ACCOUNTING AND FISCAL TREATMENT OF VALUE-ADDED TAX FOR CONSTRUCTION SERVICES

In this article we aim to approach the influence of the recent changes in the fiscal legislation regarding VAT on the accounting and taxing treatments of the construction contracts. In the case studies, we are considering the application of the "VAT upon collection" system, as well as the split VAT payment mechanism, according to the Law 227/2015 regarding the Fiscal Code and the Law 275/2017 for the approval of Government Ordinance 23/2017

Biocatalyst-artificial metalloenzyme cascade based on alcohol dehydrogenase

© The Royal Society of Chemistry. Chemo-enzymatic cascades of enzymes with transition metal catalysts can offer efficient synthetic strategies, but their development is challenging due to the incompatibility between proteins and transition metal complexes. Rhodium catalysts can be combined with alcohol dehydrogenases to regenerate nicotinamide cofactors using formate as the hydride donor. However, their use is limited, due to binding of the metals to residues on the enzyme surface, leading to mutual enzyme and catalyst inactivation. In this work, we replaced the zinc from Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) with Rh(iii) catalysts possessing nitrogen donor ligands, by covalent conjugation to the active site cysteine, to create artificial metalloenzymes for NADP+ reduction. TbADH was used as protein scaffold for both alcohol synthesis and the recycling of the cofactor, by combination of the chemically modified species with the non-modified recombinant enzyme. Stability studies revealed that the incorporation of the catalysts into the TbADH pocket provided a shielding environment for the metal catalyst, resulting in increased stability of both the recycling catalyst and the ADH. The reduction of a representative ketone using this novel alcohol dehydrogenase-artificial formate dehydrogenase cascade yielded better conversions than in the presence of free metal catalyst

Rational design of thermostable carbonic anhydrase mutants using molecular dynamics simulations

The stability of enzymes is critical for their application in industrial processes, which generally require different conditions from the natural enzyme environment. Both rational and random protein engineering approaches have been used to increase stability, with the latter requiring extensive experimental effort for the screening of variants. Moreover, some general rules addressing the molecular origin of protein thermostability have been established. Herein, we demonstrate the use of molecular dynamics simulations to gain molecular level understanding of protein thermostability and to engineer stabilizing mutations. Carbonic anhydrase (CA) is an enzyme with a high potential for biotechnological carbon capture applications, provided it can be engineered to withstand the high temperature process environments, inevitable in most gas treatment units. In this study, we used molecular dynamics simulations at 343, 353, and 363 K to study the relationship between structure flexibility and thermostability in bacterial α-CAs and applied this knowledge to the design of mutants with increased stability. The most thermostable α-CA known, TaCA from Thermovibrio ammonificans, had the most rigid structure during molecular dynamics simulations, but also showed regions with high flexibility. The most flexible amino acids in these regions were identified from root mean square fluctuation (RMSF) studies, and stabilizing point mutations were predicted based on their capacity to improve the calculated free energy of unfolding. Disulfide bonds were also designed at sites with suitable geometries and selected based on their location at flexible sites, assessed by B-factor calculation. Molecular dynamics simulations allowed the identification of five mutants with lower RMSF of the overall structure at 400 K, compared to wild-type TaCA. Comparison of free-energy landscapes between wild-type TaCA and the most promising mutants, Pro165Cys–Gln170Cys and Asn140Gly, showed an increased conformational stability of the mutants at 400 K

Iron-catalyzed indolizine synthesis from pyridines, diazo compounds, and alkynes

The iron (III) catalyzed synthesis of indolizines from commercially available alkyne, pyridine, and diazo precursors is reported. This mild, expedient method is tolerant of various solvents and proceeds with as little as 0.25 mol % [Fe(TPP)Cl]. Significantly, this multicomponent reaction is compatible with electrophilic alkynes; control experiments demonstrate the importance of the catalyst in promoting pyridinium ylide formation over background reactivity

Computationally driven design of an artificial metalloenzyme using supramolecular anchoring strategies of iridium complexes to alcohol dehydrogenase

