Enhanced Synthesis of Alkyl Galactopyranoside by <i>Thermotoga naphthophila</i> β‑Galactosidase Catalyzed Transglycosylation: Kinetic Insight of a Functionalized Ionic Liquid-Mediated System

Green synthesis is of pivotal importance for environmental sustainability. This work reports a novel approach to synthesize an array of alkyl galactopyranosides using thermophilic β-galactosidase from <i>Thermotoga naphthophila</i> RKU-10 (TN1577) as biocatalyst and milk processing waste lactose as galactosyl donor. Ammoeng 102 (only 2.5% addition of total reaction volume), a functionalized ionic liquid (IL) containing tetraaminum cation with C₁₈ acyl and oligoethylene glycol, is identified as the most promising one from a variety of structurally diverse ILs, affording a 2.37-fold increase in octyl galactopyranoside yield compared to the buffer system. Up to 18.2 g L^–1 octyl galactopyranoside could be produced in 7 h, which is significantly higher than any previous report in terms of time-space efficiency. Kinetic study and COSMO-RS <i>in silico</i> predictions elucidate that the thermophilic nature of TN1577 β-galactosidase, increased solubility of substrate, suppression of hydrolysis, and excellent biocompatibility of Ammoeng 102 with enzyme (allowing TN1577 β-galactosidase to perform optimal catalysis up to 95 °C) are the main driving forces. The general applicability of the Ammoeng 102 system is verified, by which a series of alkyl galactopyranosides are successfully synthesized with <i>n</i>-butanol to <i>n</i>-tetradecanol as alkyl acceptors and lactose as galactosyl donor

Effect of Covalent-Functionalized Graphene Oxide with Polymer and Reactive Compatibilization on Thermal Properties of Maleic Anhydride Grafted Polypropylene

The covalent functionalization of graphene oxide (GO) with bis­(3-aminopropyl)-terminated poly­(ethylene glycol) (NH₂–PEG–NH₂) and subsequent grafting with maleic anhydride grafted polypropylene (MAPP) oligomer matrix using reactive compatibilization were carefully analyzed and verified through detailed investigations. Improvements in the compatibility between the modified GO and the matrix, thermal stability, flame properties, and crystallization properties were achieved through the addition of a small amount of GO-grafted MAPP (PP-<i>g</i>-GO). Results of thermogravimetric and microscale combustion calorimetry analyses revealed an increase in <i>T</i>_max by 51 °C and reductions in the total heat release and peak heat release rate by 44.4% and 38.9%, respectively, upon the addition of 2.0 wt % PP-<i>g</i>-GO relative to pure MAPP. The approach used in this work is an efficient strategy for improving the thermal behavior of polypropylene oligomer with a view toward extending its use in advanced technological applications

The Second Intracellular Loop of the Human Cannabinoid CB2 Receptor Governs G Protein Coupling in Coordination with the Carboxyl Terminal Domain

<div><p>The major effects of cannabinoids and endocannabinoids are mediated via two G protein-coupled receptors, CB1 and CB2, elucidation of the mechanism and structural determinants of the CB2 receptor coupling with G proteins will have a significant impact on drug discovery. In the present study, we systematically investigated the role of the intracellular loops in the interaction of the CB2 receptor with G proteins using chimeric receptors alongside the characterization of cAMP accumulation and ERK1/2 phosphorylation. We provided evidence that ICL2 was significantly involved in G protein coupling in coordination with the C-terminal end. Moreover, a single alanine substitution of the Pro-139 in the CB2 receptor that corresponds to Leu-222 in the CB1 receptor resulted in a moderate impairment in the inhibition of cAMP accumulation, whereas mutants P139F, P139M and P139L were able to couple to the G_s protein in a CRE-driven luciferase assay. With the ERK activation experiments, we further found that P139L has the ability to activate ERK through both G_i- and G_s-mediated pathways. Our findings defined an essential role of the second intracellular loop of the CB2 receptor in coordination with the C-terminal tail in G protein coupling and receptor activation.</p></div

Functional characterization of cannabinoid receptor chimeras and mutants.

