Roberto Bovara

Resolution of (±)-trans-sobrerol by lipase PS-catalyzed transesterification and effects of organic solvents on enantioselectivity

Abstract Resolution of the mucolytic drug (±)- trans -sobrerol ( 1 ) was achieved by transesterification with vinyl acetate in organic media, catalyzed by free or immobilized Lipase PS. The enantioselectivity of the enzyme was markedly influenced by the nature of the organic solvent, but there was no correlation between enantiomeric ratio values (70–500) and either the hydrophobicity or the dielectric constant of the medium. With the enzyme immobilized onto Hyflo Super Cell and t -amyl alcohol as the solvent, the selectivity of Lipase PS for (−)- 1 was extremely high and, at 50 % conversion both (−)- trans -sobrerol and (+)- trans -sobrerol monoacetate were obtained in practically 100 % optical purity

Effects of water activity on Vmax and KM of lipase catalyzed transesterification in organic media

SummaryThe Vmax and KM of various forms of lipase from Pseudomonas cepacia (powder, adsorbed onto Celite or covalently linked to polyethylene glycol) were determined in organic solvents preequilibrated to water activities (aw) from <0.1 to 0.84. The model reaction was the transesterification between n-octanol and vinyl butyrate. It was found that KM for the nucleophile increased with increasing aw for all three lipase forms. Vmax increased with increasing aw for polyethylene glycol-lipase, whereas there was an optimum at intermediate aw values (0.11 – 0.38) for lipase powder and Celite-immobilized lipase.

Water activity does not influence the enantioselectivity of lipase PS and lipoprotein lipase in organic solvents

SummaryThe activity and enantioselectivity of Lipase PS from Pseudomonas cepacia and lipoprotein lipase from Pseudomonas sp. were investigated in organic solvents preequilibrated to water activities ranging from <0.1 to 0.53, using as a model reaction the transesterification between (±)-sulcatol and vinyl acetate. Variations of water activity markedly influenced the transesterification rate but did not modify the enantioselectivity of the two enzymes.

Chemiluminescent flow sensor for the determination of Paraoxon and Aldicarb pesticides

Abstract A chemiluminescence-based flow method for the determination of some organophorus and carbamate pesticides based on inhibition of acetylcholinesterase was developed. Acetylcholinesterase in solution or immobilized on methacrylate beads (Eupergit C) was coupled to choline oxidase and peroxidase immobilized on Eupergit C. In this system choline formed by acetylcholinesterase was oxidized by choline oxidase and the H 2 O 2 produced was determined via the luminol/peroxidase luminescent reaction. The detection limits (3 σ) for Paraoxon and Aldicarb were 0.75 μg 1 −1 and 4 μg 1 −1 , respectively, when soluble acetylcholinesterase was used under the following optimized experimental conditions: 56 μM luminol in working solution, sample volume 60 μl, flow-rate 0.3 ml min −1 and 60 min incubation time. The flow sensor device using all the three enzymes in the immobilized form had a higher detection limit of 125 μg 1 −1 for Paraoxon. The mid-range relative standard deviation ( n =10) using 1 mM standard substrate solution was 3.7%. The recovery from contaminated samples (soil, vegetables) varied from 81 to 108%. The results obtained by the developed methods were in good agreement with those obtained by a commonly used colorimetric test.

Enzymatic oxidoreduction of steroids in two-phase systems: Effects of organic solvents on enzyme kinetics and evaluation of the performance of different reactors

Abstract The preparative-scale regio- and stereospecific oxidoreduction of the hydroxyl-keto groups of a variety of steroids, catalysed by several hydroxysteroid dehydrogenases, has been carried out in water—organic solvent two-phase systems. The organic solvents employed were ethyl and butyl acetate. The steroid transformations were specific, complete, and one-step. In the systems containing ethyl acetate, the transformation rates were different from those obtained in butyl acetate because of the different effects of the two solvents on such parameters as K m , V max , product inhibition constant, and the partition coefficient of steroids. Various reactors (free enzymes in shaken vessels, immobilized enzymes in shaken vessels, and fixed bed reactors) have been made and their performance compared. The suitability to two-phase systems of several enzymatic NAD(P)(H)-regenerating systems has also been studied.

Bioluminescent flow sensor for D-(-)-lactate

Abstract Previously, a bioluminescent flow sensor was developed for the determination of the content of l -lactate in biological fluids (serum, plasma, ventricular fluid) by monitoring the reduced form of nicotinamide adenine dinucleotide (NADH), produced by immobilized lactate dehydrogenase (LDH), with bacterial bioluminescent enzymes immobilized on a separate nylon coil. Based on a similar scheme, a sensor has now been developed for d -(−)-lactate. The co-immobilization of alanine aminotransferase (ALT) with d -LDH improved the lactate transformation to 40–60%. The response was linear from 1 to 100 μmol 1 −1 at 25 °C for the LDH-ALT reactor. The intra- and inter-assay relative standard deviations were less than 75% and the recoveries ranged from 93 to 106%. The results agreed well with those obtained with a spectrophotometric method and applications to d -(−)-lactate determination in serum, milk and microorganism extracts are reported together with those for l -(+)-lactate.

