

Production of 2-phenylethanol and 2-phenylethylacetate from L-phenylalanine by coupling whole-cell biocatalysis with organophilic pervaporation.

An integrated bioprocess for the production of the natural rose-like aroma compounds, 2-phenylethanol (2-PE) and 2-phenylethylacetate (2-PEAc), from L-phenylalanine (L-phe) with yeasts was investigated. The hydrophobicity of the products leads to product inhibition, which can be compensated by in situ product removal (ISPR). An organophilic pervaporation unit, equipped with a polyoctylmethylsiloxane (POMS) membrane, was coupled via a bypass to a bioreactor and proved to be a suitable technique for the in situ removal of high-boiling products from culture broth. With batch cultures of the thermotolerant yeast Kluyveromyces marxianus CBS 600 in a standard medium at 35 degrees C, the use of pervaporation resulted in a double 2-PE concentration (2.2 g/L) and 1.3 g/L 2-PEAc, which only accumulated transiently in low concentrations during cultivation without ISPR. Using a previously optimized medium, the variation of the temperature from 30 degrees C to 40 degrees C caused an increase in the total conversion yield from 63% to 79%, corresponding to total product concentrations of 5.23 and 5.85 g/L, respectively. In the 40 degrees C batch experiment, the volumetric productivity (2-PE + 2-PEAc) during the exponential phase was 5.2 mmol/L h. While for 2-PE, there is still potential for further optimization, the more hydrophobic 2-PEAc was nearly completely removed from the aqueous culture broth (enrichment factor >400), resulting in highly aroma-enriched permeates. Due to the temperature-correlated performance of the pervaporation, the bioconversion was still efficient even at 45 degrees C (conversion yield: 69%). Surprisingly, at 45 degrees C, the molar ratio of the two products inverted and 2-PEAc turned out to be the main product (4.0 g/L), which opens easy control of the reaction's selectivity by external means. Retrofitting the process with interim heating and cooling equipment to use different temperature levels for cultivation and pervaporation resulted in a decreased yield and product concentration caused by multiple stress factors. The medium composition affected the pervaporation efficiency with molasses acting detrimental.

Acetates↗
PubMed2006

A model system for increasing the intensity of whole-cell biocatalysis: investigation of the rate of oxidation of D-sorbitol to L-sorbose by thin bi-layer latex coatings of non-growing Gluconobacter oxydans.

We developed a novel <50-microm thick nano-porous bi-layer latex coating for preserving Gluconobacter oxydans, a strict aerobe, as a whole cell biocatalyst. G. oxydans was entrapped in an acrylate/vinyl acetate co-polymer matrix (T (g) approximately 10 degrees C) and cast into 12.7-mm diameter patch coatings (cellcoat) containing approximately 10(9) CFU covered by a nano-porous topcoat. The oxidation of D-sorbitol to L-sorbose was used to investigate the coating catalytic properties. Intrinsic kinetics was studied in microbioreactors using a pH 6.0 D-sorbitol, phosphate, pyruvate (SPP) non-growth medium at 30 degrees C, and the Michaelis-Menten constants determined. By using a diffusion cell, cellcoat and topcoat diffusivities, optimized by arresting polymer particle coalescence by glycerol and/or sucrose addition, were determined. Cryo-FESEM images revealed a two-layer structure with G. oxydans surrounded by <40-nm pores. Viable cell density, cell leakage, and oxidation kinetics in SPP medium for >150 h were investigated. Even though the coatings were optimized for permeability, approximately 50% of G. oxydans viability was lost during cellcoat drying and further reduction was observed as the topcoat was added. High reaction rates per unit volume of coating (80-100 g/L x h) were observed which agreed with predictions of a diffusion-reaction model using parameters estimated by independent experiments. Cellcoat effectiveness factors of 0.22-0.49 were observed which are 20-fold greater than any previously reported for this G. oxydans oxidation. These nano-structured coatings and the possibility of improving their ability to preserve G. oxydans viability may be useful for engineering highly reactive adhesive coatings for multi-phase micro-channel and membrane bioreactors to dramatically increase the intensity of whole-cell oxidations.

Bioreactors↗