Figure 2
Expression and characterization of recombinant PfXyn5 in P. pastoris (a) SDS-PAGE gel of the supernatant of PfXyn5 produced by P. pastoris over 96 h period of induction. Lanes 1–4 refers to induced cultures of PfXyn5 taken at 24, 48, 72 and 96 h time point while lane 5 refers to wild type culture of P. pastoris X-33 at 96 h time point. (b) SDS-PAGE gel of purified fractions of PfXyn5 by anion exchange chromatography using Q-Sepharose Fast Flow resin. Lane 1 refers to the crude enzyme while lanes 2 and 3 refer to fractions eluted with 25 and 50 mM NaCl gradient. (c) HPLC profile of hydrolysed products of beechwood xylan without PfXyn5 (control). (d) HPLC profile of hydrolysed products of beechwood xylan with PfXyn5. X1, X2, X3, X4, X5 and X6 refers to xylose, xylobiose, xylotriose, xylotetrose, xylopentaose and xylohexaose, respectively. (e) Effects of pH on PfXyn5 activity and stability. The relative activity (%) was calculated relative to the optimum activity obtained at pH 4.5 which was taken as 100%. (f) Effects of temperature on PfXyn5 activity. The relative activity (%) was calculated relative to the optimum activity obtained at 50 °C which was taken as 100% (g) Thermostability profile of PfXyn5. Stability at 40 °C, 50 °C, 60 °C, 70 °C and 80 °C were determined by incubating enzyme at specified temperatures for 300 min while samples were withdrawn every 30 min before assaying for residual activity according to the standard assay procedure.
