In this paper, a new approach for the separation of amino acids on the electrophoresis chip-based low-voltage-driven electrophoresis was reported in detail. This low-voltage-driven electrophoresis process could be realized by powering directly the arrayed electrode pairs with low direct current (DC) voltage to generate a moving electric field along the separation microchannel, which could maintain enough electric field strength for electrophoresis. The proposed microfluidic electrophoresis chip was bonded directly with silicon-on-insulator (SOI) substrate and polydimethylsiloxane (PDMS) cover plate at room temperature. The microfluidic channels and the arrayed electrodes were etched on SOI wafer by silicon microelectromechanical system technology. A specially integrated circuit was proposed to power a 30-60-V DC voltage to particular sets of these electrode pairs in a controlled sequence such that the moving electric field could be formed, and the low-voltage-driven electrophoresis could be realized in the microchannel. In the experiments, with 10(-4) mol/L phenylalanine and lysine as analytes, the separation of amino acids on the low-voltage-driven electrophoresis microchip was conducted by homemade integrated control circuit; a method for separating amino acids was well established. It was also shown that the phenylalanine and lysine mixture was effectively separated in less than 7 min and with a resolution of 2.0. To the best of our knowledge, the low-voltage-driven microchip electrophoresis device could be of potential prospective in the fields of integrated and miniaturized biochemical analysis system.
The chemical composition and biological activities of the essential oils from the leaves, stems, and roots of Kadsura coccinea (K. coccinea) were investigated. The essential oils were extracted by hydro distillation and analyzed by gas chromatography mass spectrometry (GC-MS) and gas chromatography with flame ionization detector (GC-FID). Antioxidant activities of the essential oils were examined with DPPH radical scavenging assay, ABTS cation radical scavenging assay, and ferric reducing antioxidant power assay. Antimicrobial activities were evaluated by determining minimum inhibitory concentrations (MIC) and minimum microbiocidal concentrations (MMC). Acetylcholinesterase and butyrylcholinesterase inhibitory activity of the essential oils were also tested. A total of 46, 44, and 47 components were identified in the leaf, stem, and root oils, representing 95.66%, 97.35%, and 92.72% of total composition, respectively. The major compounds of three essential oils were α-pinene (16.60–42.02%), β-pinene (10.03–18.82%), camphene (1.56–10.95%), borneol (0.50–7.71%), δ-cadinene (1.52–7.06%), and β-elemene (1.86–4.45%). The essential oils were found to have weak antioxidant activities and cholinesterase inhibition activities. The essential oils showed more inhibitory effects against Staphylococcus aureus (S. aureus) than those of other strains. The highest antimicrobial activity was observed in the root oil against S. aureus, with MIC of 0.78 mg/mL. Therefore, K. coccinea essential oils might be considered as a natural antibacterial agent against S. aureus with potential application in food and pharmaceutical industries.
Lindera glauca is a shrub or small tree mostly distributed in China, Japan and Korea. However, reports on the biological activities of Lindera glauca fruit essential oil (LGFEO) are limited. The study on its chemical composition, and antioxidant and cholinesterase inhibitory activities were performed, along with molecular docking of six selected compounds. The LGFEO was extracted by hydro distillation and analyzed by GC-MS and GC-FID. Antioxidant activities of LGFEO were evaluated by three methods with different mechanisms. Acetylcholinesterase and butyrylcholinesterase inhibitory activities of LGFEO were tested. A total of 48 components were identified representing 95.74% of the total composition of LGFEO in which the major compounds were (E)-β-ocimene (41.53%), α-copaene (13.17%), δ-cadinene (6.20%), 3-carene (5.89%) and eucalyptol (3.57%). Weak antioxidant activities of LGFEO in three assays (9.52, 11.36 and 38.98 μmol TE/g, respectively) were observed. LGFEO showed obvious cholinesterase inhibitory activities at the final concentrations of 50 and 20 μg/mL. IC50 values for acetylcholinesterase and butyrylcholinesterase were 46.48 and 34.85 μg/mL, respectively. Molecular docking revealed that geranyl acetate, β-caryophyllene and limonene had lower binding affinities in the range of −7.1 to −6.1 kcal/mol through hydrophobic interactions and hydrogen bond. Six compounds including 3-carene, limonene, eucalyptol, (E)-β-ocimene, geranyl acetate and β-caryophyllene could contribute together to cholinesterase inhibitory activities of LGFEO. This essential oil indicated low potential as natural antioxidant, but it could be potentially used as cholinesterase inhibitor with possible application in food, aromatherapy and pharmaceutical industries.
Objective The objective was to develop a convenient strategy for co-expressing multiple proteins in Komagataella phaffii via the Cre/loxP system without introducing any markers.Results A plasmid in this strategy was generated from pPICZαA with integration of lox71-Sh ble-lox66. Firstly, the plasmid was inserted with one target protein gene and then transformed into K. phaffii KM71. Secondly, the auxiliary plasmid pPICZαA/cre/his4 containing CRE recombinase gene was further chromosomally inserted to Sh ble gene therein. Finally, methanol induction was conducted to produce CRE for Cre/loxP-mediated recombination, and consequently, the sequence between lox71 and lox66 was deleted, leading to recovery of ZeoR and His− markers. Then the resulted strain expressing the one target protein was used as the host to which another target protein gene could be inserted by the same procedures.Conclusions With easy manipulation, the method was effective in co-expressing multiple proteins with rescue of markers in this yeast.
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