Optimization of hydrolase efficiency in organic solvents

The application of hydrolases in organic solvents for synthetic purposes is a procedure routinely adopted in organic chemistry, especially for the preparation of chiral building blocks. Numerous studies have shed light on several aspects of the mechanism of hydrolase action in low-water environments. Procedures suitable to improve the catalytic efficiency of enzymes and productivity of the synthetic processes have been reported. These fundamental and applied investigations have made hydrolase-catalyzed reactions in organic solvents of industrial interest. In this article we describe and discuss various approaches adopted to optimize the performance of hydrolases in organic media, with special emphasis on the formulation of the biocatalysts which, under proper conditions, can display an activity equal to that displayed in aqueous buffers.     

Studies on the Enzymatic Acylation of Quinic Acid,Shikimic Acid,Shikimic Acid,and Their Derivatives in Organic Solvents

Quinic acid ( 1a ), shikimic acid ( 2 ), and their derivatives were acylated in organic solvents by several lipases and by the protease subtilisin Carlsberg. The most satisfactory results were obtained with methyl (or benzyl) quinate ( 7a (or 8a )) and lipase from Chromobacterium viscosum adsorbed on Celite, which showed an overshelming preference towards the acylation of OH–C(4). Under optimized conditions, the syntehtically useful 4-O -acetylquinate 8d was isolated in ca. 90% yield. On the other hand, acylation of methyl shikimate ( 10a ) showed no regioselectivity with any of the enzymes tested. A possible rationale for the different behavior of Chromobacterium viscosum lipase towards 7a and 10a is given, comparing the conformations of these two molecules, as deducted from ¹H-NMR and molecular-mechanics calculation.     

Modelling of Enzyme Properties in Organic Solvents

Summary.  In this article we review how molecular modeling techniques can be used to shed some light on the influence of organic solvents on the molecular characteristics of proteins and enzymes. Molecular dynamic simulations on bovine pancreas trypsin inhibitor, chymotrypsin, and subtilisin make it possible to get a deeper understanding into how increased intramolecular interactions improve conformational rigidity, thus explaining the lower reactivity and the higher thermostability of enzymes in non-aqueous media. The application of thermodynamics-based models allows first qualitative predictions on the selectivity of many reaction types; however, the application of quantum mechanical/molecular mechanical methods is required for the development of quantitative models of actual reactivity patterns. Received November 18, 1999. Accepted (revised) February 8, 2000     

The Candida antarctica lipase B catalysed kinetic resolution of seudenol in non-aqueous media of controlled water activity

For further investigation of the kinetic resolutions in transesterification reactions with the highly enantioselective Candida antarctica lipase B, an easy to study model reaction with one typical substrate, the Douglas Fir Beetle pheromone 3-methyl-2-cyclohexen-1-ol (Seudenol), was developed. The influence of the nature of the solvent and thermodynamic water activity was studied. Initial rates showed constant or progressively increasing values with increasing water activity. Enantioselectivity depended on the choice of solvent and descended in most cases with increasing water activity. No general correlations of enantioselectivity or activity with physicochemical constants of the solvent were found. However, at a water activity of 0.11 a tendency toward optimum hydrophobicity (i.elog P ≈ for enantioselectivity was observed. Enantiomeric ratios were in the range 8–32.     

A sensitive continuous-flow bioluminescent system for determining ethanol in serum and saliva

A continuous-flow system, based on NAD(P)H:FMN oxidoreductase and bacterial luciferase co-immobilized on a nylon coil placed in front of a photomultiplier, and alcohol dehydrogenase separately immobilized on a second nylon coil, is described for the assay of ethanol in serum and saliva. The flow is air-segmented and 5–50-μl samples are required. The sample throughput is 25–30 h^?1 with no carryover. The detection limit is 1 μmol l^?1 ethanol (50 pmol injected) and the response is linear between 50 and 2500 pmol of ethanol. The relative standard deviation is 3–10% for both within-day and between-day assays, and serum and saliva can be analyzed directly. The immobilized enzymes are satisfactorily stable and up to 900 samples can be analyzed with one enzyme reactor.     

Bioluminescence flow system for determination of branched-chain l-amino acids in serum and urine

A continuous-flow bioluminescence method for determinations of branched-chain l-amino acids in serum and urine is described. Serum can be analyzed directly after simple filtration. Response is linear for 20-2000 pmol in the biological matrix. Leucine dehydrogenase is immobilized onto a nylon coil separated from the reactor coil containing bacterial bioluminescence enzymes. The stability of the immobilized is high (lifetime > two months) and more than 900 samples can be analyzed with the use of a few mg of enzymes. The results obtained agree well with those obtained by ion-exchange chromatography (amino acid analyzer).     

Kinetics of chalcone oxidation by peroxide anion catalysed by poly-l-leucine

An insight into the kinetics, mechanism and optimum reaction conditions of the Julia-Colonna epoxidation has been gained using a soluble polyleucine catalyst.     

Enzyme-Mediated Regioselective Acylations of Flavonoid Disaccharide Monoglycosides

Flavonoid disaccharide monoglycosides have been acylated by the catalytic action of the protease subtilisin in anhydrous pyridine. The effects of the nature of the sugars and of the interglycosidic bonds on the regioselectivity of the reactions have been analyzed. The selectivity was excellent with rutin ( 1 ), hesperidin ( 2 ), naringin ( 6 ), and quercetin 3-O-[O-(β-D -glucopyranosyl)-(1→4)-α-L -rhamnoside] ( 9 ), giving single monoesters on their glucose moieties (see la , 2a , 6a , and 9b , resp.); quite interestingly, in the last compound, acylation did not occur at the free primary OH group but at the secondary OH? C(3?). On the other hand, a mixture of mono- and diesters was obtained with the flavonoid peltatoside ( 7 ).     

Alkylation of Adenine,Adenosine, and NAD+ with 1,3-Propanesultone. Synthesis of N6-(3-sulfonatopropyl)-NAD+, a new NAD+ derivative with substantial coenzyme activity

The reactivity of 1,3-propanesultone with adenine, adenosine, and NAD⁺ was studied in order to prepare N⁶-(3-sulfonatopropyl)-NAD⁺ ( 3b ), a new NAD⁺ derivative substituted at the purine moiety with sunstantial coanzyme activity for several dehydrogenases. The regiochemistry of the alkylation at the purione nucleus was investigated by UV, ¹H-NMR, and FAB-MS proved to be a powerful tool for determining the molecular weight of these polar and poorly volatile compounds. In addition, regular framgmentation of 3b and other NAD⁺ derivatives was observed.