Fig. 2
Analysis of the expression of the RBD from PPEPCK and PAOX1 in shake flask cultures of K. phaffii. A Strategy for the generation of KpPAOX1RBD and KpPPEPCKRBD expressing His-tagged RBD from PAOX1 and PPEPCK, respectively. KpPPEPCKRBD was further transformed with pPEPCKA-RBD to generate KpPPEPCKRBD* (Table 1), in which the PPEPCK-GFP expression cassette is integrated at the PEPCK and HIS4 loci of the genome. B SDS‒PAGE analysis of RBD secreted into medium by cells cultured for 24 h in YNBM or YNB-MSG as indicated. RBD was purified from the culture medium of cells equivalent to 50 A600 units. His-tagged RBD bound to Ni-agarose beads was subjected to SDS‒PAGE. M, molecular weight markers (kDa). C Quantification of RBD in the gel by densitometric scanning using ImageJ. Expression in KpPAOX1RBD was taken as 100%. Error bars in the graphs indicate the mean ± SD of 3 biological replicates. The p value was obtained from Student’s t test and is mentioned on the bar. *p < 0.05; ns, not significant. D Protein profile of RBD purified from KpPPEPCKRBD* cultured for 24 h, 48 h and 72 h in BMY-MSG as visualized by SDS‒PAGE. His tagged RBD present in the culture medium of cells equivalent to 50 A600 units was bound to Ni–NTA agarose beads and visualized by SDS‒PAGE. M, molecular weight markers (kDa). E Schematic diagram of ammonia production from the GDH2-catalyzed reaction and its efflux in the culture medium, resulting in alkalization. F Colorimetric detection of ammonia in the culture medium using Nessler’s reagent containing K2HgI4 and KOH. A reddish‐brown complex formed by the reaction of ammonia with iodide and mercury ions under alkaline conditions is detected spectrophotometrically by measuring absorbance at 420 nm. The pH of the culture medium measured at different time intervals is indicated. G Effect of maintenance of extracellular pH at ~ 7.0 by the addition of H2SO4 to BMY-MSG on RBD levels M, molecular weight markers (kDa). H Effect of H2SO4 addition on the growth of KpPPEPCKRBD* cultured in BMY-MSG for up to 72 h. Data are the average of two independent experiments