A comparison of the Arthrobacter sp. 32c β-D-galactosidase gene sequence with those from the NCBI database showed that it was most closely related to the Arthrobacter sp. FB24 gene (77.13% sequence identity) and to the A. aurescens TC1 gene (71.8% sequence identity) (Fig. 1B). The deduced amino acid sequence from Arthrobacter sp. 32c β-D-galactosidase gene was also used to compare with other amino Vorinostat price acid sequences deposited in the NCBI database. The Arthrobacter sp. 32c β-D-galactosidase was found to be a member of the glycoside hydrolase family 42 and contained

an A4 beta-galactosidase fold. The enzyme shares 84% of identity and 91% of similarity to the sequence of the Arthrobacter sp. FB24, 74% identity and 84% similarity to the sequence of the Arthrobacter aurescens TC1 and only 51% identity and 65% similarity to the sequence of the Janibacter sp. HTCC2649 β-D-galactosidase. Overexpression and purification of recombinant Arthrobacter sp. 32c β-D-galactosidase In order to produce and investigate the biochemical properties of Arthrobacter sp. 32c β-D-galactosidase, we constructed bacterial and yeast expression systems. The recombinant arabinose-inducible MK-2206 price pBAD-Myc-HisA-β-gal32c plasmid was used for the expression of the Arthrobacter sp. 32c β-D-galactosidase gene in E. coli LMG194/plysN [29]. The highest enzyme biosynthesis

yields were achieved by adding arabinose to the final concentration of 0.02% w/w, at A600 0.5 and by further cultivation for 5 h. After purification a single protein migrating near 70 kDa was observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with Coomassie blue (Fig. 2A, lane 3). It was in good agreement with the molecular mass deduced from the nucleotide sequence (75.9 kDa). The applied overexpression system was quite efficient, giving 27 mg (Table 1) of purified β-D-galactosidase from 1 L of induced culture. The relative molecular mass of native enzyme estimated by gel filtration on a column of SB-3CT Superdex 200 HR 10/30, previously calibrated with protein molecular mass standards, was 195,550 Da. Hence, it is assumed that the purified Arthrobacter sp. 32c β-D-galactosidase is probably a trimeric protein. Table 1 Purification of recombinant

Arthrobacter sp. 32c β-D-galactosidase. Purification step Volume (ml) Protein (mg) Specific activity (U mg-1) Total activity (U) Purification (fold) Recovery (%) E. coli LMG plysN pBADMyc-HisA-32cβ-gal Cell extract 30 580 13.8 8004 1.0 100 Affinity chromatography 3.2 27 155.9 4209 21.0 53 P. pastoris GS115 pPICZαA-32cβ-gal Broth 1000 3400 28.7 97580 1.0 100 Protein precipitation 54 340 136.1 46274 10.0 47 Affinity chromatography 11 137 154.7 21194 24.8 22 P. pastoris GS115 pGAPZαA-32cβ-gal Broth 1000 5200 16.2 84240 1.0 100 Protein precipitation 46 450 102.7 46215 11.6 55 Affinity chromatography 10 97 153.1 14851 53.6 18 Figure 2 SDS-PAGE analysis of the expression and purification steps of the Arthrobacter sp. 32c β-D-galactosidase expressed by E.