A new β-galactosidase from Paenibacillus wynnii with potential for industrial applications

Abstract

Commercial β-galactosidases exhibit undesirable kinetic properties regarding substrate affinity (KM for lactose) and product inhibition (Ki for galactose). An in silico screening of gene sequences was done and identified a putative β-galactosidase (BgaPw) from the psychrophilic bacterium Paenibacillus wynnii. The cultivation of the wild-type P. wynnii strain resulted in very low β-galactosidase activities of a maximum of 150 nkatoNPGal/Lmedium. The recombinant production of BgaPw in Escherichia coli BL21(DE3) increased the yield about 9,000-fold. Here, a volumetric activity of 1350.18 ± 11.82 µkatoNPGal/Lculture was achieved in a bioreactor cultivation. The partly purified BgaPw showed a pH optimum at 7.0, a temperature maximum at 40°C and an excellent stability at 8°C with a half-life of 77 d. Kinetic studies with BgaPw were done in milk or in milk-imitating synthetic buffer, so-called Novo buffer, respectively. Remarkably, the KM value of BgaPw with lactose was as low as 0.63 ± 0.045 mM in milk. It was found that the resulting products of lactose hydrolysis, namely, galactose and glucose, did not inhibit the β-galactosidase activity of BgaPw but instead showed a striking activating effect in both cases (up to 144%). In a comparison study in milk, lactose was completely hydrolyzed by BgaPw in 72 h at 8°C, whereas 2 other known β-galactosidases were less powerful and converted only about 90% of lactose in the same time. Finally, the formation of galactooligosaccharides (GOS) was demonstrated with the new BgaPw, starting with pharma-lactose (400 g/L). A GOS production of about 144 g/L was achieved after 24 h (36.0% yield).