Guidelines for reporting of biocatalytic reactions.

Enzymes and whole cells are being increasingly applied in research and industry, but the adoption of biocatalysis relies strongly on useful scientific literature. Unfortunately, too many published papers lack essential information needed to reproduce and understand the results. Here, members of the scientific committee of the European Federation of Biotechnology Section on Applied Biocatalysis (ESAB) provide practical guidelines for reporting experiments.

Preparative-scale separation by simulated moving bed chromatography of biocatalytically produced regioisomeric lactones.

The separation of enantiomerically pure, but regioisomeric compounds by chromatography is attractive for the production of chiral compounds, if the process leads to high-purity products in high yield with excellent throughput and reduced solvent use. The biocatalytic Baeyer-Villiger oxidation of racemic ketones gives rapid access to an equimolar mixture of regioisomeric lactones with excellent enantiomeric purity. The existing separation methods have so far represented a bottleneck for preparative applications of this technology.

Can an inactivating agent increase enzyme activity in organic solvent? Effects of 18-crown-6 on lipase activity, enantioselectivity, and conformation.

Lipase from Burkholderia cepacia (lipase BC) and lipase B from Candida antarctica (CALB) show an increase of the transesterification activity in toluene (up to 2.4- and 1.7-fold, respectively), when lyophilized with 18-crown-6.

Exploitation of the alcohol dehydrogenase-acetone NADP-regeneration system for the enzymatic preparative-scale production of 12-ketochenodeoxycholic acid.

The performance of a new NADP-regeneration system, based on the use of alcohol dehydrogenase (ADH)-acetone, has been investigated for the regioselective oxidation of cholic acid (1) to 12-ketochenodeoxycholic acid (2). Enzymes stabilities and substrate and/or product inhibitory effects under defined synthetic reaction conditions have been evaluated. The optimized system, based on a 4% w/v solution of 1 in a reaction mixture containing 25% v/v acetone, allowed the preparative scale transformation of 1 into 2 with a 92% conversion.

Temperature-induced conformational change at the catalytic site of Sulfolobus solfataricus alcohol dehydrogenase highlighted by Asn249Tyr substitution. A hydrogen/deuterium exchange, kinetic, and fluorescence quenching study.

A combination of hydrogen/deuterium exchange, fluorescence quenching, and kinetic studies was used to acquire experimental evidence for the crystallographically hypothesized increase in local flexibility which occurs in thermophilic NAD(+)-dependent Sulfolobus solfataricus alcohol dehydrogenase (SsADH) upon substitution Asn249Tyr. The substitution, located at the adenine-binding site, proved to decrease the affinity for both coenzyme and substrate, rendering the mutant enzyme 6-fold more active when compared to the wild-type enzyme [Esposito et al. (2003) FEBS Lett.

Polyamino acids as synthetic enzymes: mechanism, applications and relevance to prebiotic catalysis.

Polyamino acids, such as polyleucine, behave as synthetic enzymes in the asymmetric epoxidation of chalcone and other electron-deficient alkenes (the Julià-Colonna reaction). The influences of reaction conditions, of the molecular structure of the catalysts and of the scaling-up of the process on the enantioselectivity of the reaction have been determined. The kinetics and mechanism have been investigated using a soluble PEG-polyleucine conjugate, which behaves in a similar way to an enzyme, showing saturation kinetics for both chalcone and HOO-.

Mono- and disaccharides enhance the activity and enantioselectivity of Burkholderia cepacia lipase in organic solvent but do not significantly affect its conformation.

Sucrose, trehalose, and mannitol were colyophilized with lipase from Burkholderia cepacia and their effects on the activity and enantioselectitivity of the enzyme evaluated using as model reactions the transesterification between n-octanol or 6-methyl-5-hepten-2-ol with vinyl acetate. The lipase co-lyophilized with sugars showed an activity which was up to 4.7-fold higher (at a sugar/lipase ratio >or= 20) than that observed without sugar.

[Cp*Rh(bpy)(H2O)]2+ as a coenzyme substitute in enzymatic oxidations catalyzed by Baeyer-Villiger monooxygenases.

[Cp*Rh(bpy)(H2O)]2+ was applied as a flavin regenerating reagent in BVMO catalyzed oxidations of organic sulfides to chiral sulfoxides.

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.

Activity and enantioselectivity of wildtype and lid mutated Candida rugosa lipase isoform 1 in organic solvents.

The activity and enantioselectivity of lipase 1 from Candida rugosa and of a chimera enzyme obtained by replacing the lid of isoform 1 with the lid of isoform 3 were compared in organic solvents. The alcoholysis of chloro ethyl 2-hydroxy hexanoate with methanol and of vinyl acetate with 6-methyl-5-hepten-2-ol were used as model reactions in different reaction conditions. The chimera enzyme was less active and enantioselective than the wildtype in all the conditions tested.

Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes.

The fungus Candida rugosa produces multiple lipase isoenzymes (CRLs) with distinct differences in substrate specificity, in particular with regard to selectivity toward the fatty acyl chain length. Moreover, isoform CRL3 displays high activity towards cholesterol esters. Lipase isoenzymes share over 80% sequence identity but diverge in the sequence of the lid, a mobile loop that modulates access to the active site.

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.

Towards large-scale synthetic applications of Baeyer-Villiger monooxygenases.

Biocatalysis is coming of age, with an increasing number of reactions being scaled-up and developed. The diversity of reactions is also increasing and oxidation reactions have recently been considered for scale-up to commercial processes. One important chemical conversion, which is difficult to achieve enantio- or enantiotopo- selectively, is the Baeyer-Villiger (BV) oxidation of ketones.

Separation of proteins in a multicompartment electrolyzer with chambers defined by a bed of gel beads.

Multicompartment electrolyzers (MEs) with isoelectric membranes were introduced in 1989 for purifying proteins in an electric field. At the basis of ME technology there are membranes consisting of cross-linked copolymers of acrylamide and acrylamido monomers bearing protolytic groups. The technology employed for casting the membranes is an extension of the isoelectric focusing in immobilized pH gradient technique for which specific acrylamido monomers, known with the trade name of Immobiline, have been developed.

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.

Modification of the enantioselectivity of two homologous thermophilic carboxylesterases from Alicyclobacillus acidocaldarius and Archaeoglobus fulgidus by random mutagenesis and screening.

The esterase genes est2 from Alicyclobacillus acidocaldarius and AF1716 from Archaeoglobus fulgidus were subjected to error-prone PCR in an effort to increase the low enantioselectivity of the corresponding enzymes EST2 and AFEST, respectively. The model substrate ( RS)- p-nitrophenyl-2-chloropropionate was chosen to produce ( S)-2-chloropropionic acid, an important intermediate in the synthesis of some optically pure compounds, such as the herbicide mecoprop. In the case of EST2, a single mutant, Leu212Pro, was obtained showing a slightly enhanced preference toward the ( S) substrate; in the case of AFEST, a double mutant, Leu101Ile/Asp117Gly, was obtained showing an increased preference in the opposite direction.

Modeling enzyme reactivity in organic solvents and water through computer simulations.

In this article, we review how molecular modeling techniques can be used to shed light on how water and organic solvents influence the reactivity of enzymes. The application of thermodynamics-based models allowed the first qualitative predictions on the selectivity of many reaction types. However, it was with the application of quantum mechanical/molecular mechanical (QM/MM) methods that quantitative models of actual reactivity patterns could be realistically formulated.