The bifidobacterial β-galactosidase (BbgIV) was produced in E. coli DH5α at 37 and 30 °C in a 5 L bioreactor under varied conditions of dissolved oxygen (dO2) and pH. The yield of soluble BbgIV was significantly (P < 0.05) increased once the dO2 dropped to 0-2% and remained at such low values during the exponential phase. Limited dO2 significantly (P < 0.05) increased the plasmid copy number and decreased the cells growth rate. Consequently, the BbgIV yield increased to its maximum (71-75 mg per g dry cell weight), which represented 20-25% of the total soluble proteins in the cells. In addition, the specific activity and catalytic efficiency of BbgIV were significantly (P < 0.05) enhanced under limited dO2 conditions. This was concomitant with a change in the enzyme secondary structure, suggesting a link between the enzyme structure and function. The knowledge generated from this work is very important for producing BbgIV as a biocatalyst for the development of a cost-effective process for the synthesis of prebiotic galactooligosaccharides from lactose.