Exploring 1-alkene biosynthesis in bacterial antagonists and jeotgalicoccus sp. ATCC 8456.

Terminal olefins are important platform chemicals, drop-in compatible hydrocarbons and also play an important role as biocontrol agents of plant pathogens. Currently, 1-alkenes are derived from petroleum, although microbial biosynthetic routes are known. Jeotgalicoccus sp.

The Effect of Accessibility of Insoluble Substrate on the Overall Kinetics of Enzymatic Degradation.

The enzymatic reaction kinetics on cellulose and other solid substrates is limited by the access of the enzyme to the reactive substrate sites. We introduce a general model in which the reaction rate is determined by the active surface area, and the resulting kinetics consequently reflects the evolving relationship between the exposed substrate surface and the remaining substrate volume. Two factors influencing the overall surface-to-volume ratio are considered: the shape of the substrate particles, characterized by a single numerical parameter related to its dimensionality, and the distribution of the particle sizes.

Magnetically Responsive Enzyme and Hydrogen-Bonded Organic Framework Biocomposites for Biosensing.

The one-pot synthesis of multicomponent hydrogen-bonded organic framework (HOF) biocomposites is reported. The co-immoblization of enzymes and magnetic nanoparticles (MNPs) into the HOF crystals yielded biocatalysts (MNPs-enzyme@BioHOF-1) with dynamic localization properties. Using a permanent magnet, it is possible to separate the MNPs-enzyme@BioHOF-1 particles from a solution.

Keeping the Distance: Activity Control in Solid-Supported Sucrose Phosphorylase by a Rigid α-Helical Linker of Tunable Spacer Length.

Enzyme immobilization into carrier materials has broad importance in biotechnology, yet understanding the catalysis of enzymes bound to solid surfaces remains challenging. Here, we explore surface effects on the catalysis of sucrose phosphorylase through a fusion protein approach. We immobilize the enzyme via a structurally rigid α-helical linker [EAK] of tunable spacer length due to the variable number of pentapeptide repeats used ( = 6, 14, 19).

Silicone-based AC102-loaded cochlear implant coatings protect residual hearing in an animal model of cochlear implantation.

Cochlear implant users with residual hearing benefit synergistically from combined electrical stimulation via the cochlear implant and preserved residual hearing after surgery. However, direct mechanical trauma and subsequent inflammation may deteriorate hearing function. AC102, a novel otoprotective pyridoindole with anti-apoptotic and anti-oxidative properties significantly improved hearing recovery following cochlear implantation when administered intratympanically prior to surgery.

Expanding the high-pH range of the sucrose synthase reaction by enzyme immobilization.

The glycosylation of an alcohol group from a sugar nucleotide substrate involves proton release, so the reaction is favored thermodynamically at high pH. Here, we explored expansion of the alkaline pH range of sucrose synthase (SuSy; EC 2.4.

Electrochemically Induced Nanoscale Stirring Boosts Functional Immobilization of Flavocytochrome P450 BM3 on Nanoporous Gold Electrodes.

Enzyme-modified electrodes are core components of electrochemical biosensors for diagnostic and environmental analytics and have promising applications in bioelectrocatalysis. Despite huge research efforts spanning decades, design of enzyme electrodes for superior performance remains challenging. Nanoporous gold (npAu) represents advanced electrode material due to high surface-to-volume ratio, tunable porosity, and intrinsic redox activity, yet its coupling with enzyme catalysis is complex.

Enzyme Machinery for Bacterial Glucoside Metabolism through a Conserved Non-hydrolytic Pathway.

The flexible acquisition of substrates from nutrient pools is critical for microbes to prevail in competitive environments. To acquire glucose from diverse glycoside and disaccharide substrates, many free-living and symbiotic bacteria have developed, alongside hydrolysis, a non-hydrolytic pathway comprised of four biochemical steps and conferred from a single glycoside utilization gene locus (GUL). Mechanistically, this pathway integrates within the framework of oxidation and reduction at the glucosyl/glucose C3, the eliminative cleavage of the glycosidic bond and the addition of water in two consecutive lyase-catalyzed reactions.

Discovery, characterization, and comparative analysis of new UGT72 and UGT84 family glycosyltransferases.

