pastoris GS115 pPICZαA-32cβ-gal methanol induced variant (B) and P. pastoris GS115 pGAPZαA-32cβ-gal constitutive variant (C). Lanes 1 – protein weight marker. Panel A: lane 2 – cell selleck inhibitor extract after expression, lane

3 – purified β-D-galactosidase after affinity chromatography. Panel B and C: lane 2 – broth after protein expression, lane 3 – protein precipitate, lane 4 – purified β-D-galactosidase after affinity chromatography. In the P. pastoris expression system the methanol induced and constitutive biosynthesis variants for larger scale production of the enzyme were tested. By cloning the gene in the form of translational fusion with the S. cerevisiae α-factor leader sequence under the control of either the methanol induced promoter AOX1 or under the constitutive promoter GAP, pPICZαA-32cβ-gal and pGAPZαA-32cβ-gal recombinant expression plasmids were constructed. P. pastoris GS115 strain was transformed with linearized pPICZαA-32cβ-gal or pGAPZαA-32cβ-gal plasmids. The obtained P. pastoris GS115 recombinant strains harbouring pGAPZαA-32cβ-gal or pPICZαA-32cβ-gal recombinant plasmids were used for extracellular production of the Arthrobacter sp. 32c β-D-galactosidase (Fig. 2B, lane 2 and Fig. 2C, lane 2). The applied overexpression systems were efficient, C188-9 cost giving see more approximately 137 and 97 mg (Table 1) of purified β-D-galactosidase (Fig. 2B and 2C, lanes 4) from 1 L of induced culture for the AOX1 and constitutive system, respectively. Noteworthy

is the fact that all attempts in extracellular expression of β-D-galactosidase from Pseudoalteromonas sp.22b [10, 11] previously described by us did not succeed (data not shown). pheromone The corresponded β-D-galactosidase is a tetramer composed of 115 kDa subunits. All the amount

of produced protein with fused secretion signal was accumulated in the cells. We also tried to produce the Pseudoalteromonas sp. 22b β-D-galactosidase in the form of fusion protein with other secretion sequences: PHO5 and STA2. All attempts gave negative results. It seems that molecular mass of desired recombinant protein is limited for extracellular production by P. pastoris host. Characterization of Arthrobacter sp. 32c β-D-galactosidase The temperature profiles of the hydrolytic activity of the recombinant Arthrobacter sp. 32c β-D-galactosidase showed that the highest specific activity with ONPG was at 50°C (155 U/mg). Lowering or raising temperature from 50°C resulted in the reduction of β-D-galactosidaseactivity. Recombinant β-D-galactosidase exhibited 15% of the maximum activity even at 0°C and approximately 60% at 25°C (Fig. 3). In order to determine the optimum pH for recombinant β-D-galactosidase, we measured the enzyme activity at various pH values (pH 4.5–9.5) at 0–70°C, using ONPG as a substrate. β-D-galactosidase exhibited maximum activity in pH 6.5 and over 90% of its maximum activity in the pH range of 6.5–8.5 (Fig. 3). Figure 3 Effect of temperature on activity of recombinant Arthrobacter sp.