<p>The values are expressed as the mean ± SEM (<i>n = </i>3 experiments). % of maximal, the value of cAMP level percentage of the value obtained upon 10 µM forskolin treated only. Fold increase, the valve of cAMP level related to basal activity.</p><p>ND, not detectable.</p>α<p>The values were obtained in the absence of forskolin.</p>b<p>The values were obtained in the presence of 2 µM WIN55,212-2.</p

Valorizing Dairy Waste: Thermophilic Biosynthesis of a Novel Ascorbic Acid Derivative

l-Ascorbic acid (l-AA) is an essential nutrient that is extremely unstable and cannot be synthesized by the human body. Therefore, attempts have been performed to develop biologically active l-AA derivatives with improved stability. This work presents a facile, scalable, and efficient enzymatic transgalactosylation of lactose to l-AA using β-glucosidase (TN0602) from Thermotoga naphthophila RKU-10. β-Glucosidase TN0602 displays high transgalactosylation activity at pH 5.0, 75 °C, and l-AA/lactose ratio of 2:1 to form a novel l-AA derivative [2-<i>O</i>-β-d-galactopyranosyl-l-ascorbic acid (l-AA-Gal)] with a maximal productivity of 138.88 mmol L^–1 in 12 h, which is higher than most reports of enzymatic synthesis of l-AA-α-glucoside. Synthetic l-AA-Gal retains most l-AA antioxidant capability and presents dramatically higher stability than l-AA in an oxidative environment (Cu²⁺). In conclusion, this work reports a new way to valorize dairy waste lactose into a novel molecule l-AA-Gal, which could be a promising l-AA derivative to be used in a wide range of applications

Comparison of effects of G_i inhibitor and PKA inhibitor on the activation of ERK in wild-type CB2 and P139L expressing cells.

<p>(<b>A</b>) Transiently transfected HEK293 cells were pretreated with or without 100 ng/mL <i>Pertussis toxin</i> (PTX) for 12 h or pretreated with or without 10 µM H89 for 30 min, and then stimulated with increasing concentrations of WIN55,212-2. (<b>B</b>) ERK signals were quantified by densitometry and expressed as a ratio of activated over total ERK. The maximal phosphorylation of ERK obtained in control cells at 5 min of stimulation with WIN55,212-2 in the absence of inhibitors were arbitrarily chosen as 100%. Each data point represents the mean ± SEM from three independent experiments. ***<i>P</i><0.001 compared with the entire control curve.</p

Effects of multi-domains in the CB2 receptor on G_s- and G_i-dependent signaling.

<p>(<b>A</b>) Schematic diagram of composition of CB2 chimeras with multi-domains substitution. The overall composition of individual cannabinoid receptor chimeras is shown schematically. Numbers indicate the amino acid residues corresponding to the parental cannabinoid receptors. The CB1 receptor sequence is shown in black, and the CB2 receptor sequence is in dark grey. (<b>B</b>) ELISA analysis of CB2 receptors expression. HEK293 cells were transiently transfected with Flag epitope-tagged receptors and the cell surface expression was measured by ELISA analysis, as described under <i>Methods</i>. The results represent the mean ± SEM of three independent experiments, each done in triplicate. (<b>C</b>) Effects of cAMP accumulation for multi-chimeric receptors upon agonist stimulation. For cAMP measurements, cells were incubated with 10 µM forskolin or with various concentrations of WIN55,212-2 plus 10 µM forskolin, except the chimera CB2-ICL2Cter in the absence of forskolin. For CB2-ICL2ICL3, CB2-ICL2ICL3Cter and CB2 wild-type, values are expressed as percentage of forskolin stimulation. For CB2-ICL2Cter, values are expressed as percentage of basal activity. i2i3, CB2-ICL2ICL3 chimera receptor; i2i3cter, CB2-ICL2ICL3Cter chimera receptor; i2Cter, CB2-ICL2Cter chimera receptor.</p

Effects of key residues in the ICL2 of the CB2 receptor on selectively G_i and G_s coupling.