Activity, stability, and conformation of methoxypoly(ethylene glycol)-subtilisin at different concentrations of water in dioxane

The transesterification activity, autolysis, thermal stability and conformation of subtilisin Carlsberg, made soluble in dioxane by covalent linking to methoxypoly(ethylene glycol) (PEG), were investigated as a function of the concentration of water in the medium. Electrospray mass spectrometry showed that the modified enzyme preparation was a mixture of proteins containing from 2 to 5 covalently linked PEG chains per subtilisin molecule. PEG-subtilisin catalyzed transesterification between vinyl butyrate and 1-hexanol was optimum at 0.55 MH(2)O, while hydrolysis prevailed above 2 MH(2)O. There was a decrease in the overall enzyme activity with increasing water concentration because of autolysis and denaturation of the enzyme. Subtilisin powder and celite-immobilized subtilisin were more stable and less susceptible to autolysis than the PEG-modified enzyme. Circular dichroism and intrinsic protein-fluorescence studies showed that the conformation of PEG-subtilisin did not change as a function of water concentrations between 0 and 9 M. The K(m,app) value of PEG-subtilisin for 1-hexanol was highly influenced by water, which behaved as a competitive inhibitor in the transesterification reaction with an affinity for the enzyme similar to that of the alcohol. The K(m,app) for the acylating agent was not significantly modified by water. Lyoprotectants such as sorbitol and free PEG did not influence the activity of PEG-subtilisin but notably increased the activity of subtilisin powder and celite-immobilized subtilisin. The addition of 1.7-5.5 M water, however, rendered enzyme preparations containing no additives as active as those containing the lyoprotectants.

Preparation of 12-ketochenodeoxycholic acid from cholic acid using coimmobilized 12α-hydroxysteroid dehydrogenase and glutamate dehydrogenase with NADP+ cycling at high efficiency

Abstract 12-Ketochenodeoxycholic acid, an essential intermediate in the synthesis of chenodeoxycholic acid, has been enzymatically prepared from cholic acid. The specific oxidation of the 12α-hydroxyl group of cholic acid with NADP + was catalysed by 12α-hydroxysteroid dehydrogenase (12α-hydroxysteroid: NAD + oxidoreductase, EC 1.1.1.176), and the regeneration of NADP + was obtained through the glutamate dehydrogenase ( l -glutamate:NADP + oxidoreductase, EC 1.4.1.4) catalysed reduction of α-ketoglutarate. The two enzymes were immobilized onto Sepharose CL-4B activated with tresyl chloride. The coimmobilized enzymes showed a cycling efficiency for the coenzyme similar to that of the free enzymes. High concentrations of cholic acid (up to 4%, w/v) were completely and specifically transformed into the 12-keto derivative using amounts of cofactor about 1600 times lower on a molar basis. The immobilized enzymes maintained ∼70% of the initial activity after 2 months of continuous use.

Continuous flow analyses of nadh using bacterial bioluminescent enzymes immobilized on nylon

Abstract A continuous flow bioluminescent method for NADH analysis has been developed using nylon 6 as a solid support. Bacterial luciferase and NAD:FMN oxidoreductase are covalently co-immobilized to a nylon coil (1 m × 1.0 mm ID). The reactor (nylon coil) is placed in front of a photomultiplier tube inside a luminometer linked to an air segmented continuous flow system. Response is linear from 1 to 2500 pmol/tube allowing determination of NADH at picomolar levels. Inter and intra assay precision of the method is satisfactory (5-10%). Range of sample volume injected is 5-100 μl. More than 20 samples per hour can be analyzed and no carryover was observed. The stability of the nylon immobilized enzymes is high (over 2 months) and over 500 samples can be analyzed with a few mg of enzymes.

Activity and selectivity of some hydrolases in enantiomeric solvents

SummaryThe activity and the regio- and enantioselectivity of five lipases and one protease were investigated in the two enantiomeric solvents (R)-carvone and (S)-carvone. It was found that in all cases enzyme activity changed as a function of solvent configuration and that, for the same enzyme, it was higher in (R)-carvone or in (S)-carvone depending on the nature of the substrate. Instead, no significative variation of regio- and enantioselectivity was observed moving from one enantiomeric solvent to the other.