A Study on the Regioselective Acetylation of Flavonoid Aglycons Catalyzed by Immobilized Lipases.

This study aimed to explore the capacity of immobilized lipases on the acetylation of six aglycon flavonoids, namely myricetin, quercetin, luteolin, naringenin, fisetin and morin. For this purpose, lipase B from (CaLB) and lipase from (TLL) were immobilized onto the surface of ZnOFe nanoparticles derived from an aqueous olive leaf extract. Various factors affecting the conversion of substrates and the formation of monoesterified and diesterified products, such as the amount of biocatalyst and the molar ratio of the substrates and reaction solvents were investigated.

Identification and characterization of a novel thermostable PL7 alginate lyase from a submarine volcanic metagenomic library.

Seaweed biomass is as an abundant and renewable source of complex polysaccharides, including alginate which has a variety of applications. A sustainable method for exploiting alginate towards the production of valuable oligosaccharides is through enzymatic processing, using alginate lyases. Industrial refinement methods demand robust enzymes.

Comparative Study of Immobilized Biolipasa-R for Second Generation Biodiesel Production from an Acid Oil.

The primary objective of this work is to develop a sustainable biocatalytic transesterification process for low-grade oils, aligning with EU green technology requirements for the shift to second generation biodiesel. Thus, we investigated the immobilization and subsequent application of the lipase Biolipasa-R on transesterification processes to produce fatty acid methyl esters (FAMEs) from both a sunflower oil and an acid oil which is a bioproduct of the biodiesel industry. The lipase was immobilized on biomaterials, such as diatomaceous earth, with a yield of 60 %, and commercial carriers such as methacrylic resins with a yield of 100 %.

Optimization of Enzymatic Transesterification of Acid Oil for Biodiesel Production Using a Low-Cost Lipase: The Effect of Transesterification Conditions and the Synergy of Lipases with Different Regioselectivity.

This study describes the enzymatic production of second-generation biodiesel using low-quality acid oil as a substrate. Biolipasa-R, a commercially available and low-cost lipase, was employed for enzymatic transesterification. Response surface methodology was applied to optimize the enzymatic transesterification process.

Enzymatic Photometric Assays for the Selective Detection of Halides.

Halides are substrates and products of a number of biotechnologically important enzymes like dehalogenases, halide methyltransferases, and halogenases. Therefore, the determination of halide concentrations in samples is important. The classical methods based on mercuric thiocyanate are very dangerous, produce hazardous waste, and do not discriminate between chloride, bromide, and iodide.

Recovery of Hydroxytyrosol from Olive Mill Wastewater Using the Promiscuous Hydrolase/Acyltransferase PestE.

Olive mill wastewater (OMWW) is produced annually during olive oil extraction and contains most of the health-promoting 3-hydroxytyrosol of the olive fruit. To facilitate its recovery, enzymatic transesterification of hydroxytyrosol (HT) was directly performed in an aqueous system in the presence of ethyl acetate, yielding a 3-hydroxytyrosol acetate rich extract. For this, the promiscuous acyltransferase from Pyrobaculum calidifontis VA1 (PestE) was engineered by rational design.

Rational engineering of ()-selective amine transaminase for the acceptance of bulky substrates.

Despite the plethora of information on ()-selective amine transaminases, the ()-selective ones are still not well-studied; only a few structures are known to date, and their substrate scope is limited, apart from a few stellar works in the field. Herein, the structure of ()-selective amine transaminase is elucidated to facilitate engineering towards variants active on bulkier substrates. The V37A variant exhibited increased activity towards 1-phenylpropylamine and to activity against 1-butylamine.

From Natural Methylation to Versatile Alkylations Using Halide Methyltransferases.

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).

Directed Evolution of a Halide Methyltransferase Enables Biocatalytic Synthesis of Diverse SAM Analogs.

Biocatalytic alkylations are important reactions to obtain chemo-, regio- and stereoselectively alkylated compounds. This can be achieved using S-adenosyl-l-methionine (SAM)-dependent methyltransferases and SAM analogs. It was recently shown that a halide methyltransferase (HMT) from Chloracidobacterium thermophilum can synthesize SAM from SAH and methyl iodide.

Activity and specificity studies of the new thermostable esterase EstDZ2.

In this paper, we study the activity and specificity of EstDZ2, a new thermostable carboxyl esterase of unknown function, which was isolated from a metagenome library from a Russian hot spring. The biocatalytic reaction employing EstDZ2 proved to be an efficient method for the hydrolysis of aryl p-, o- or m-substituted esters of butyric acid and esters of secondary alcohols. Docking studies revealed structural features of the enzyme that led to activity differences among the different substrates.

The challenge of using isopropylamine as an amine donor in transaminase catalysed reactions.

Amine transaminases (ATAs) propose an appealing alternative to transition metal catalysts as they can provide chiral amines of high purity from pro-chiral compounds by asymmetric synthesis. Industrial interest on ATAs arises from the fact that chiral amines are present in a wide spectrum of pharmaceutical and other high value-added chiral compounds and building blocks. Despite their potential as useful synthetic tools, several drawbacks such as challenges associated with the thermodynamic equilibrium can still impede their utilization.

Cloning, expression and characterization of cold active esterase (EstN7) from Bacillus cohnii strain N1: A novel member of family IV.

