Mtp 450

Cells in a 12-well plate were washed twice with PBS and fixed with 10% formaldehyde in PBS for 10 min at room temperature. After washing with PBS, the cells were washed with 60% isopropanol and stained with 0.18% Oil Red O in 60% isopropanol for 20 min at room temperature, followed by a final washing with 60% isopropanol and then a final washing with PBS. The resultant stained lipid droplets were imaged by bright field microscopy, and then the dye in the cells was extracted with isopropanol and quantified by measuring its absorbance at 492 nm with a microplate reader (MTP-450; Corona Electric, Ibaraki, Japan).

To verify the binding ability and its structure specificity of β-1,6-glucanase variants to glucans, direct and competitive ELISA-like assays were carried out. In brief, for direct ELISA, pustulan or laminarin (0–5,000 ng/ml) in 0.1 m sodium carbonate buffer (pH 9.5) was added to a 96-well clear plate and incubated at 4 °C. The next day, the plate was washed by PBS containing 0.05% Tween 20 (PBST) and blocked with 1% BSA/PBST (BPBST) by incubating for 1 h. Solid-phased glucans were reacted with recombinant modified Neg1 in BPBST (2 μg/ml) for 1 h and washed, and the HRP-conjugated anti-His tag antibody (BioLegend) was added to the plate. After 1 h, the plate was washed, and the binding of modified enzymes to solid-phase glucans was monitored using the peroxidase substrate TMB, and color development was stopped with 1 m phosphoric acid; the optical density was measured at 450 nm using a microplate reader (MTP450; Corona Electric, Ibaraki, Japan). For competitive ELISA, various glucans (20 and 100 μg/ml, final concentrations) were mixed with Neg1–E321Q–His (0.5 μg/ml, final concentration) and preincubated for 1 h. The pustulan (0.5 μg/ml)-coated 96-well clear plate was blocked, washed, and incubated with the above mixture of E321Q–His and glucan for 1 h. After washing, the binding of E321Q–His to the immobilized pustulan was assessed as described above.

Collected whole blood was allowed to stand at room temperature for 90 min, and then centrifuged (12,000 rpm for 10 min at 4°C) to obtain serum. The enzyme activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were then measured using a Wako Transaminase CII-Test kit (Wako Pure Chemical Co.) following the manufacturer’s instructions. The color development was stopped using citric acid solution, and the optical density at 550 nm was measured using a microplate reader (MTP450; Corona Electric, Ibaraki, Japan). Lactate dehydrogenase (LDH) activity was assayed according to the instructions supplied with the commercial assay kit (NescoVL LD, Alfresa Pharma Co., Osaka, Japan). The absorbance at 340 nm was read using a Safire microplate reader (Tecan).