Novel concepts for the biocatalytic synthesis of second-generation biodiesel

Biodiesel is synthesized by the transesterification of triglycerides of oils with short-chain alcohols, such as methanol and ethanol. According to the Renewable Energy Directive guidelines (RED II 2018/2001/EU) the contribution of advanced biofuels, which do not include edible oils, towards the overall EU target, is at 1% in 2025 and at least 3.5% in 2030. Bioprocesses that valorize non-edible oils for the production of second-generation biodiesel could play a critical role in achieving this goal. Immobilized lipases, as well as other enzyme classes, such as cutinases and acyltransferases, are utilized as biocatalysts for this process. For the sustainability of the process, renewable materials can be used as immobilization matrices, or even enzymes anchored on the cells as whole-cell biocatalysts. Membrane reactors can also be employed to facilitate the enzymatic transesterification by conducting a continuous enzymatic reaction and simultaneously separate the products in a single operation. The advances on the aforementioned fast-pacing fields are presented in this work

Smectite clays as solid supports for immobilization of beta-glucosidase:Synthesis, characterization, and biochemical properties

Nanomaterials as solid supports can improve the efficiency of immobilized enzymes by reducing diffusional limitation as well as by increasing the surface area per mass unit and therefore improving enzyme loading. In this work, beta-glucosidase from almonds was immobilized on two smectite nanoclays. The resulting hybrid biocatalysts were characterized by a combination of powder X-ray diffraction, X-ray photoelectron spectroscopy, thermogravimetric. analysis, differential thermal analysis, and infrared spectroscopy. Biochemical studies showed an improved thermostability of the immobilized enzyme as well as enhanced performance at higher temperatures and in a wider pH range

Bioinformatic analysis of fold-type III PLP-dependent enzymes discovers multimeric racemases

Pyridoxal-5′-phosphate (PLP)-dependent enzymes are ubiquitous in nature and catalyze a variety of important metabolic reactions. The fold-type III PLP-dependent enzyme family is primarily comprised of decarboxylases and alanine racemases. In the development of a multiple structural alignment database (3DM) for the enzyme family, a large subset of 5666 uncharacterized proteins with high structural, but low sequence similarity to alanine racemase and decarboxylases was found. Compared to these two classes of enzymes, the protein sequences being the object of this study completely lack the C-terminal domain, which has been reported important for the formation of the dimer interface in other fold-type III enzymes. The 5666 sequences cluster around four protein templates, which also share little sequence identity to each other. In this work, these four template proteins were solubly expressed in Escherichia coli, purified, and their substrate profiles were evaluated by HPLC analysis for racemase activity using a broader range of amino acids. They were found active only against alanine or serine, where they exhibited Michaelis constants within the range of typical bacterial alanine racemases, but with significantly lower turnover numbers. As the already described racemases were proposed to be active and appeared to be monomers as judged from their crystal structures, we also investigated this aspect for the four new enzymes. Here, size exclusion chromatography indicated the presence of oligomeric states of the enzymes and a native-PAGE in-gel assay showed that the racemase activity was present only in an oligomeric state but not as monomer. This suggests the likelihood of a different behavior of these enzymes in solution compared to the one observed in crystalline form

Bioinformatic analysis of fold-type III PLP-dependent enzymes discovers multimeric racemases

Pyridoxal-5′-phosphate (PLP)-dependent enzymes are ubiquitous in nature and catalyze a variety of important metabolic reactions. The fold-type III PLP-dependent enzyme family is primarily comprised of decarboxylases and alanine racemases. In the development of a multiple structural alignment database (3DM) for the enzyme family, a large subset of 5666 uncharacterized proteins with high structural, but low sequence similarity to alanine racemase and decarboxylases was found. Compared to these two classes of enzymes, the protein sequences being the object of this study completely lack the C-terminal domain, which has been reported important for the formation of the dimer interface in other fold-type III enzymes. The 5666 sequences cluster around four protein templates, which also share little sequence identity to each other. In this work, these four template proteins were solubly expressed in Escherichia coli, purified, and their substrate profiles were evaluated by HPLC analysis for racemase activity using a broader range of amino acids. They were found active only against alanine or serine, where they exhibited Michaelis constants within the range of typical bacterial alanine racemases, but with significantly lower turnover numbers. As the already described racemases were proposed to be active and appeared to be monomers as judged from their crystal structures, we also investigated this aspect for the four new enzymes. Here, size exclusion chromatography indicated the presence of oligomeric states of the enzymes and a native-PAGE in-gel assay showed that the racemase activity was present only in an oligomeric state but not as monomer. This suggests the likelihood of a different behavior of these enzymes in solution compared to the one observed in crystalline form

PCSK9 Inhibitors for the Management of Dyslipidemia in People with Type 2 Diabetes: How Low is Too Low?

