KnowVolution: Redesigning enzymes for innovations

Directed evolution has matured in academia and industry to a routinely applied algorithm to tailor enzyme properties1 to match especially demands in synthesis and material science. In order to free directed enzyme evolution from methodological restraints and to efficiently explore its potential, one has to balance time requirements for a directed evolution campaign, the number of generated enzyme variants, and limitations in state of the art screening technologies. For instance, saturation mutagenesis of six amino acid positions in an enzyme, which usually consists of \u3e50 amino acids, yields 64 million (206) different enzyme variants. The latter represents the upper throughput for activity-based screening systems2. In essence, protein engineers have to accept that they will not be able to sample through the theoretical obtainable sequence space of enzyme variants and smarter strategies are required for efficient directed enzyme evolution. The KnowVolution (Knowledge gaining direct evolution)3 approach represents such a directed evolution 2.0 strategy, which identifies in four phase with limited screening efforts, significantly improved enzymes variants and ensures a molecular understanding of improved enzyme properties. Three out of six in a review reported KnowVolution campaigns3 were commercialized by industrial partners; thereby limiting the number of substitutions turned out to be a key prerequisite for maintaining thermal resistance, process stability and selectivity. In addition, directed enzyme evolution by random mutagenesis will be compared to improvements that are obtainable with a variant library that contains all natural possible diversity with ONE amino acid exchange (SSM library)4. The comparison of 3000 mutations from random mutagenesis libraries with the SSM library taught us how many of the natural occurring beneficial positions are obtainable or unobtainable by state of art methodologies in directed evolution and provided first insights on general design principles to improve enzymatic resistance in organic cosolvents4 and ionic liquids4. References: (1) a.Shivange, A. V., Marienhagen, J., Mundhada, H., Schenk, A., Schwaneberg, U. (2009). Curr. Opin. Chem. Biol. 13, 19. b.Ruff, A. J., Dennig, A., Schwaneberg, U. (2013). FEBS J. 280, 2961. (2) a.Körfer, G., Pitzler, C., Vojcic, L., Martinez, R., Schwaneberg, U. (2016). Scientific Reports, 6, 1-12. b.Lülsdorf, N., Pitzler, C., Biggel, M., Martinez, R., Vojcic, L., Schwaneberg, U. (2015). Chem. Commun. 51, 8679. c.Ruff, A. J., Dennig, A., Wirtz, G., Blanusa, M., Schwaneberg, U. (2012). ACS Catalysis 2, 2724. (3) Cheng, F., Zhu, L., Schwaneberg, U. (2015). Chem. Commun. 51, 9760. a.Zhao, J., Frauenkron-Machedjou, V. J., Kardashliev, T., Ruff, A. J., Zhu, L., Bocola, M., Schwaneberg, U. (2017). Appl. Microbiol. Biotechnol., 2017, DOI: 10.1007/s00253-016-8035-1. b.Frauenkron-Machedjou, V. J., Fulton, A., Zhu, L., Bocola, M., Zhu, L., Jaeger, K.-E., Schwaneberg, U. (2015). ChemBioChem, 16, 937-945. c.Zhao, J., Kardashliev, T., Ruff, A. J., Bocola, M., Schwaneberg, U. (2014). Biotechnol. Bioeng. 111, 2380

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

Особенности применения автодинных КВЧ модулей для сканирующей зондовой микроволновой микроскопии

Представлен анализ структуры КВЧ микроскопа, конструкции зондового элемента и использования КВЧ автодинных модулей как варианта реализации микроволной сканирующей микроскопии. Показано существенное упрощение реализации КВЧ микроскопа при использовании автодинных датчиков

Мониторинг лесных угодий на основе материалов дистанционного зондирования Обь-Томского междуречья в границах Тимирязевского лесничества

В результате выполнения выпускной квалификационной работы был проведен информационно-аналитический анализ нормативно-правовой документации, изучен лесохозяйственный регламент Тимирязевского лесничества Обь-Томского междуречья Томской области, выбраны источники космической информации с достаточной разрешающей способностью для целей лесного мониторинга, а именно снимки, полученные со спутников Landsat 7, Sentinel 2A. Также рассмотрена методика оценки состояния лесных массивов с использованием ГИС-технологий, определены методы автоматизированного дешифрирования для обработки информации и получения результатов, выполнена классификация растительного покрова по снимкам Landsat 7.As a result of the final qualification work, an information and analytical analysis of legal documentation was conducted, the forestry regulations of the Timiryazev forest district of the Ob-Tomsk interfluve of the Tomsk region were studied, and sources of space information with sufficient resolution for forest monitoring were selected, namely images obtained from Landsat 7 and Sentinel 2A satellites. The method of assessing the state of woodlands using GIS technologies is also considered, methods of automated decryption for processing information and obtaining results are defined, and vegetation cover is classified according to Landsat 7 images

