Molecular cloning and high-level expression of a β-galactosidase gene from Paecilomyces aerugineus in Pichia pastoris

A β-galactosidase gene (designated PaGalA) was cloned for the first time from Paecilomyces aerugineus and expressed in Pichia pastoris under the control of the AOX1 promoter. The coding region of 3036 bp encoded a protein of 1011 amino acids with a deduced molecular mass of 108.7 kDa. The PaGalA without the signal peptide was cloned into a vector pPIC9K and was expressed successfully in P. pastoris as active extracellular β-galactosidase. The recombinant β-galactosidase (PaGalA) was secreted into the medium at an extremely high levels of 22 mg ml−1 having an activity of 9500 U ml−1 from high density fermentation culture, which is by far the highest yield obtained for a β-galactosidase. The purified enzyme with a high specific activity of 820 U mg−1 had a molecular mass of 120 kDa on SDS-PAGE. PaGalA was optimally active at pH 4.5 and a temperature of 60 °C. The recombinant β-galactosidase was able to hydrolyze lactose efficiently at pH 5.0 and 50 °C. It also possessed transglycosylation activities at high concentrations of lactose. PaGalA exhibited better lactose hydrolysis efficiency in whey than two other widely used commercial lactases. The extremely high expression levels coupled with favorable biochemical properties make this enzyme highly suitable for commercial purposes in the hydrolysis of lactose in milk or whey.

Graphical abstractA β-galactosidase gene (designated PaGalA) was cloned for the first time from Paecilomyces aerugineus and expressed in Pichia pastoris. The extremely high expression levels coupled with favorable biochemical properties make this enzyme highly suitable for commercial purposes in the hydrolysis of lactose in milk or whey.Figure optionsDownload full-size imageDownload as PowerPoint slideResearch highlights▶ High-level expression of a β-galactosidase gene was achieved in Pichia pastoris. ▶ The β-galactosidase was optimally active at pH 4.5 and a temperature of 60 °C. ▶ The enzyme efficiently hydrolyzed lactose lactose present in acid- and sweet-whey.