Novel high-performance metagenome β-galactosidases for lactose hydrolysis in the dairy industry
| Autor: | Timo Stressler, Lutz Fischer, Frank Niehaus, Nico Böhmer, Sarah Erich, Thilo Schwarz, Jürgen Eck, Sabine Lutz-Wahl, Jacob Ewert, Beatrice Kuschel |
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| Rok vydání: | 2015 |
| Předmět: |
β-Galactosidase
Bioengineering Lactose Applied Microbiology and Biotechnology chemistry.chemical_compound Hydrolysis Bioreactors Kluyveromyces Enzyme Stability Bioreactor Escherichia coli Animals Food Industry Food science Gene Library Kluyveromyces lactis Lactose hydrolysis Galactosidases biology General Medicine biology.organism_classification beta-Galactosidase Recombinant Proteins Kinetics Milk chemistry Biochemistry Product inhibition Galactose Metagenome Lactose-free milk Biotechnology |
| Zdroj: | Journal of Biotechnology. 210:27-37 |
| ISSN: | 0168-1656 |
| DOI: | 10.1016/j.jbiotec.2015.06.411 |
| Popis: | 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 |
| Databáze: | OpenAIRE |
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