Esterases and lipases from extremophiles have attracted great attention due to their unique characteristics and wide applications. In the present study, an open reading frame (ORF) encoding a novel cold active esterase (EstN7) from Bacillus cohnii strain N1 was cloned and expressed in Escherichia coli. The full-length esterase gene encoding a protein of 320 amino acids with estimated molecular weight of 37.

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

-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.

Mutations of PKA cyclic nucleotide-binding domains reveal novel aspects of cyclic nucleotide selectivity.

Cyclic AMP and cyclic GMP are ubiquitous second messengers that regulate the activity of effector proteins in all forms of life. The main effector proteins, the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), are preferentially activated by cAMP and cGMP, respectively. However, the molecular basis of this cyclic nucleotide selectivity is still not fully understood.

A coupled photometric assay for characterization of S-adenosyl-l-homocysteine hydrolases in the physiological hydrolytic direction.

S-Adenosyl-l-homocysteine hydrolases (SAHases) are important metabolic enzymes and their dysregulation is associated with some severe diseases. In vivo they catalyze the hydrolysis of S-adenosyl-l-homocysteine (SAH), the by-product of methylation reactions in various organisms. SAH is a potent inhibitor of methyltransferases, thus its removal from the equilibrium is an important requirement for methylation reactions.

Amine Transaminase Engineering for Spatially Bulky Substrate Acceptance.

Amine transaminase (ATA) catalyzing stereoselective amination of prochiral ketones is an attractive alternative to transition metal catalysis. As wild-type ATAs do not accept sterically hindered ketones, efforts to widen the substrate scope to more challenging targets are of general interest. We recently designed ATAs to accept aromatic and thus planar bulky amines, with a sequence-based motif that supports the identification of novel enzymes.

Protein-engineering of an amine transaminase for the stereoselective synthesis of a pharmaceutically relevant bicyclic amine.

Application of amine transaminases (ATAs) for stereoselective amination of prochiral ketones represents an environmentally benign and economically attractive alternative to transition metal catalyzed asymmetric synthesis. However, the restrictive substrate scope has limited the conversion typically to non-sterically demanding scaffolds. Recently, we reported on the identification and design of fold class I ATAs that effect a highly selective asymmetric synthesis of a set of chiral aromatic bulky amines from the corresponding ketone precursors in high yield.

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.

Identification of (S)-selective transaminases for the asymmetric synthesis of bulky chiral amines.

The use of transaminases to access pharmaceutically relevant chiral amines is an attractive alternative to transition-metal-catalysed asymmetric chemical synthesis. However, one major challenge is their limited substrate scope. Here we report the creation of highly active and stereoselective transaminases starting from fold class I.

Graphene oxide derivatives with variable alkyl chain length and terminal functional groups as supports for stabilization of cytochrome c.

In this study we report the ability of reduced and non-reduced graphene oxide-based nanomaterials (GONs), modified with variable alkyl chain length and terminal functional groups, to act as effective scaffolds for the immobilization of cytochrome c (cyt c) using different immobilization procedures. The GONs/cyt c conjugates are characterized by a combination of techniques, namely atomic force microscopy, X-ray photoelectron and FT-IR spectroscopies as well as thermo-gravimetric and differential thermal analysis. The effect of the structure of functional groups and the surface chemistry of GONs on the immobilization efficiency, the peroxidase activity and the stability of the cyt c was investigated and correlated with conformational changes on the protein molecule upon immobilization.

Highly efficient and easy protease-mediated protein purification.

As both research on and application of proteins are rarely focused on the resistance towards nonspecific proteases, this property remained widely unnoticed, in particular in terms of protein purification and related fields. In the present study, diverse aspects of protease-mediated protein purification (PMPP) were explored on the basis of the complementary proteases trypsin and proteinase K as well as the model proteins green fluorescent protein (GFP) from Aequorea victoria, lipase A from Candida antarctica (CAL-A), a transaminase from Aspergillus fumigatus (AspFum), quorum quenching lactonase AiiA from Bacillus sp., and an alanine dehydrogenase from Thermus thermophilus (AlaDH).

Structure of product-bound SMG1 lipase: active site gating implications.

Monoacylglycerol and diacylglycerol lipases are industrially interesting enzymes, due to the health benefits that arise from the consumption of diglycerides compared to the traditional triglyceride oils. Most lipases possess an α-helix (lid) directly over the catalytic pocket which regulates the activity of the enzyme. Generally, lipases exist in active and inactive conformations, depending on the positioning of this lid subdomain.

Conversion of a Mono- and Diacylglycerol Lipase into a Triacylglycerol Lipase by Protein Engineering.

Despite the fact that most lipases are believed to be active against triacylglycerides, there is a small group of lipases that are active only on mono- and diacylglycerides. The reason for this difference in substrate scope is not clear. We tried to identify the reasons for this in the lipase from Malassezia globosa.

Simultaneous use of in silico design and a correlated mutation network as a tool to efficiently guide enzyme engineering.

In order to improve the efficiency of directed evolution experiments, in silico multiple-substrate clustering was combined with an analysis of the variability of natural enzymes within a protein superfamily. This was applied to a Pseudomonas fluorescens esterase (PFE I) targeting the enantioselective hydrolysis of 3-phenylbutyric acid esters. Data reported in the literature for nine substrates were used for the clustering meta-analysis of the docking conformations in wild-type PFE I, and this highlighted a tryptophan residue (W28) as an interesting target.