Rational Guidelines for the Two-Step Scalability of Enzymatic Polycondensation: Experimental and Computational Optimization of the Enzymatic Synthesis of Poly(glycerolazelate).

The lipase-catalyzed polycondensation of azelaic acid and glycerol is investigated according to a Design-of-Experiment approach that helps to elucidate the effect of experimental variables on monomer conversion, M and regioselectivity of acylation of glycerol. Chemometric analysis shows that after 24 h the reaction proceeds regardless of the presence of the enzyme. Accordingly, the biocatalyst was removed after a first step of synthesis and the chain elongation continued at 80 °C.

Evolving biocatalysis to meet bioeconomy challenges and opportunities.

The unique selectivity of enzymes, along with their remarkable catalytic activity, constitute powerful tools for transforming renewable feedstock and also for adding value to an array of building blocks and monomers produced by the emerging bio-based chemistry sector. Although some relevant biotransformations run at the ton scale demonstrate the success of biocatalysis in industry, there is still a huge untapped potential of catalytic activities available for targeted valorization of new raw materials, such as waste streams and CO. For decades, the needs of the pharmaceutical and fine chemistry sectors have driven scientific research in the field of biocatalysis.

Investigating the Role of Conformational Effects on Laccase Stability and Hyperactivation under Stress Conditions.

Fungal laccase from Steccherinum ochraceum 1833 displays remarkable stability under different harsh conditions: organic/buffer mixtures, thermal treatment, and microwave radiation. The behavior is particularly significant in the light of the sharp inactivation observed for two different fungal laccases. Laccase from S.

BioGPS descriptors for rational engineering of enzyme promiscuity and structure based bioinformatic analysis.

A new bioinformatic methodology was developed founded on the Unsupervised Pattern Cognition Analysis of GRID-based BioGPS descriptors (Global Positioning System in Biological Space). The procedure relies entirely on three-dimensional structure analysis of enzymes and does not stem from sequence or structure alignment. The BioGPS descriptors account for chemical, geometrical and physical-chemical features of enzymes and are able to describe comprehensively the active site of enzymes in terms of "pre-organized environment" able to stabilize the transition state of a given reaction.

Efficient immobilisation of industrial biocatalysts: criteria and constraints for the selection of organic polymeric carriers and immobilisation methods.

Efficient immobilisation protocols are the result of perfect matching of factors depending on the enzyme, the process and the support for immobilisation. Physical-chemical phenomena, such as partition, solvation and diffusion, strongly affect the efficiency of the biocatalyst in each specific reaction system. Therefore, tailored solutions must be developed for each specific process of interest.

Lipases immobilization for effective synthesis of biodiesel starting from coffee waste oils.

Immobilized lipases were applied to the enzymatic conversion of oils from spent coffee ground into biodiesel. Two lipases were selected for the study because of their conformational behavior analysed by Molecular Dynamics (MD) simulations taking into account that immobilization conditions affect conformational behavior of the lipases and ultimately, their efficiency upon immobilization. The enzymatic synthesis of biodiesel was initially carried out on a model substrate (triolein) in order to select the most promising immobilized biocatalysts.

Endo- and exo-inulinases: enzyme-substrate interaction and rational immobilization.

Three-dimensional models of exoinulinase from Bacillus stearothermophilus and endoinulinase from Aspergillus niger were built up by means of homology modeling. The crystal structure of exoinulinase from Aspergillus awamori was used as a template, which is the sole structure of inulinase resolved so far. Docking and molecular dynamics simulations were performed to investigate the differences between the two inulinases in terms of substrate selectivity.

Synbeads porous-rigid methacrylic support: application to solid phase peptide synthesis and characterization of the polymeric matrix by FTIR microspectroscopy and high resolution magic angle spinning NMR.

Porous and rigid methacrylic Synbeads were optimized and applied efficiently to the solid phase peptide synthesis with the objective of improving significantly volumetric yields (0.33 mol/L calculated on the basis of maximum chemical accessibility, i.e.

Volsurf computational method applied to the prediction of stability of thermostable enzymes.

A computational model for the quantitative prediction of protein thermostability has been developed by means of the Volsurf method. A data set of 22 enzymes of reported thermostability in water systems, for the most part coming from thermophilic and hyperthermophilic organisms, has been built up. Molecular descriptors of the protein surface have been calculated and their role in the stabilization of the macromolecule has been analyzed by a multivariate statistical approach.

Computational methods to rationalize experimental strategies in biocatalysis.

Computational methods are more and more widely applied in biocatalysis to gain rational guidelines, to orient experimental planning and, ultimately, to avoid expensive and time-consuming experiments. In this respect, molecular modelling, multivariate statistical analysis and chemometrics in general are useful computational tools, although they follow completely different investigative approaches.

Optimized polymer-enzyme electrostatic interactions significantly improve penicillin G amidase efficiency in charged PEGA polymers.

Hydrolytic yields as high as 80% were obtained by using penicillin G amidase (PGA) on substrates anchored on optimized positively charged PEGA polymers. By increasing the amount of permanent charges inside the polymer, electrostatic interactions between the positively charged PEGA(+) and the negatively charged PGA (pI = 5.2-5.

An innovative application of the "flexible" GRID/PCA computational method: study of differences in selectivity between PGAs from Escherichia coli and a Providentia rettgeri mutant.

The original GRID/PCA technique was adapted for the development of a tool potentially useful for the plan of a research strategy in rational enzyme design. The use of the MOVE directive of GRID made it possible to partially take into account protein flexibility, and the multivariate analysis was used as an instrument for focusing only on relevant information related to the differences in enzyme substrate selectivities. The comparison of two different penicillin G acylases, from Escherichia coli and from Providentia rettgeri, was used as a case study; these enzymes are very similar and their reported selectivities differ only for a couple of mutations around the active site.

Nonswelling macroporous synbeads for improved efficiency of solid-phase biotransformations.

An application of novel, highly porous nonswelling resins (Synbeads) for enzymatic catalysis on solid supports is reported. These new resins combine easy handling of the beads, chemical stability, improved accessibility of proteins and higher productivity relative to swelling polymers. The present study demonstrates that the resin porosity greatly affects the efficiency in solid-phase biotransformations and that Synbead resins are valuable alternatives to swelling polymers for solid-phase chemistry and biocatalysis.

A homology model of penicillin acylase from Alcaligenes faecalis and in silico evaluation of its selectivity.

A three-dimensional model of the relatively unknown penicillin acylase from Alcaligenes faecalis (PA-AF) was built up by means of homology modeling based on three different crystal structures of penicillin acylase from various sources. An in silico selectivity study was performed to compare this homology model to the structure of the Escherichia coli enzyme (PA-EC) in order to find any selectivity differences between the two enzymes. The program GRID was applied in combination with the principal component analysis technique to identify the regions of the active sites where the PAs potentially engage different interactions with ligands.

GRID/tetrahedral intermediate computational approach to the study of selectivity of penicillin G acylase in amide bond synthesis.

Molecular modelling was used to investigate the catalytic site of penicillin G acylase (PGA) by building up a simple enzyme-ligand model able to describe and predict the enzyme selectivity. The investigation was based on a double computational approach: first, the GRID computational procedure was applied to gain a qualitative description of the chemical features of the PGA active site; second, a classical "transition state approach" was used to simulate the tetrahedral intermediates and to evaluate their energies. GRID calculations employed different probes which gave a complete description of the chemical interactions occurring upon binding of different ligands, thus indicating those structures having good affinity with the active site of the enzyme.