Figure 6

Transglutaminase and deglycosylation activity measurements in various strains developed.(A) TG activity was measured in wild type, Ax2 (WT), overexpresser, [act15/png-eyfp]/Ax2] (PNG O/E) and knockout, png^-/Ax2 (pngKO) cells. Activity in WT was taken as 100%. No significant decrease in activity in the pngKO cells was observed. (B) Deglycosylation activity using denatured ovalbumin as a substrate was measured using commercially available pure PNGase F and lysates of [act15/png-eyfp]/Ax-2 and (png^-/Ax2) cells at various time points (0 h, 1 h, 3 h, 5 h and 24 h) by Western blot analysis. The glycosylated (+CHO) band was seen in all the lanes and was comparable to that observed in lane 1 and 7 where ovalbumin was incubated without any enzyme for overnight. When purified enzyme (lanes 12–16) and lysate from PNGase overexpressing strain was used, a time dependent increase in the lower deglycosylated (−CHO) band was observed (lanes 2–6). The absence of this -CHO band shows the loss of deglycosylation activity in the knock out strain (lanes 8–11). +CHO and -CHO indicate glycosylated and deglycosylated forms of ovalbumin respectively. [Ov-ovalbumin; P-purified PNGase F; O-[act15/png-eyfp]/Ax-2; K- (png^-/Ax2); 0–0 h reaction;1-1 h reaction; 3–3 h reaction; 5-5 h reaction; 24-24 h reaction].