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-. Enantioselective catalysis is achieved with peptides with as few as five residues and scalemic catalysts show high chiral amplification. Here, we discuss the relevance of these-enzyme like catalysts to prebiotic processes, such as the role of small peptides in the formation of optically active cyanohydrins.

[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.

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. Using cyclohexanone monooxygenase to catalyse the reaction produces optically pure esters and lactones with exquisite enantiomeric excess values. Recently, these enzymes and their many applications in synthetic chemistry have been explored. The scale-up of these conversions has been examined with the idea of implementing the first commercial Baeyer-Villiger monooxygenase-based process. Here, we review the state-of-the-art situation for the scale-up and exploitation of these enzymes.

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. In the active enzyme conformation, the open lid participates in the substrate-binding site and contributes to substrate recognition. To address the role of the lid in CRL activity and specificity, we substituted the lid sequences from isoenzymes CRL3 and CRL4 in recombinant rCRL1, thus obtaining enzymes differing only in this stretch of residues. Swapping the CRL3 lid was sufficient to confer to CRL1 cholesterol esterase activity. On the other hand, a specific shift in the chain-length specificity was not observed. Chimeric proteins displayed different sensitivity to detergents in the reaction medium.

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.

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.

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. The document embraces the recommendations of the STRENDA initiative (Standards for Reporting Enzymology Data) in the context of pure enzymology and provides further guidelines and explanations on topics of crucial relevance for biocatalysis. In particular, guidelines are given on issues such as the selectivity, specificity, productivity and stability of biocatalysts, as well as on methodological problems related to reactions in multiphase systems. We believe that adoption and use of these guidelines could greatly increase the value and impact of published work in biocatalysis, and hence promote the further growth of applications.

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. Simulated moving bed chromatography is described in this work as an efficient and scalable separation technology able to meet the goals of both high efficiency as well as reduced solvent use. The main factors influencing method optimisation were identified and the parameters of temperature, pressure, weak adsorption, separation factor and robustness were examined with the aim to resolve bottlenecks. Under optimised conditions, the productivity of the process was 0.026g/(g(stationary)(phase) day) of pure regioisomers and the solvent consumption was 0.363L/g(feed).

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. Nevertheless, the increase was observed only for low (less than 100) 18-crown-6/lipase molar ratio, while at higher ratios, the activity decreased for both enzymes to values lower than those obtained in the absence of the additive. In 1,4-dioxane, the activation is lower for lipase BC (1.7-fold) and for CALB (1.5-fold). Concerning enantioselectivity, tested in the kinetic resolution of 6-methyl-5-hepten-2-ol, only in the case of CALB, an effect of the additive (the E value varied from about 120 to 280) was observed. In water, 4% (w/w) of 18-crown-6 caused a loss of activity in the hydrolysis of p-nitrophenyl laurate of about 88 and 99.75%, compared to that observed in the absence of the crown ether for CALB and lipase BC, respectively. These data and the conformational analysis of both lipases, carried out by FT/IR spectroscopy indicate that the enzyme inactivation in water and in organic solvents at 18-crown-6/lipase molar ratios, higher than 100 might be due to conformational changes caused by the additive. Instead, at molar ratios lower than 100, 18-crown-6 might increase the activity - particularly, in toluene - thanks to the fact that in its presence, the enzyme has an hydrogen bonds pattern, more similar to that in water. This suggests that the additive would be able to provide the enzyme with more water.

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.

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. Analogously, lipase enantioselectivity, expressed as the enantiomeric ratio, increased up to 2.8-fold in the presence of sugars. The conformation of the lipase was investigated by means of Fourier transform infrared spectroscopy (FT/IR) in water and as lyophilized powder. The infrared spectra of lyophilized lipase in the presence and, even more so, in the absence of sugars were different from that of the enzyme in water. In particular, the band at around 1,654/cm, typically assigned to alpha-helix, was less intense in the lyophilized samples. Nevertheless, the enzyme in the presence of sugars showed a decrease of the bands at 1,614-1,620/cm and at 1,680-1,695/cm that indicates a lower content of intermolecular beta-sheets (typical of protein aggregates). Additionally the increase of the component at 1,546/cm in the amide II region is consistent with a hydrogen bond pattern of the enzyme more similar to that shown in water. These results suggest that although sugars are not able to fully preserve the native secondary structure, they might contribute to reduce the conformational changes caused by protein/protein interactions. These factors in combinations with others (e.g., ability to reduce deleterious interactions between the enzyme and inert supports) make sugars (both mono- and disaccharides) an interesting class of additives for improving the performance of biocatalysts in organic solvents.

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. 539, 14-18]. The amide H/D exchange data show that the wild-type and mutant enzymes have similar global flexibility at 22 and 60 degrees C. However, the temperature dependence of the Stern-Volmer constant determined by acrylamide quenching shows that the increase in temperature affects the local flexibility differently, since the K(SV) increment is significantly higher for the wild-type than for the mutant enzyme over the range 18-45 degrees C. Interestingly, the corresponding van't Hoff plot (log K(SV) vs 1/T) proves nonlinear for the apo and holo wild-type and apo mutant enzymes, with a break at approximately 45 degrees C in all three cases due to a conformational change affecting the tryptophan microenvironment experienced by the quencher molecules. The Arrhenius and van't Hoff plots derived from the k(cat) and K(M) thermodependence measured with cyclohexanol and NAD(+) at different temperatures display an abrupt change of slope at 45-50 degrees C. This proves more pronounced in the case of the mutant enzyme compared to the wild-type enzyme due to a conformational change in the structure rather than to an overlapping of two or more rate-limiting reaction steps with different temperature dependencies of their rate constants. Three-dimensional analysis indicates that the observed conformational change induced by temperature is associated with the flexible loops directly involved in the substrate and coenzyme binding.

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.

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. However, the use of continuous membranes presents several disadvantages. Due to the mechanical characteristics of polyacrylamide, the gel must physically adhere onto a rigid support, which prevents it from collapsing. The support must have a highly porous structure in order to be permeable to proteins. The mechanical fragility of the membranes is one of the main problems that hinders the industrial scale application of ME separators. In order to overcome this problem, we propose to substitute the continuous membranes with a bed of gel beads of identical comonomer composition, obtained by an inverse emulsion polymerization process.

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.

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. A rationale for such decreases could be that the chimera lipase has a lower proportion of enzyme molecules in the open form. This might lead to a hindered access to the enzyme active site, thus affecting the catalytic activity.

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. The 3-D structures of the EST2 and AFEST enzymes were analyzed by molecular modeling to determine the effects of the mutations. Mutations were positioned differently in the structures, but in both cases caused small modifications around the active site and in the oxyanion loop.