Diabetic patients are considered to be at high risk for the development of atherosclerotic disease. Management of hypercholesterolemia is of paramount importance to optimize cardiovascular outcomes in this subset of patients and statins are regarded as the mainstay of treatment. However, the recent advent of PCSK-9 inhibitors has provided a useful alternative in the management of dyslipidemia, especially when statins cannot be tolerated or when hypolipidemic targets cannot be achieved. In this review, we discuss current trends in their use, and we focus on their role in the management of diabetic dyslipidemia

S-Adenosyl-L-Homocysteine Hydrolase Inhibition by a Synthetic Nicotinamide Cofactor Biomimetic

S-adenosyl-L-homocysteine (SAH) hydrolases (SAHases) are involved in the regulation of methylation reactions in many organisms and are thus crucial for numerous cellular functions. Consequently, their dysregulation is associated with severe health problems. The SAHase-catalyzed reaction is reversible and both directions depend on the redox activity of nicotinamide adenine dinucleotide (NAD+) as a cofactor. Therefore, nicotinamide cofactor biomimetics (NCB) are a promising tool to modulate SAHase activity. In the present in vitro study, we investigated 10 synthetic truncated NAD+ analogs against a SAHase from the root-nodulating bacterium Bradyrhizobium elkanii. Among this set of analogs, one was identified to inhibit the SAHase in both directions. Isothermal titration calorimetry (ITC) and crystallography experiments suggest that the inhibitory effect is not mediated by a direct interaction with the protein. Neither the apo-enzyme (i.e., deprived of the natural cofactor), nor the holo-enzyme (i.e., in the NAD+-bound state) were found to bind the inhibitor. Yet, enzyme kinetics point to a non-competitive inhibition mechanism, where the inhibitor acts on both, the enzyme and enzyme-SAH complex. Based on our experimental results, we hypothesize that the NCB inhibits the enzyme via oxidation of the enzyme-bound NADH, which may be accessible through an open molecular gate, leaving the enzyme stalled in a configuration with oxidized cofactor, where the reaction intermediate can be neither converted nor released. Since the reaction mechanism of SAHase is quite uncommon, this kind of inhibition could be a viable pharmacological route, with a low risk of off-target effects. The NCB presented in this work could be used as a template for the development of more potent SAHase inhibitors

Air Flow Study around Isolated Cubical Building in the City of Athens under Various Climate Conditions

This study focuses on the airflow and pollutant dispersion around an isolated cubical building located in a warm Mediterranean climate, taking into account the local microclimate conditions (of airflow, albedo of building and soil, and air humidity) using a large-eddy simulation (LES) numerical approach. To test the reliability of computations, comparisons are made against the SILSOE cube experimental data. Three different scenarios are examined: (a) Scenario A with adiabatic walls, (b) Scenario B with the same constant temperature on all the surfaces of the building, and (c) Scenario C using convective and radiative conditions imposed by the local microclimate. For the first two cases the velocity and temperature fields resulting are almost identical. In the third case, the resulting temperature on the surfaces of the building is increased by 19.5%, the center (eye) of the wake zone is raised from the ground and the maximum pollutant concentration is drastically reduced (89%)

Air Flow Study around Isolated Cubical Building in the City of Athens under Various Climate Conditions

This study focuses on the airflow and pollutant dispersion around an isolated cubical building located in a warm Mediterranean climate, taking into account the local microclimate conditions (of airflow, albedo of building and soil, and air humidity) using a large-eddy simulation (LES) numerical approach. To test the reliability of computations, comparisons are made against the SILSOE cube experimental data. Three different scenarios are examined: (a) Scenario A with adiabatic walls, (b) Scenario B with the same constant temperature on all the surfaces of the building, and (c) Scenario C using convective and radiative conditions imposed by the local microclimate. For the first two cases the velocity and temperature fields resulting are almost identical. In the third case, the resulting temperature on the surfaces of the building is increased by 19.5%, the center (eye) of the wake zone is raised from the ground and the maximum pollutant concentration is drastically reduced (89%)

From Natural Methylation to Versatile Alkylations Using Halide Methyltransferases

Abstract Halide methyltransferases (HMTs) enable the enzymatic synthesis of S‐adenosyl‐l‐methionine (SAM) from S‐adenosyl‐l‐homocysteine (SAH) and methyl iodide. Characterisation of a range of naturally occurring HMTs and subsequent protein engineering led to HMT variants capable of synthesising ethyl, propyl, and allyl analogues of SAM. Notably, HMTs do not depend on chemical synthesis of methionine analogues, as required by methionine adenosyltransferases (MATs). However, at the moment MATs have a much broader substrate scope than the HMTs. Herein we provide an overview of the discovery and engineering of promiscuous HMTs and how these strategies will pave the way towards a toolbox of HMT variants for versatile chemo‐ and regioselective biocatalytic alkylations