Lc 10a hplc system

The molecular weight (M_w, M_n) of aAAP-1 was measured by using Shimadzu LC-10AVP HPLC system (Shimadzu Corporation, Kyoto, Japan), equipped with BRT 105-104-102 Tandem gel column (Φ × h, 8.0 mm× 300 mm, Borui Saccharide, Biotech. Co. Ltd., YangZhou, China) and a Parallax detector. After being centrifuged (12000 rpm, 10 min) and filtered with a 0.45-μm filter, the (5.0 mg/mL) supernatant was injected into the detector. The 0.05 M NaCl was regarded as the flow phase, and the elution rate was 0.6 mL/min. The standard curve, where the elution time was plotted against the logarithm of the molecular weight, was made using dextran as a standard (1152, 11,600, 23,800, 48,600, 80,900, 148,000, 273,000, and 409,800 kDa).
Monosaccharide composition analysis of aAAP-1 was performed using a Shimadzu LC-10A HPLC system (Shimadzu Corporation, Tokyo, Japan) with an Agilent eclipse plus C18 column (Φ × h, 4.5 mm × 150 mm, 5 μm, Agilent Technologies Inc., Santa Clara, CA, USA). The detection wavelength was set at 254 nm. The temperature of the column was maintained at 35 °C. The mobile phase consisted of acetonitrile (A) and NaH₂PO₄ buffer (B: 0.45 g NaH₂PO₄ + 900 mL purified water + 1.0 mL TEA + 100 mL acetonitrile) with a flow rate of 1.0 mL/min. A gradient program was conducted by 94% B + 6% A (V:V), 4 min and 88% B + 12% A(V:V), 55 min.

Before analyses of soluble Sb and acetate, the collected samples were filtered through a 0.2 μm cellulose acetate membrane filter (Advantec). Inductively coupled plasma-atomic emission spectrometry (SPS7800, SII Nano Technology, Tokyo, Japan) was used to determine the concentration of total soluble Sb. The speciation of Sb(V) and Sb(III) was performed using a Shimadzu LC-20A high-performance liquid chromatography system (HPLC; Shimadzu, Kyoto, Japan) coupled with hydride generation-atomic fluorescence spectrometry (HG Millennium Excalibur System; P S Analytical, Orpington, UK). A PRP-X100 anion-exchange HPLC column (Hamilton, Reno, USA) and a mobile phase of 200 mM ammonium tartrate (pH 5.0) were used for the separation of Sb(V) and Sb(III), and the separated Sb species were detected with a P802SF hollow cathode lamp for Sb (Photron Pty, Narre Warren, Australia). The acetate concentration was determined with a Shimadzu LC-10A HPLC system (Shimadzu) equipped with an Aminex HPX-87H ion exclusion column (Bio-Rad Laboratories, Hercules, California, USA) using a mobile phase of 5.0 mM sulfuric acid.
The OD₆₀₀ of the culture was measured with a UV-1850 UV–vis spectrophotometer (Shimadzu). The protein concentration was determined using a BCA Protein Assay kit (Takara Bio) and an Epoch 2 microplate spectrophotometer (BioTek, Winooski, Vermont, USA).

The cells were harvested at 36 h of the fermentation and prepared according to the methods of previous reports for amino acid analyses [15 (link), 41 (link)]. The levels of SAM, Met, Ser and Asp were measured by a Shimadzu LC10A HPLC system (Shimadzu, Kyoto, Japan) equipped with a Megres C18 column (5 μm, 4.6 mm × 250 mm) (Hanbon Sci. & Tech., China). Peak area analysis was performed based on the standard calibration curves of SAM, Met, Ser and Asp (Sangon, Shanghai, China) according to the methods of previous reports [15 (link), 44 (link), 45 ]. For the measurement of Cys, the precolumn derivatization high-performance liquid chromatographic method was performed according to previous reports [46 (link)].