Jlc 500 v2

The gastrocnemius muscle was homogenized in five volumes of ice-cold 5% 5-sulfosalicylic acid using a bead homogenizer (TAITEC) and centrifuged at 10,000×g for 10 min at 4 °C^{53 (link)}. Measurement of amino acid concentration in the supernatant was outsourced to Organization for Research Initiative and Promotion at Tottori University (Tottori, Japan) and measured using an automatic amino acid analyzer (JLC-500/V2, JEOL, Ltd., Tokyo, Japan).

Yeast cells were cultured in SD+Am medium for 2 days at 30°C, then inoculated into the same medium at an OD₆₀₀ of 0.1. After cultivation for 48 h at 30°C under shaking, the yeast cells were harvested by centrifugation and washed twice. The resulting yeast pellet was used for measurement of intracellular amino acid contents. In the case of yeast cells expressing ILV1^WT and ILV1^H480Y, the separated supernatant was used for quantification of extracellular fusel alcohol contents (in “Quantification of extracellular fusel alcohols content”). Next, harvested cells were resuspended in sterilized water and the suspension was adjusted to an OD₆₀₀ = 40. Consequently, amino acids in an aliquot (0.5 ml) of the cell suspension were extracted by boiling water at 100°C for 20 min. After centrifugation (5 min at 15,000 × g), the amino acid content in each supernatant was subsequently quantified with an amino acid analyzer with ion-exchange chromatography and post-column ninhydrin derivatization (JLC-500/V2, JEOL, Tokyo, Japan). The content of each amino acid was represented as μmol per g dry cell weight (DCW).

The free amino acid content was determined using an automatic amino acid analyser (JLC-500/V2, Jeol, Tokyo, Japan) according to the post-label ninhydrin method. Briefly, the collected supernatants were appropriately diluted, and the pH values were adjusted to the range of 2 to 3, followed by filtering using 0.45 μm filters. Then, the filtered supernatants were subjected to the amino acid analyser. The separation of free amino acids was performed using the cation exchange resin in a high separation mode with a lithium citrate buffer system. Then, the separated free amino acids were derivatized with the ninhydrin reagent and detected with a visible light detector. The open Type AN-II and Type B (Wako Pure Chemical, Osaka, Japan) were used as the standards.

Yeast cells were cultured in the SD + Am medium for 2 days at 30°C and then inoculated into the same medium at an optical density at 600 nm (OD₆₀₀) of 0.1. After cultivation for 48 h at 30°C under shaking, yeast cells were harvested by centrifugation and washed twice. The resulting yeast pellet was resuspended in sterilized water, and the suspension was adjusted to an OD₆₀₀ = 300. Consequently, amino acids in 0.4 mL of the cell suspension were extracted by boiling water at 100°C for 20 min. After centrifugation (5 min at 15,000 × g), the amino acid content in each supernatant was subsequently quantified with an amino acid analyzer with ion-exchange chromatography and post-column ninhydrin derivatization (JLC-500/V2, JEOL, Tokyo, Japan). The content of each amino acid was represented as µmol per gram dry cell weight (DCW). In the case of strains WT and HNV-5, we also measured intracellular and extracellular Thr contents of these yeast cells cultivated in the YPD medium as follows. Strains WT and HNV-5 were inoculated into the YPD medium starting from an OD₆₀₀ of 0.1. After incubation for 24 h at 30°C under shaking, yeast cells and the culture medium were separated by centrifugation. Amino acids in the collected cells were extracted using the same method as described above. Thr content in yeast cells and the culture medium was quantified with the amino acid analyzer.

Test substances were desalted using ultrafiltration via an ultrafiltration filter unit, and 20 μg aliquots were hydrolyzed with 2.5 mol/L trifluoroacetic acid at 100°C for 3 h. The samples were then cooled, dried, and reconstituted in L-rhamnose solution. Neutral monosaccharide was analyzed using a Shin-pack ISA-07/S2504 column (0.7 μm ID and 250 mm in length, Shimadzu) with a 50-min linear gradient of 0.1–0.4 mol/L potassium buffer (pH 8.0–9.0) by the Reducing Sugar Analysis System (Shimadzu). For amino sugar determination, test substances were hydrolyzed with 4 mol/L HCl at 100°C for 3 h, cooled, dried, and reconstituted in 0.02 mol/L HCl. An amino acid analyzer JLC-500/V2 (JEOL, Tokyo, Japan) was used for the analysis. For sialic acid analysis, test substances were treated with 0.1 mol/L NaOH at 37°C for 30 min, neutralized with HCl, and then reacted with 0.035 mol/L HCl at 70°C for 140 min to release sialic acids. The released sialic acids were fluorescently labeled with 1,2-diamino-4,5-methylenedioxybenzene at 50°C for 150 min. The solutions were analyzed by RP-HPLC using a COSMOSIL 5C18-PAQ column (4.6 mm ID and 150 mm in length, Nacalai Tesque, Kyoto, Japan) with a methanol/acetonitrile/water solvent system and fluorescence detection (Ex: 373 nm, Em: 448 nm). Quantification was performed relative to the appropriate monosaccharide standard curve.

S. cerevisiae transformants expressing the WT and ScLys20 variants were precultured for 2 days at 30°C and then inoculated into the same medium at an OD₆₀₀ of 0.1. After cultivation for 72 h at 30°C, yeast cells were collected by centrifugation and then washed twice with sterilized water. Harvested cells were resuspended in sterilized water, and the suspension was adjusted to an OD₆₀₀ of 20. Consequently, intracellular amino acids in an aliquot (0.5 ml) of the cell suspension were extracted by boiling water at 100°C for 20 min. After centrifugation, each supernatant was subsequently quantified with an amino acid analyzer by ion-exchange chromatography and postcolumn ninhydrin derivatization (JLC-500/V2; JEOL). The content of each amino acid was represented as μmol per mg dry cell weight (DCW).