2-(4-Methoxyphenyl)Ethyl-2-Acetamido-2-Deoxy-ß-D-Pyranoside Exerts a Neuroprotective Effect through Regulation of Energy Homeostasis and O-GlcNAcylation.

Dysfunction of energy metabolism exerts a central role in triggering neuron death following cerebral ischemia. Neuronal energy metabolism is highly dependent on glucose. O-GlcNAcylation, a post-translational modification, is a novel pro-survival pathway that modulates glucose homeostasis in ischemic stroke. Here, we explored whether activation O-GlcNAcylation and maintaining energy homeostasis mediated the neuroprotective effect of 2-(4-methoxyphenyl)ethyl-2-acetamido-2-deoxy-ß-D-pyranoside, a synthetic salidroside analog (named SalA-4 g) which was previously developed in our laboratory. For in vivo analyses, SalA-4 g improved the outcome after transient middle cerebral artery occlusion (MCAO). 18F-FDG PET/MRI indicated that SalA-4 g accelerated the recovery of energy metabolism in the ipsilateral hippocampus in MCAO rats. In vitro analyses showed that glucose uptake was markedly increased, and O-GlcNAcylation was also activated by SalA-4 g in hippocampal neurons under both normal and oxygen glucose deprivation (OGD) conditions. Moreover, SalA-4 g exerted obvious neuroprotective effects in hippocampal neurons against moderate OGD injury. Our study indicates that boosting a pro-survival pathway-GlcNAcylation-and regulating energy homeostasis are important biochemical mechanisms responsible for SalA-4 g neuroprotection.

Green Synthesis of Conjugated Linoleic Acids from Plant Oils Using a Novel Synergistic Catalytic System.

A novel and efficient method has been developed for converting plant oil into a specific conjugated linoleic acid (CLA) using a synergistic biocatalytic system based on immobilized Propionibacterium acnes isomerase (PAI) and Rhizopus oryzae lipase (ROL). PAI exhibited the greatest catalytic activity when immobilized on D301R anion-exchange resin under optimal conditions (PAI dosage of 12 410 U of PAI/g of D301R, glutaraldehyde concentration of 0.4%, and reaction conditions of pH 7.0, 25 °C, and 60 min). Up to 109 g/L trans-10,cis-12-CLA was obtained after incubation of 200 g/L sunflower oil with PAI (1659 U/g of oil) and ROL (625 mU/g of oil) at pH 7.0 and 35 °C for 36 h; the corresponding conversion ratio of linoleic acid (LA) to CLA was 90.5%. This method exhibited the highest proportion of trans-10,cis-12-CLA yet reported and is a promising method for large-scale production.

Characterization of Glycerol Dehydrogenase from Thermoanaerobacterium thermosaccharolyticum DSM 571 and GGG Motif Identification.

Glycerol dehydrogenases (GlyDHs) are essential for glycerol metabolism in vivo, catalyzing its reversible reduction to 1,3-dihydroxypropranone (DHA). The gldA gene encoding a putative GlyDH was cloned from Thermoanaerobacterium thermosaccharolyticum DSM 571 (TtGlyDH) and expressed in Escherichia coli. The presence of Mn(2+) enhanced its enzymatic activity by 79.5%. Three highly conserved residues (Asp(171), His(254), and His(271)) in TtGlyDH were associated with metal ion binding. Based on an investigation of glycerol oxidation and DHA reduction, TtGlyDH showed maximum activity towards glycerol at 60°C and pH 8.0 and towards DHA at 60°C and pH 6.0. DHA reduction was the dominant reaction, with a lower Km(DHA) of 1.08 ± 0.13 mM and Vmax of 0.0053 ± 0.0001 mM/s, compared with glycerol oxidation, with a Km(glycerol) of 30.29 ± 3.42 mM and Vmax of 0.042 ± 0.002 mM/s. TtGlyDH had an apparent activation energy of 312.94 kJ/mol. The recombinant TtGlyDH was thermostable, maintaining 65% of its activity after a 2-h incubation at 60°C. Molecular modeling and site-directed mutagenesis analyses demonstrated that TtGlyDH had an atypical dinucleotide binding motif (GGG motif) and a basic residue Arg(43), both related to dinucleotide binding.

Biochemical properties of a novel thermostable and highly xylose-tolerant ß-xylosidase/α-arabinosidase from Thermotoga thermarum.

BACKGROUND: ß-Xylosidase is an important constituent of the hemicellulase system and it plays an important role in hydrolyzing xylooligosaccharides to xylose. Xylose, a useful monose, has been utilized in a wide range of applications such as food, light, chemical as well as energy industry. Therefore, the xylose-tolerant ß-xylosidase with high specific activity for bioconversion of xylooligosaccharides has a great potential in the fields as above. RESULTS: A ß-xylosidase gene (Tth xynB3) of 2,322 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum DSM 5069 that encodes a protein containing 774 amino acid residues, and was expressed in Escherichia coli BL21 (DE3). The phylogenetic trees of ß-xylosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. The phylogeny and amino acid analysis indicated that the Tth xynB3 ß-xylosidase was a novel ß-xylosidase of GH3. The optimal activity of the Tth xynB3 ß-xylosidase was obtained at pH 6.0 and 95°C and was stable over a pH range of 5.0-7.5 and exhibited 2 h half-life at 85°C. The kinetic parameters Km and Vmax values for p-nitrophenyl-ß-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside were 0.27 mM and 223.3 U/mg, 0.21 mM and 75 U/mg, respectively. The kcat/Km values for p-nitrophenyl-ß-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside were 1,173.4 mM-1 s-1 and 505.9 mM-1 s-1, respectively. It displayed high tolerance to xylose, with Ki value approximately 1000 mM. It was stimulated by xylose at higher concentration up to 500 mM, above which the enzyme activity of Tth xynB3 ß-xylosidase was gradually decreased. However, it still remained approximately 50% of its original activity even if the concentration of xylose was as high as 1000 mM. It was also discovered that the Tth xynB3 ß-xylosidase exhibited a high hydrolytic activity on xylooligosaccharides. When 5% substrate was incubated with 0.3 U Tth xynB3 ß-xylosidase in 200 µL reaction system for 3 h, almost all the substrate was biodegraded into xylose. CONCLUSIONS: The article provides a useful and novel ß-xylosidase displaying extraordinary and desirable properties: high xylose tolerance and catalytic activity at temperatures above 75°C, thermally stable and excellent hydrolytic activity on xylooligosaccharides.