Yun Hee Choi

Optimum SFE condition for lignans ofSchisandra chinensis fruits

SummaryThe fruits ofSchisandra chinensis were extracted with supercritical carbon dioxide under various conditions of temperature (40–80 °C) and pressure (13.6–34.0 MPa) and each extract was analyzed by high-performance liquid chromatography for the main bioactive lignans schisandrol A (1), schisandrol B (2), schisandrin A (3), schisandrin B (4), and schisandrin C (5). When the extraction time was sufficient (30 min/100 mg plant material) the temperature and pressure of the supercritical carbon dioxide had no distinct effect on the extraction yields of the targetted lignans. If, however, the extraction time was 6 min, increasing the pressure greatly enhanced the yields of the lignans. The extraction efficiency of supercritical fluid extraction (SFE) was compared with that of conventional extraction with organic solvent. The efficiency of extraction ofS. chinensis lignans by SFE was approximately 80% that by methanol extraction but was superior to that achievable by use of other solvents such as chloroform-methanol (2:1),n-hexane, and petroleum ether. The results obtained suggest that in terms of efficiency and selectivity SFE could be used as an alternative to conventional extraction with organic solvent.

Optical measurements of gas temperatures in atmospheric pressure RF cold plasmas

This work was supported by grant No. R01-2000- 00254 fromthe Korea Science and Engineering Foundation.

Solubilization and Iterative Saturation Mutagenesis of α1,3-fucosyltransferase from Helicobacter pylori to enhance its catalytic efficiency.

α1,3‐Fucosyltransferase (α1,3‐FucT) is essential for the biosynthesis of biologically active α1,3‐fucosyloligosacchairdes (3‐FOs) from human milk oligosaccharides (HMO), particularly 3‐fucosyllactose (3‐FL) trisaccharide. α1,3‐FucT from Helicobacter pylori 26695 (FutA) accepts lactose and LacNAc as glycan acceptors and has a very low level of expression in Escherichia coli, and it shows a low catalytic activity for lactose in the large‐scale synthesis of 3‐FL. To overcome the poor solubility of FutA, codon optimization, and systematic truncation of the protein at the C‐terminus with only one heptad repeat remaining (Δ52 FutA) were conducted to yield 150–200 mg/L of soluble protein of FutA and resulting in more than an 18‐fold increase in the 3‐FL yield. To improve the low level of enzyme catalytic activity for lactose, focused directed evolution was attempted using a semi‐rational approach that combines structure‐guided computational analysis and subsequent iterative saturation mutagenesis (ISM). In order to select the functional residues in active site/substrate binding site, docking simulation was used together with HotSpot Wizard to target evolutionarily variable amino acid positions. A128 site was selected from the key residue located in the active site, and A128N mutant displayed a 3.4‐fold higher catalytic activity than wild‐type Δ52 FutA. Considering that the A128N mutation is located in the deep cleft of the lactose binding site, the residues within the substrate binding sites, especially on the two α‐helices for lactose and one α‐helix for GDP‐fucose, were subjected to structure‐guided ISM. The selected residues from each helix were clustered, and ISM was performed for each cluster in parallel. In particular, the mutant with triple mutations (A128N/H129E/Y132I) located on the α5 helix exhibited a 9.6‐fold improvement in specific activity when compared to wild‐type Δ52 FutA. When such clustered mutations on two α‐helices (α5 and α2/loop) were combined, mutants with triple (A128N/H129E/S46F) and quadruple mutations (A128N/H129E/Y132I/S46F) were generated, which showed the synergistic effects, that is 14.5‐ and 15.5‐fold improvement in specific activity relative to wild‐type Δ52 FutA, respectively. The mutations increased their binding affinity for lactose and kcat values for lactose and GDP‐fucose. The Δ52 FutA quadruple mutant (A128N/H129E/Y132I/S46F) was successfully applied to in vitro synthesis of 3‐FL with an improved yield and productivity (>96% yield based on 5 mM of GDP‐Fuc within 1 h). Biotechnol. Bioeng. 2016;113: 1666–1675.

Implementing bacterial acid resistance into cell-free protein synthesis for buffer-free expression and screening of enzymes

Cell-free protein synthesis utilizes translational machinery isolated from the cells for in vitro expression of template genes. Because it produces proteins without gene cloning and cell cultivation steps, cell-free protein synthesis can be used as a versatile platform for high-throughput expression of enzyme libraries. Furthermore, the open nature of cell-free protein synthesis allows direct integration of enzyme synthesis with subsequent screening steps. However, the presence of high concentration of chemical buffers in the conventional reaction mixture makes it difficult to streamline cell-free protein synthesis with pH-based assay of the synthesized enzymes. In this study, we have implemented an enzyme-assisted bacterial acid resistance mechanism into an Escherichia coli (E.coli) extract-based cell-free protein synthesis system in place of chemical buffers. When deployed in the reaction mixture for cell-free synthesis of enzymes, through proton-consuming conversion of glutamate into γ-aminobutyric acid (GABA), an engineered glutamate decarboxylase (GADβ) was able to maintain the pH of reaction mixture during enzyme synthesis. Because the reaction mixture becomes free of buffering capacity upon the depletion of glutamate, synthesized enzyme could be directly assayed without purification steps. The designed method was successfully applied to the screening of mutant library of sialyltransferase genes to identify mutants with improved enzymatic activity.

Helium concentration measurements in an atmospheric micro-plasmas using visible emission spectroscopy

Summary form only given, as follows. A simple in-situ gas monitoring system can be developed with micro discharge plasma sources based on optical emission spectroscopy. Helium concentrations are estimated from the visible line emission of atmospheric plasmas generated with various micro plasma discharges. With this technique, volumetric helium concentrations are accurately measured even down to a percent. Since selected emission lines are not affected by moisture, this measuring system can be operated even without sampling and drying processes, which is a crucial element of in-situ monitoring system. The compact in-situ gas monitoring system will be presented, and the system is expected to be applicable to measuring other elements than helium.