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

The industrially utilised ß-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 ß-galactosidases with more favourable kinetic properties. Six novel metagenome ß-galactosidases (M1-M6) were found with an improved lactose hydrolysis performance in original milk when directly compared to the commercial ß-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 ß-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 ß-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 ß-galactosidase from K. lactis (GODO-YNL2). Lactose was completely (>99.99%) hydrolysed by M1 and to 99.6% (w/v) by K. lactis ß-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".

A novel manganese starvation-inducible expression system for Lactobacillus plantarum.

A novel expression system for Lactobacillus plantarum was developed. This system is based on the manganese starvation-inducible promoter from specific manganese transporter of L. plantarum NC8, which was cloned for the first time. The expression of a ß-glucosidase from Pyrococcus furiosus (CelB) was achieved by cultivating L. plantarum NC8 at low manganese concentrations with MRS medium and the pmntH2-CelB expression vector. A CelB activity of 8.52 µkatoNPGal L(-1) was produced in a bioreactor (4 L). The advantages of the novel expression system are that no addition of an external inducing agent was required, and additionally, no further introduction of regulatory genes was necessary. The new promoter meets the general demands of a food-grade expression system.

Recombinant expression, purification and characterisation of the native glutamate racemase from Lactobacillus plantarum NC8.

Glutamic acid racemases (MurI, E.C. 5.1.1.3) catalyse the racemisation of L- and D-glutamic acid. MurIs are essential enzymes for bacterial cell wall synthesis, which requires d-glutamic acid as an indispensable building block. Therefore these enzymes are suitable targets for antimicrobial drugs as well as for the potential design of auxotrophic selection markers. A high expression system in Escherichia coli BL21 (DE3) was constructed to produce and characterise the biochemical properties of the MurI from Lactobacillus plantarum NC8. In a 4-L-bioreactor cultivation, 3266 nkat(D-Glu)/mg(protein) of specific enzyme activity was produced. The recombinant, tag-free Murl was purified by an innovative affinity chromatography method using L-glutamic acid as the relevant docking group, followed by an anion exchange chromatography step (purification factor 9.2, yield 11%). This two-step purification strategy resulted in a Murl sample with a specific activity of 34,060 nkat(D-Glu)/mg(protein), comprising a single protein band in SDS-PAGE. The purified Murl possessed an assay temperature optimum of 50 °C, but it was not stable at this temperature. The half-lives of the purified Murl were 162 h at 20 °C and only 1.9 h at 40 °C. The Murl activity was maximum between pH 7 and 10, resulting in a maximal half-life of 287 h at pH 7. Only D- and L-glutamic acid were recognised as substrates for the Murl with similar K(cat)/K(M) ratios of 3.6s(-1)/mM for each enantiomer.