Optimizing the phosphorus cycle in the sugar beet production process by phytase supplement

Phosphate stewardship and ultimately recycling is one of the great challenges of humankind. Depleting phosphorus (P) resources demand new strategies for an efficient use of this essential nutrient. Therefore, especially phosphorus cycles in agriculture have to be closed. Against this backdrop we propose a new value chain to recover phosphate from plant waste material in sugar production. The approach is based on naturally occurring enzymes that free the phosphate bound in an organic form (mainly phytate in sugar beet slices). Thereby, the currently implemented value chain of phosphate rock mining, production of phosphoric acid, chemical synthesis of polyphosphates, and after use phosphate disposal into waste water, rivers and finally into the ocean will be extended and in the long run disrupted. In sugar production processes this could be achieved by supplementing thermally resistant phytases to leach the phosphate bound as phytate form sugar beet slices. With this procedure the P concentration in sugar beet slices and the export of phosphorous with fodder to areas with high animal density and in consequence P-excess in fields will be reduced. Instead, isolated phosphorus will be transferred into spent lime and subsequently back to the sugar beet fields. The BioSC collaboration project PhytaPhoS assesses and evaluates the potential of P recovery employing phytase, its feasibility and economic approaches from lab scale to field application. Phytases are extremely highly active phosphatases (\u3e1000 U/mg), mobilizing inorganic phosphate from plant based phytate, which is a natural plant phosphate reservoir [1]. Aim in the project is improving specific activity and thermal resistance of a selected phytase by directed evolution [2,3,4] and optimizing the phytase production by employing a signal peptide library from Bacillus subtilis [5]. Please click Additional Files below to see the full abstract

Rapid and Robust Coating Method to Render Polydimethylsiloxane Surfaces Cell-Adhesive

Polydimethylsiloxane (PDMS) is a synthetic material with excellent properties for biomedical applications because of its easy fabrication method, high flexibility, permeability to oxygen, transparency, and potential to produce high-resolution structures in the case of lithography. However, PDMS needs to be modified to support homogeneous cell attachments and spreading. Even though many physical and chemical methods, like plasma treatment or extracellular matrix coatings, have been developed over the last decades to increase cell surface interactions, these methods are still very time-consuming, often not efficient enough, complex, and can require several treatment steps. To overcome these issues, we present a novel, robust, and fast one-step PDMS coating method using engineered anchor peptides fused to the cell-adhesive peptide sequence (glycine-arginine-glycine-aspartate-serine, GRGDS). The anchor peptide attaches to the PDMS surface predominantly by by simply dipping PDMS in a solution containing the anchor peptide, presenting the GRGDS sequence on the surface available for cell adhesion. The binding performance and kinetics of the anchor peptide to PDMS are characterized, and the coatings are optimized for efficient cell attachment of fibroblasts and endothelial cells. Additionally, the applicability is proven using PDMS-based directional nanotopographic gradients, showing a lower threshold of 5 mu m wrinkles for fibroblast alignment

A 96-multiplex capillary electrophoresis screening platform for product based evolution of P450 BM3

The main challenge that prevents a broader application of directed enzyme evolution is the lack of high-throughput screening systems with universal product analytics. Most directed evolution campaigns employ screening systems based on colorimetric or fluorogenic surrogate substrates or universal quantification methods such as nuclear magnetic resonance spectroscopy or mass spectrometry, which have not been advanced to achieve a high-throughput. Capillary electrophoresis with a universal UV-based product detection is a promising analytical tool to quantify product formation. Usage of a multiplex system allows the simultaneous measurement with 96 capillaries. A 96-multiplexed capillary electrophoresis (MP-CE) enables a throughput that is comparable to traditional direct evolution campaigns employing 96-well microtiter plates. Here, we report for the first time the usage of a MP-CE system for directed P450 BM3 evolution towards increased product formation (oxidation of alpha-isophorone to 4-hydroxy-isophorone; highest reached total turnover number after evolution campaign: 7120 mol4-OH molP450−1). The MP-CE platform was 3.5-fold more efficient in identification of beneficial variants than the standard cofactor (NADPH) screening system