Smart engineering of various enzymes for asymmetric synthesis of chiral molecules on industrial scale

The commercialization of sustainable enzymatic and microbial catalysis technology is gaining increasing priority for the synthesis of chiral compounds from achiral precursors that require high selectivity and high substrate load. Thus, the fast and cost-effective development of novel biocatalytic processes using an integrated platform offers significant opportunities to fine chemical, pharmaceutical, food & feed, material and related industries. Please click Additional Files below to see the full abstract

Sustainable biocatalytic synthesis of β-hydroxyl-α-amino acids on an industrial scale

The development and commercialization of sustainable enzymatic and microbial catalysis technologies is gaining increasing priority to reduce the environmental impact of chemical and related industries. Enzymes offer, as common platform, significant opportunities for innovations to enhance production capabilities to meet these new reduced impact demands, whilst retaining product quality and keeping costs down. To enable cost efficient biocatalytic processes, however, high selectivity, high activity, high substrate loadings and tolerance to organic solvents are required. This is in general not sufficiently displayed by natural enzymes, despite providing high selectivity and activity on native substrates under physiological conditions. As a solution, enzyme engineering allows us to optimize any enzyme to a powerful catalyst that overcomes these boundaries and can be conveniently applied under industrial conditions. As an industrial example, we will discuss the synthesis of β-hydroxyl-α-amino acids, important chiral building blocks in pharmaceutical and fine chemical industry. Current chemical synthesis protocols employ hazardous and environmentally unfriendly methods, thus, a replacement by safe and green biocatalysis is desired. However, wild type aldolases do not meet the anticipated target criteria for selectivity, activity and solvent tolerance under process conditions. Nonetheless, smart bioinformatics guided directed evolution, enabled us to develop an enzyme variant which meets the industrial target for the sustainable (βR)-β-Hydroxy-4-nitro-L-phenylalanine production at multi ton scale, which is a precursor for chloramphenicol. Remarkably, this new synthetic route to chloramphenicol, enabled by our engineered aldolase, is 5 steps shorter than the traditional chemical route, avoiding the use of various hazardous and environmentally unfriendly reactants. Please click Additional Files below to see the full abstract

Wide-field compensation of monochromatic eye aberrations: expected performance and design trade-offs

Contiene: fórmulas y 6 ilustraciones.The optical quality of the human eye varies across the visual field. Hence an exact compensation of the eye aberration for a given field point can give rise to a less-than-optimum compensation in neighboring field regions. We have studied some aspects of this problem and present here an approach to design wide-field (,10°) optically thin correcting elements, e.g., phase plates, deformable mirrors, and liquid-crystal displays. Their expected performance is assessed using actual eye aberration data. Particular attention is given to the design of elements providing a minimum averaged rms residual aberration and those providing a nearly uniform rms residual aberration across a given field.Work supported by the Spanish Ministerio de Ciencia y Tecnología, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I 1 D 1 I), DPI2002-04370-C02. - This paper was published in Journal of the Optical Society of America A, and is made available as an electronic reprint with the permission of OSA. The paper can be found at the following URL on the OSA website: http://www.opticsinfobase.org/abstract.cfm?URI=josaa-20-1-1. Systematic or multiple reproduction or distribution to multiple locations via electronic or other means is prohibited and is subject to penalties under law.Peer reviewe

CaLB Catalyzed Conversion of ε-Caprolactone in Aqueous Medium. Part 1: Immobilization of CaLB to Microgels

The enzymatic ring-opening polymerization of lactones is a method of increasing interest for the synthesis of biodegradable and biocompatible polymers. In the past it was shown that immobilization of Candida antarctica lipase B (CaLB) and the reaction medium play an important role in the polymerization ability especially of medium ring size lactones like ε-caprolactone (ε-CL). We investigated a route for the preparation of compartmentalized microgels based on poly(glycidol) in which CaLB was immobilized to increase its esterification ability. To find the ideal environment for CaLB, we investigated the acceptable water concentration and the accessibility for the monomer in model polymerizations in toluene and analyzed the obtained oligomers/polymers by NMR and SEC. We observed a sufficient accessibility for ε-CL to a toluene like hydrophobic phase imitating a hydrophobic microgel. Comparing free CaLB and Novozym® 435 we found that not the monomer concentration but rather the solubility of the enzyme, as well as the water concentration, strongly influences the equilibrium of esterification and hydrolysis. On the basis of these investigations, microgels of different polarity were prepared and successfully loaded with CaLB by physical entrapment. By comparison of immobilized and free CaLB, we demonstrated an effect of the hydrophobicity of the microenvironment of CaLB on its enzymatic activity

