Heterologous expression in the yeast double knockout mutant

The AmCDase coding sequence was subcloned into the yeast expression vector pYES2/CT, which was regulated by the Gal1 promoter, and the construct was transformed into the double knockout mutant of yeast Δypc1 Δydc1 by the lithium acetate method [4, 19, 20]. The yeast strains containing pYES2-AmCDase were grown in synthetic basic culture medium with plenty of glucose and uracil-deficient supplementation. The yeast transformed with empty pYES2/CT vector was assigned to the control group. Then, the yeast was cultured in 2% SC-Ura^-medium (w/v) with galactose and the cells were collected at different culture time points. To identify the best time for the induction of AmCDase, the amount of recombinant AmCDase was determined by Western blotting using anti-His antibody. The yeasts at different culture time points were solubilized in SDS/PAGE sample buffer, and the cell lysates were isolated as mentioned previously [21]. The homogenates were assayed for the following tests in which D-erythro-C12-NBD-ceramide was used as a substrate and its content was analysed by reverse-phase HPLC (High Performance Liquid Chromatography) with DAD detector (Determination conditions: Sino Chrom C18 reversed-phase column, Agilent 1100 chromatographic system, detector wavelength of 230 nm, column temperature 25 °C, acetonitrile as the mobile phase, sample size 10 μl) and the standard curve was consequently obtained. To test the biochemical properties of recombinant AmCDase, , the samples were initialized with the same amount of D-e rythro-C12-NBD-ceramide as a substrate, but the different amounts of recombinant AmCDase that reacted with the substrate were added into these samples. D-erythro-C12-NBD-ceramide was diluted at a concentration of 100 μM (3.13 mol %) in 100 mM phosphate buffer, pH 5.7, containing 0.2% Triton X-100, and 5 mM MgCl₂ in a total volume of 100 μl. The enzymatic reaction lasted 1 h at 37 °C, and was terminated by the addition of chloroform/methanol (1:1, v/v) [9]. The enzymatic reaction was started by the addition of the same amount of cell lysate, and a remnant amount of D-erythro-C12-NBD-ceramide in different samples after the reaction was analysed under the same conditions.