title: 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 35 L), resulting in a total β-galactosidase M1 activity of about 1100 μkatoNPGal,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=21.8 d), a desirably low apparent KM,lactose,8 °C value of 3.8±0.7 mM and a high apparent KI,galactose,8 °C value of 196.6±55.5 mM. 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 25 h process time. Thus, M1 was able to achieve the limit of <100 mg lactose per litre milk, which is recommended for dairy products labelled as "lactose-free".
authors.affiliation: University of Hohenheim, Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, Garbenstr. 25, 70599 Stuttgart, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Erich
authors.affiliation: University of Hohenheim, Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, Garbenstr. 25, 70599 Stuttgart, Germany.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Kuschel
authors.affiliation: University of Hohenheim, Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, Garbenstr. 25, 70599 Stuttgart, Germany.
authors.forename: Thilo
authors.initials: T
authors.lastname: Schwarz
authors.affiliation: University of Hohenheim, Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, Garbenstr. 25, 70599 Stuttgart, Germany.
authors.forename: Jacob
authors.initials: J
authors.lastname: Ewert
authors.affiliation: University of Hohenheim, Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, Garbenstr. 25, 70599 Stuttgart, Germany.
authors.forename: Nico
authors.initials: N
authors.lastname: Böhmer
authors.affiliation: B.R.A.I.N. Biotechnology Research and Information Network AG, Darmstaedter Str. 34-36, 64673, Zwingenberg, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Niehaus
authors.affiliation: B.R.A.I.N. Biotechnology Research and Information Network AG, Darmstaedter Str. 34-36, 64673, Zwingenberg, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Eck
authors.affiliation: University of Hohenheim, Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, Garbenstr. 25, 70599 Stuttgart, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Lutz-Wahl
authors.affiliation: University of Hohenheim, Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, Garbenstr. 25, 70599 Stuttgart, Germany.
authors.forename: Timo
authors.initials: T
authors.lastname: Stressler
authors.affiliation: University of Hohenheim, Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, Garbenstr. 25, 70599 Stuttgart, Germany. Electronic address: lutz.fischer@uni-hohenheim.de.
authors.forename: Lutz
authors.initials: L
authors.lastname: Fischer
mesh_terms: D000818:Animals; D019149:Bioreactors; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D019649:Food Industry; D015723:Gene Library; D006868:Hydrolysis; D007700:Kinetics; D007785:Lactose / Q000737:chemistry*; D054892:Metagenome; D008892:Milk / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D001616:beta-Galactosidase; D007785:Lactose
keywords: Lactose hydrolysis; Lactose-free milk; Metagenome; β-Galactosidase