Novel high-performance metagenome β-galactosidases for lactose hydrolysis in the dairy industry

Abstract

The industrially utilised β-galactosidases from Kluyveromyces spp. and Aspergillus spp. feature undesirable kinetic properties in praxis, such as an unsatisfactory lactose affinity (KM) and product inhibition (KI) by galactose. In this study, a metagenome library of about 1.3 million clones was investigated with a three-step activity-based screening strategy in order to find new β-galactosidases with more favourable kinetic properties. Six novel metagenome β-galactosidases (M1–M6) were found with an improved lactose hydrolysis performance in original milk when directly compared to the commercial β-galactosidase from Kluyveromyces lactis (GODO-YNL2). The best metagenome candidate, called “M1”, was recombinantly produced in Escherichia coli BL21(DE3) in a bioreactor (volume 35L), resulting in a total β-galactosidase M1 activity of about 1100μkatoNPGal,37°C L−1. Since milk is a sensitive and complex medium, it has to be processed at 5–10°C in the dairy industry. Therefore, the β-galactosidase M1 was tested at 8°C in milk and possessed a good stability (t1/2=21.8d), a desirably low apparent KM,lactose,8°C value of 3.8±0.7mM and a high apparent KI,galactose,8°C value of 196.6±55.5mM. A lactose hydrolysis process (milk, 40 nkatlactose mLmilk,8°C−1) was conducted at a scale of 0.5L to compare the performance of M1 with the commercial β-galactosidase from K. lactis (GODO-YNL2). Lactose was completely (>99.99%) hydrolysed by M1 and to 99.6% (w/v) by K. lactis β-galactosidase after 25h process time. Thus, M1 was able to achieve the limit of <100mg lactose per litre milk, which is recommended for dairy products labelled as “lactose-free”.