Paenibacillus polymyxa is one of the bacteria carrying the gene encoding β-glucosidase (Bgl) enzyme. The β-glucosidase enzyme can hydrolyze lactose and synthesize galactooligosaccharides through the transglycosylation process. Galactooligosaccharides are sugar that is recognized as prebiotics. Our study using heterologous expression of the β-glucosidase enzyme in Escherichiacheria coli BL21 (DE3) has been carried out previously. This study aims to further characterize the purity of a recombinant β-glucosidase enzyme at various temperature and pH. Enzyme purification was carried out by a one-step purification method using a 6x-His Tag system. The purity of β-glucosidase was evaluated using SDS-PAGE electrophoresis and its specific was activity analyzed by p-nitrophenol beta-D-glucopyranoside (pNPG) assay (at 400 nm) and Bradford assay (at 595 nm) using spectrophotometry. The β-glucosidase enzyme was successfully purified by one-step purification. A single band at 52 kDa appeared on the SDS-PAGE electrophoresis gel, revealing the purity of β-glucosidase protein molecular weight. Pure β-glucosidase had a specific activity of 4.1 U/mg after being measured in pNPG assay and Bradford assay. The optimum pH and temperature for the pure β-glucosidase were 6.0 and 55°C, respectively. The enzyme activity is affected by the addition of metal ions mainly Zn2+. The result shows a similar and same condition with the β-glucosidase enzyme from different bacterial sources. Further study will explore the transglycosylation potential of the recombinant β-glucosidase enzyme from Paenibacillus polymyxa. [ABSTRACT FROM AUTHOR]