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".

Updating the metagenomics toolbox.

Metagenomics--the application of the genomics suit of technologies to uncultivated microorganisms--is coming of age. Sophisticated technologies are being developed and adapted to this promising genetic resource to make increasing use of the seemingly boundless molecular and functional diversity. Particular progress has been made in the areas of randomly proliferating limited-source DNA, massively parallel sequencing without cloning, isolating specific target sequences from highly complex template mixtures, high-throughput assay systems targeting metabolic pathways, artificial transcriptional regulators activating reporter genes to indicate enzymatic substrate conversion and cDNA cloning from extracted mRNA to directly clone actively expressed genes from a microbial consortium. However, challenges still lie ahead. Most prominently, the efficient heterologous expression of a plethora of potentially interesting enzymes from unknown source organisms is not readily achieved.

Development of a compression molding process for three-dimensional tailored free-form glass optics.

Because of the limitation of manufacturing capability, free-form glass optics cannot be produced in a large volume using traditional processes such as grinding, lapping, and polishing. Very recently compression molding of glass optics became a viable manufacturing process for the high-volume production of precision glass optical components. An ultraprecision diamond-turning machine retrofitted with a fast tool servo was used to fabricate a free-form optical mold on a nickel-plated surface. A nonuniform rational B-spline trajectory generator was developed to calculate the computer numerical control machine tool path. A specially formulated glass with low transition temperature (Tg) was used, since the nickel alloy mold cannot withstand the high temperatures required for regular optical glasses. We describe the details of this process, from optical surface geometry, mold making, molding experiment, to lens measurement.

Metagenomics: an inexhaustible access to nature's diversity.

The chemical industry has an enormous need for innovation. To save resources, energy and time, currently more and more established chemical processes are being switched to biotechnological routes. This requires white biotechnology to discover and develop novel enzymes, biocatalysts and applications. Due to a limitation in the cultivability of microbes living in certain habitats, technologies have to be established which give access to the enormous resource of uncultivated microbial diversity. Metagenomics promises to provide new and diverse enzymes and biocatalysts as well as bioactive molecules and has the potential to make industrial biotechnology an economic, sustainable success.

Biocatalytic deuterium- and hydrogen-transfer using over-expressed ADH-'A': enhanced stereoselectivity and 2H-labeled chiral alcohols.

Employing the over-expressed highly organic solvent tolerant alcohol dehydrogenase ADH-'A' from Rhodococcus ruber DSM 44541, versatile building blocks, which were not accessible by the wild type catalyst, were obtained in > 99% e.e.; furthermore, employing d8-2-propanol as deuterium source, stereoselective biocatalytic deuterium transfer was made feasible to furnish enantiopure deuterium labeled sec-alcohols on a preparative scale employing a single enzyme.

Screening for novel enzymes for biocatalytic processes: accessing the metagenome as a resource of novel functional sequence space.

Historically, biotechnology has missed up to 99% of existing microbial resources by using traditional screening techniques. Strategies of directly cloning 'environmental DNA' comprising the genetic blueprints of entire microbial consortia (the so-called 'metagenome') provide molecular sequence space that along with ingenious in vitro evolution technologies will act synergistically to bring a maximum of available sequence-space into biocatalytic application.