Glycosylated derivatives of natural product polyphenols display a spectrum of biological activities, rendering them critical for both nutritional and pharmacological applications. Their enzymatic synthesis by glycosyltransferases is frequently constrained by the limited repertoire of characterized enzyme-catalyzed transformations. Here, we explore the glycosylation capabilities and substrate preferences of newly identified plant uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) within the UGT72 and UGT84 families, with particular focus on natural polyphenol glycosylation from UDP-glucose.

Interplay of structural preorganization and conformational sampling in UDP-glucuronic acid 4-epimerase catalysis.

Understanding enzyme catalysis as connected to protein motions is a major challenge. Here, based on temperature kinetic studies combined with isotope effect measurements, we obtain energetic description of C-H activation in NAD-dependent UDP-glucuronic acid C4 epimerase. Approach from the ensemble-averaged ground state (GS) to the transition state-like reactive conformation (TSRC) involves, alongside uptake of heat (  = 54 kJ mol), significant loss in entropy (  = 20 kJ mol; 298 K) and negative activation heat capacity (  = -0.

Efficient Synthetic Access to Stable Isotope Labelled Pseudouridine Phosphoramidites for RNA NMR Spectroscopy.

Here we report the efficient synthetic access to C/N-labelled pseudouridine phosphoramidites, which were incorporated into a binary H/ACA box guide RNA/product complex comprising 77 nucleotides (nts) in total and into a 75 nt E. coli tRNA. The stable isotope (SI) labelled pseudouridines were produced via a highly efficient chemo-enzymatic synthesis.

Bottom-Up Synthesized Glucan Materials: Opportunities from Applied Biocatalysis.

Linear d-glucans are natural polysaccharides of simple chemical structure. They are comprised of d-glucosyl units linked by a single type of glycosidic bond. Noncovalent interactions within, and between, the d-glucan chains give rise to a broad variety of macromolecular nanostructures that can assemble into crystalline-organized materials of tunable morphology.

Mechanochemical Coupling of Catalysis and Motion in a Cellulose-Degrading Multienzyme Nanomachine.

The cellulosome is a megadalton-size protein complex that functions as a biological nanomachine of cellulosic fiber degradation. We show that the cellulosome behaves as a Brownian ratchet that rectifies protein motions on the cellulose surface into a propulsion mechanism by coupling to the hydrolysis of cellulose chains. Movement on cellulose fibrils is unidirectional and results from "macromolecular crawl" composed of dynamic switches between elongated and compact spatial arrangements of enzyme subunits.

Limits of Non-invasive Enzymatic Activation by Local Temperature Control.

Enzymatic activity depends on and can therefore be regulated by temperature. Selective modulation of the activity of different enzymes in one reaction pot would require temperature control local to each type of enzyme. It has been suggested previously that immobilization of enzyme on magnetic nanoparticles and exposing them to alternating magnetic field can enhance the reaction rate.

Carrier-based immobilization of Aerococcus viridansl-lactate oxidase.

l-Lactate oxidase has important applications in biosensing and finds increased use in biocatalysis. The enzyme has been characterized well, yet its immobilization has not been explored in depth. Here, we studied immobilization of Aerococcus viridansl-lactate oxidase on porous carriers of variable matrix material (polymethacrylate, polyurethane, agarose) and surface functional group (amine, Ni-loaded nitrilotriacetic acid (NiNTA), epoxide).

CO-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16.

Background: Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C.

Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase.

Background: In whole-cell bio-catalysis, the biosystems engineering paradigm shifts from the global reconfiguration of cellular metabolism as in fermentation to a more focused, and more easily modularized, optimization of comparably short cascade reactions. Human milk oligosaccharides (HMO) constitute an important field for the synthetic application of cascade bio-catalysis in resting or non-living cells. Here, we analyzed the central catalytic module for synthesis of HMO-type sialo-oligosaccharides, comprised of CMP-sialic acid synthetase (CSS) and sialyltransferase (SiaT), with the specific aim of coordinated enzyme co-expression in E.

Pushing the boundaries of phosphorylase cascade reaction for cellobiose production II: Model-based multiobjective optimization.

