Journal
JOURNAL OF BIOTECHNOLOGY
Volume 210, Issue -, Pages 27-37Publisher
ELSEVIER
DOI: 10.1016/j.jbiotec.2015.06.411
Keywords
beta-Galactosidase; Metagenome; Lactose hydrolysis; Lactose-free milk
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The industrially utilised beta-galactosidases from Kluyveromyces spp. and Aspergillus spp. feature undesirable kinetic properties in praxis, such as an unsatisfactory lactose affinity (K-M) and product inhibition (K-I) 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 beta-galactosidases with more favourable kinetic properties. Six novel metagenome beta-galactosidases (M1-M6) were found with an improved lactose hydrolysis performance in original milk when directly compared to the commercial beta-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 beta-galactosidase M1 activity of about 1100 mu kat(0NPGal,37 degrees C) L-1. Since milk is a sensitive and complex medium, it has to be processed at 5-10 degrees C in the dairy industry. Therefore, the beta-galactosidase M1 was tested at 8 degrees C in milk and possessed a good stability (t(1/2) = 21.8 d), a desirably low apparent K-M,K- lactose,K- 8 degrees C value of 3.8+/-0.7mM and a high apparent K-I,K- galactose,K- 8 degrees C value of 196.6+/-55.5mM. A lactose hydrolysis process (milk, 40 nkat(lactose) mL(milk, 8 degrees C)(-1)) was conducted at a scale of 0.5 L to compare the performance of M1 with the commercial beta-galactosidase from K. lactis (GODO-YNL2). Lactose was completely (> 99.99%) hydrolysed by M1 and to 99.6% (w/v) by K. lactis beta-galactosidase after 25 h 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. (C) 2015 The Authors. Published by Elsevier B.V.
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