<p>(<b>A</b>) Structures of CB2 mutations within the second intracellular loop as well as the C-terminal. (<b>B</b>) ELISA analysis of CB2 receptors expression. HEK293 cells were transiently transfected with Flag epitope-tagged receptors and the cell surface expression was measured by ELISA analysis, as described under <i>Methods</i>. The results represent the mean ± SEM of three independent experiments, each done in triplicate. (<b>C</b>) Dose response curve of cAMP accumulation for the CB2P139A and CB2P139L upon WIN55,212-2 stimulation. For cAMP measurements, cells were incubated with various concentrations of WIN55,212-2 for P139L and with various concentrations of WIN55,212-2 plus 10 µM forskolin for P139A and wild-type for 4 h. Values were expressed as percentage of forskolin stimulation for CB2P139A and CB2 wild-type, and as percentage of basal activity for CB2P139L. (<b>D</b>) Effects of substitutions of P139 with various kinds of amino acids in the CB2 receptor on WIN55,212-2-induced cAMP formation. HEK293 cells were treated with 2 µM WIN55,212-2, and cAMP production was measured as described in the <i>materials and methods</i>. The resulting increases in cAMP were expressed as fold increase above basal. Data are expressed as the mean ± SEM and are representative of three independent experiments.</p

Effects of key domains in the CB2 receptor on G_i-dependent signaling.

<p>(<b>A</b>) Schematic diagram of composition of cannabinoid CB2 receptor chimeras. The overall composition of individual cannabinoid receptor chimeras is shown schematically. Numbers indicate the amino acid residues corresponding to the parental cannabinoid receptors. The CB2 receptor sequence is shown in dark grey, and the CB1 receptor sequence is in black. (<b>B</b>) ELISA analysis of CB2 receptors expression. HEK293 cells were transiently transfected with Flag epitope-tagged receptors and the cell surface expression was measured by ELISA analysis, as described under <i>Methods</i>. The results represent the mean ± SEM of three independent experiments, each done in triplicate. (<b>C</b>) Dose-response curve of cAMP accumulation for the CB2 chimeric receptors upon agonist stimulation. For cAMP measurements, cells were incubated with various concentrations of WIN55,212-2 plus 10 µM forskolin for 4 h. cAMP measurements were carried out as described in the <i>Materials and Methods.</i> Data are expressed as the percent cAMP activity over forskolin. Data shown are expressed as the mean ± SEM and are representative of three independent experiments. (<b>D</b>) Basal cAMP accumulation of CB2 wild type and chimera receptors. For basal cAMP measurements, cells were incubated for 48 h after transfection and then directly lysed for cAMP assay. (E) Effects of PTX on cAMP accumulation of CB2 wild-type and chimera receptors. Cells were transiently transfected with receptors and pCRE-luc. 48 h later, PTX (100 ng/ml) was added to the reseeded cells in FBS-free medium and cells were incubated for another 12 h. Then, cells were incubated with 10 µM forskolin or 2 µM WIN55,212-2 plus 10 µM forskolin for 4 h and lysed for cAMP assay. Data shown are expressed as the mean ± SEM and are representative of three independent experiments. Data are expressed as the percent cAMP activity over wild type CB2 receptor. ***<i>p</i><0.001. i1, CB2-ICL1 chimera receptor; i2, CB2-ICL2 chimera receptor; i3, CB2-ICL3 chimera receptor; Cter, CB2-Cter chimera receptor.</p

HAS2 gene sequencing primers for various exons.

<p>HAS2 gene sequencing primers for various exons.</p