Artificial metalloenzymes (ArMs) confer non-biological reactivities to biomolecules, whilst taking advantage of the biomolecular architecture in terms of their selectivity and renewable origin. In particular, the design of ArMs by the supramolecular anchoring of metal catalysts to protein hosts provides flexible and easy to optimise systems. The use of cofactor dependent enzymes as hosts gives the advantage of both a (hydrophobic) binding site for the substrate and a cofactor pocket to accommodate the catalyst. Here, we present a computationally driven design approach of ArMs for the transfer hydrogenation reaction of cyclic imines, starting from the NADP+-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH). We tested and developed a molecular docking workflow to define and optimize iridium catalysts with high affinity for the cofactor binding site of TbADH. The workflow uses high throughput docking of compound libraries to identify key structural motifs for high affinity, followed by higher accuracy docking methods on smaller, focused ligand and catalyst libraries. Iridium sulfonamide catalysts were selected and synthesised, containing either a triol, a furane, or a carboxylic acid to provide the interaction with the cofactor binding pocket. IC50 values of the resulting complexes during TbADH-catalysed alcohol oxidation were determined by competition experiments and were between 4.410 mM and 0.052 mM, demonstrating the affinity of the iridium complexes for either the substrate or the cofactor binding pocket of TbADH. The catalytic activity of the free iridium complexes in solution showed a maximal turnover number (TON) of 90 for the reduction of salsolidine by the triol-functionalised iridium catalyst, whilst in the presence of TbADH, only the iridium catalyst with the triol anchoring functionality showed activity for the same reaction (TON of 36 after 24 h). The observation that the artificial metalloenzymes developed here lacked stereoselectivity demonstrates the need for the further investigation and optimisation of the ArM. Our results serve as a starting point for the design of robust artificial metalloenzymes, exploiting supramolecular anchoring to natural NAD(P)H binding pockets

Oxidation of cadaverine by putrescine oxidase from Rhodococcus erythropolis

BACKGROUNDPutrescine oxidase (EC 1.4.3.10) is of interest for the microbial production of unsubstituted platform nitrogen (N-)heterocycles, because it only requires inexpensive oxygen as co-substrate. Putrescine oxidase from Rhodococcus erythropolis (Re-PuO) was shown previously to catalyze the oxidation of cadaverine; however, there is little information in the literature about the robustness of this enzyme for biotechnological applications. The aim of this study was to investigate the suitability of Re-PuO for the bioproduction of 1-piperideine from cadaverine under different reaction conditions.RESULTSThe formation of 1-piperideine catalyzed by Re-PuO was demonstrated using o-aminobenzaldehyde as a reagent to trap the cyclic imine and shift the equilibrium for cyclization. A direct assay of Re-PuO activity for cadaverine oxidation was then implemented, by monitoring oxygen consumption. Characterization of the reaction mixture by 1H NMR and mass spectrometry confirmed the presence of piperideine dimers and trimers, yet the quantification of the reaction products could not be achieved. The optimum temperature and pH conditions for enzyme activity were determined as 55 °C and 8.5, respectively. At pH 7.5, the enzyme retained its activity after 65 h incubation at 25 °C, but lost 75% of its activity after 1 h incubation at 55 °C. The enzyme showed no substrate inhibition at concentrations as high as 100 mmol L–1 cadaverine. Complete biotransformation of cadaverine was observed in whole cells at physiological conditions.CONCLUSIONSThese results successfully demonstrate the potential of putrescine oxidase for the bioproduction of N-heterocycles from cadaverine. © 2021 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI)

Engineering of Thermovibrio ammonificans carbonic anhydrase mutants with increased thermostability

Carbonic anhydrase can be used as an additive to improve the efficiency of carbon capture and utilisation processes, due to its ability to increase the rate of CO2 absorption into solvents. Successful industrial application requires robust carbonic anhydrases, able to withstand process conditions and to perform consistently over long periods of time. Tolerance of high temperatures, pH and salt concentrations are particularly desirable features. We have previously used molecular dynamics simulations to rationally design four mutants of Thermovibrio ammonificans carbonic anhydrase with increased rigidity, and we hypothesized that this will result in an increased thermostability. Herein, we report on the successful recombinant expression and characterization of these mutants. Four of the TaCA variants showed increased stability at 90 ᵒC during 1 h, compared to wild-type. Two out of the four mutations predicted by the theoretical studies resulted in marked stabilization of the protein, with up to 3-fold higher time of half-life for mutant N140 G compared to the wild-type enzyme at 60 ᵒC. A significantly 50-fold increased ester hydrolysis activity was also observed with the most thermostable variant at 95 ᵒC compared to 25 ᵒC, suggesting an increased flexibility of the active site at high temperatures