Fig. 1. (A) Map of the clone AF118226 representing the genomic organization of the barley high pI α-glucosidase and the exon specific primers used for cloning. (B) The processed α-glucosidase (α-ΔGlc) encoding expression vector used in the P. pastoris transformation.

Fig. 2. (A) Progress of α-glucosidase activity in the medium during fermentation. (B) Secreted activity as a function of cell-density. The arrow indicates the end of the un-induced stage of fermentation, i.e., the glycerol utilization phase, and the start of the methanol induced phase. (C) Silver stained SDS–PAGE (4–12% gradient) of culture representing: lane 1, molecular markers. Lane 2, 24 h of fermentation corresponding to the un-induced stage. Lanes 3 and 4 presents 20 μl of culture from the induction stage at 101 and 120 h, respectively. Arrow heads illustrate induced accumulated protein. (D) Immunoblotting on culture medium using Mab against a poly-histidine tag. Lane 1, positive control (Positope, Invitrogen). Lane 2, 24 h of fermentation. Lane 3, at harvest (120 h).

Fig. 3. Silver stained SDS–PAGE (4–12% gradient) of culture medium representing lane 1, 24 h; lane 2, 101 h; lane 3, 120 h; lane 4, cell extract of P. pastoris at 120 h of fermentation i.e., at harvest; lane 5, diafiltration retentate; lane 6, diafiltration filtrate; lane 7, ultrafiltration retentate; lane 8, ultrafiltration filtrate; lane 9, pooled fractions of Ni–NTA chromatography; and lanes 10 and 11, 40 and 4 μg protein, respectively of pooled fractions from size-exclusion chromatography.

Fig. 4. (A) Michaelis–Menten plot on the velocity of hydrolysis of maltose. (B) Lineweaver–Burk double-reciprocal plot for data in (A).

Purification of recombinant high pI barley α-glucosidase from P. pastoris culture supernatant

One unit of activity is defined as the amount of enzyme that releases 2 μmol × min⁻¹ of glucose from maltose, when using 15 mM maltose as substrate at 37 °C.