Cloning, expression, and purification of a recombinant cold-adapted β-galactosidase from antarctic bacterium Pseudoalteromonas sp. 22b

Abstract

The gram-negative antarctic bacterium Pseudoalteromonas sp. 22b, isolated from the alimentary tract of krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted β-galactosidase. The gene encoding this β-galactosidase has been PCR amplified, cloned, expressed in Escherichia coli, purified, and characterized. The enzyme is active as a homotetrameric protein, and each monomer consists of 1028 amino acid residues. The enzyme was purified to homogeneity (50% recovery of activity) by using the fast, two-step procedure, including affinity chromatography on PABTG–Sepharose. Enzymatic properties of the recombinant protein are identical to those of native Pseudoalteromonas sp. 22b β-galactosidase. The enzyme is cold-adapted and at 10 °C retains 20% of maximum activity. The purified enzyme displayed maximum activity close to 40 °C and at pH of 6.0–8.0. PNPG was its preferred substrate (58% higher activity than against ONPG). The enzyme was particularly thermolabile, losing all activities within 10 min at 50 °C. The hydrolysis of lactose in a milk assay revealed that 90% of milk lactose was hydrolyzed during 6 h at 30 °C and during 28 h at 15 °C. Because of its attributes, the recombinant Pseudoalteromonas sp. 22b β-galactosidase could be applied at refrigeration temperatures for production of lactose-reduced dairy products.