The inherent complexity of coupled biocatalytic reactions presents a major challenge for process development with one-pot multienzyme cascade transformations. Kinetic models are powerful engineering tools to guide the optimization of cascade reactions towards a performance suitable for scale up to an actual production. Here, we report kinetic model-based window of operation analysis for cellobiose production (≥100 g/L) from sucrose and glucose by indirect transglycosylation via glucose 1-phosphate as intermediate.

Pushing the boundaries of phosphorylase cascade reaction for cellobiose production I: Kinetic model development.

One-pot cascade reactions of coupled disaccharide phosphorylases enable an efficient transglycosylation via intermediary α-d-glucose 1-phosphate (G1P). Such transformations have promising applications in the production of carbohydrate commodities, including the disaccharide cellobiose for food and feed use. Several studies have shown sucrose and cellobiose phosphorylase for cellobiose synthesis from sucrose, but the boundaries on transformation efficiency that result from kinetic and thermodynamic characteristics of the individual enzyme reactions are not known.

Enzymatic β-elimination in natural product O- and C-glycoside deglycosylation.

Biological degradation of natural product glycosides involves, alongside hydrolysis, β-elimination for glycosidic bond cleavage. Here, we discover an O-glycoside β-eliminase (OGE) from Agrobacterium tumefaciens that converts the C3-oxidized O-β-D-glucoside of phloretin (a plant-derived flavonoid) into the aglycone and the 2-hydroxy-3-keto-glycal elimination product. While unrelated in sequence, OGE is structurally homologous to, and shows effectively the same Mn active site as, the C-glycoside deglycosylating enzyme (CGE) from a human intestinal bacterium implicated in β-elimination of 3-keto C-β-D-glucosides.

Fabrication and characterization of a novel zein/pectin/pumpkin seed oil Pickering emulsion and the effects of myricetin on oxidation stability.

In this study, zein/pectin/pumpkin seed oil (PSO) Pickering emulsions (ZPPEs) were fabricated loading with myricetin (MYT), and the quality control methods of oxidation stability were innovatively investigated. The microstructure and particle properties of zein-pectin particles were determined. The zein to pectin ratio of 5:3 and oil phase fraction (φ = 50 %) turned out as the most optimal conditions for the stabilization of myricetin-loaded ZPPEs.

Two Novel -Type and an Oxindole Glucuronide Alkaloid from Disclose Related Biosynthetic Pathways to .

Phytochemical investigation of the two species (Apocynaceae) . Wall. and .

Ligand-Directed Chemistry on Glycoside Hydrolases - A Proof of Concept Study.

Selective covalent labelling of enzymes using small molecule probes has advanced the scopes of protein profiling. The covalent bond formation to a specific target is the key step of activity-based protein profiling (ABPP), a method which has become an indispensable tool for measuring enzyme activity in complex matrices. With respect to carbohydrate processing enzymes, strategies for ABPP so far involve labelling the active site of the enzyme, which results in permanent loss of activity.

Solvent Engineering for Nonpolar Substrate Glycosylation Catalyzed by the UDP-Glucose-Dependent Glycosyltransferase UGT71E5: Intensification of the Synthesis of 15-Hydroxy Cinmethylin β-d-Glucoside.

Sugar nucleotide-dependent glycosyltransferases are powerful catalysts of the glycosylation of natural products and xenobiotics. The low solubility of the aglycone substrate often limits the synthetic efficiency of the transformation catalyzed. Here, we explored different approaches of solvent engineering for reaction intensification of β-glycosylation of 15HCM (a C15-hydroxylated, plant detoxification metabolite of the herbicide cinmethylin) catalyzed by safflower UGT71E5 using UDP-glucose as the donor substrate.

Chemoenzymatic Syntheses of Fluorine-18-Labeled Disaccharides from [F] FDG Yield Potent Sensors of Living Bacteria .

Chemoenzymatic techniques have been applied extensively to pharmaceutical development, most effectively when routine synthetic methods fail. The regioselective and stereoselective construction of structurally complex glycans is an elegant application of this approach that is seldom applied to positron emission tomography (PET) tracers. We sought a method to dimerize 2-deoxy-[F]-fluoro-d-glucose ([F]FDG), the most common tracer used in clinical imaging, to form [F]-labeled disaccharides for detecting microorganisms based on their bacteria-specific glycan incorporation.