In silico screening of transaminase using semi-empirical QM/MM approach

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TransCent: Computational enzyme design by transferring active sites and considering constraints relevant for catalysis

BACKGROUND: Computational enzyme design is far from being applicable for the general case. Due to computational complexity and limited knowledge of the structure-function interplay, heuristic methods have to be used. RESULTS: We have developed TransCent, a computational enzyme design method supporting the transfer of active sites from one enzyme to an alternative scaffold. In an optimization process, it balances requirements originating from four constraints. These are 1) protein stability, 2) ligand binding, 3) pKa values of active site residues, and 4) structural features of the active site. Each constraint is handled by an individual software module. Modules processing the first three constraints are based on state-of-the-art concepts, i.e. RosettaDesign, DrugScore, and PROPKA. To account for the fourth constraint, knowledge-based potentials are utilized. The contribution of modules to the performance of TransCent was evaluated by means of a recapitulation test. The redesign of oxidoreductase cytochrome P450 was analyzed in detail. As a first application, we present and discuss models for the transfer of active sites in enzymes sharing the frequently encountered triosephosphate isomerase fold. CONCLUSION: A recapitulation test on native enzymes showed that TransCent proposes active sites that resemble the native enzyme more than those generated by RosettaDesign alone. Additional tests demonstrated that each module contributes to the overall performance in a statistically significant manner

Comparison of Candida antarctica Lipase B Variants for Conversion of ε-Caprolactone in Aqueous Medium-Part 2

Enzyme-catalyzed ring-opening polymerization of lactones is a method of increasing interest for the synthesis of polyesters. In the present work, we investigated which changes in the structure of Candida antarctica lipase B (CaLB) shift the catalytic equilibrium between esterification and hydrolysis towards polymerization. Therefore, we present two concepts: (i) removing the glycosylation of CaLB to increase the surface hydrophobicity; and (ii) introducing a hydrophobic lid adapted from Pseudomonas cepacia lipase (PsCL) to enhance the interaction of a growing polymer chain to the elongated lid helix. The deglycosylated CaLB (CaLB-degl) was successfully generated by site-saturation mutagenesis of asparagine 74. Furthermore, computational modeling showed that the introduction of a lid helix at position Ala148 was structurally feasible and the geometry of the active site remained intact. Via overlap extension PCR the lid was successfully inserted, and the variant was produced in large scale in Pichia pastoris with glycosylation (CaLB-lid) and without (CaLB-degl-lid). While the lid variants show a minor positive effect on the polymerization activity, CaLB-degl showed a clearly reduced hydrolytic and enhanced polymerization activity. Immobilization in a hydrophobic polyglycidol-based microgel intensified this effect such that a higher polymerization activity was achieved, compared to the “gold standard” Novozym® 435

In silico screening of transaminase using semi-empirical QM/MM approach

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Semi-classical treatment of proton-neutron monopole interaction

We apply a time dependent variational method to a many-body Hamiltonian consisting of a spherical shelf model term, a proton-proton and neutron-neutron pairing interaction and a monopole particle-hole and particle-particle proton-neutron interaction. The variational state is a generalized BCS state where all T = 1 Cooper pairs with T-z = 0, +/- 1 are included. Stationary solutions correspond to generalized BCS equations and define the static ground state. The linearized equations of motion are of RPA type and describe small oscillations of the nuclear system around the static ground state. Numerical application is made for a one level case. In contrast to previous treatments, the proton-neutron particle-particle interaction is included first in the mean field equations, defining the quasiparticle approximation, and then the residual interaction is taken into account by the RPA approach. In this way one obtains a noncollapsing RPA ground state.Peer reviewe