KnowVolution of the Polymer-Binding Peptide LCI for Improved Polypropylene Binding

The functionalization of polymer surfaces by polymer-binding peptides offers tremendous opportunities for directed immobilization of enzymes, bioactive peptides, and antigens. The application of polymer-binding peptides as adhesion promoters requires reliable and stable binding under process conditions. Molecular modes of interactions between material surfaces, peptides, and solvent are often not understood to an extent that enables (semi-) rational design of polymer-binding peptides, hindering the full exploitation of their potential. Knowledge-gaining directed evolution (KnowVolution) is an efficient protein engineering strategy that facilitates tailoring protein properties to application demands through a combination of directed evolution and computational guided protein design. A single round of KnowVolution was performed to gain molecular insights into liquid chromatography peak I peptide, 47 aa (LCI)-binding to polypropylene (PP) in the presence of the competing surfactant Triton X-100. KnowVolution yielded a total of 8 key positions (D19, S27, Y29, D31, G35, I40, E42, and D45), which govern PP-binding in the presence of Triton X-100. The recombination of two of the identified amino acid substitutions (Y29R and G35R; variant KR-2) yielded a 5.4 ± 0.5-fold stronger PP-binding peptide compared to LCI WT in the presence of Triton X-100 (1 mM). The LCI variant KR-2 shows a maximum binding capacity of 8.8 ± 0.1 pmol/cm2 on PP in the presence of Triton X-100 (up to 1 mM). The KnowVolution approach enables the development of polymer-binding peptides, which efficiently coat and functionalize PP surfaces and withstand surfactant concentrations that are commonly used, such as in household detergents

From natural to artificial metalloproteins – Challenges and opportunities

Organometallic catalysts are used in a wide range of industrially important reactions. By immobilizing such catalysts to a protein (Fig. 1), we can bring them into aqueous media, fine-tune activities and selectivities and overcome the challenges associated with trace metal removal in the product fraction [1, 2]. Trace metal removal is particularly important for the synthesis of pharmaceutical compounds. Typically, the transition metal content should be below 10 ppm. We have employed two types of metal catalysts, a Ru-based Grubbs-Hoveyda-type catalyst for olefin metathesis and a Rh-catalyst for phenylacetylene polymerization. These catalysts were covalently attached to either nitrobindin (NB) [3] or ferric hydroxamate uptake protein component: A (FhuA) [4] β-barrel proteins, yielding biohybrid catalysts (also denoted artificial metalloproteins) that can be immersed in aqueous reaction media either in their free form or immobilized to bacterial cells. Moreover, we could show that the metal catalysts can be immobilized on surfaces consisting of silica or polypropylene via peptide-based adhesion promoters, thereby enabling “green” surface immobilization strategies with the potential of catalyst recycling [5]. Either strategy yielded highly active catalysts that show great promise for single or sequential one-pot reactions. Separation of products and the catalysts was readily achieved by extraction. With the potential to tune reaction efficiencies and selectivities by modifying either the metal catalyst or the protein surrounding, biohybrid catalysts bear great potential to amend or even substitute the repertoire of reactions available by organic synthesis and, likewise, biocatalysis. Please click Additional Files below to see the full abstract

Molecular understanding of sterically controlled compound release through an engineered channel protein (FhuA)

<p>Abstract</p> <p>Background</p> <p>Recently we reported a nanocontainer based reduction triggered release system through an engineered transmembrane channel (FhuA Δ1-160; Onaca <it>et al</it>., 2008). Compound fluxes within the FhuA Δ1-160 channel protein are controlled sterically through labeled lysine residues (label: 3-(2-pyridyldithio)propionic-acid-N-hydroxysuccinimide-ester). Quantifying the sterical contribution of each labeled lysine would open up an opportunity for designing compound specific drug release systems.</p> <p>Results</p> <p>In total, 12 FhuA Δ1-160 variants were generated to gain insights on sterically controlled compound fluxes: Subset A) six FhuA Δ1-160 variants in which one of the six lysines in the interior of FhuA Δ1-160 was substituted to alanine and Subset B) six FhuA Δ1-160 variants in which only one lysine inside the barrel was not changed to alanine. Translocation efficiencies were quantified with the colorimetric TMB (3,3',5,5'-tetramethylbenzidine) detection system employing horseradish peroxidase (HRP). Investigation of the six subset A variants identified position K556A as sterically important. The K556A substitution increases TMB diffusion from 15 to 97 [nM]/s and reaches nearly the TMB diffusion value of the unlabeled FhuA Δ1-160 (102 [nM]/s). The prominent role of position K556 is confirmed by the corresponding subset B variant which contains only the K556 lysine in the interior of the barrel. Pyridyl labeling of K556 reduces TMB translocation to 16 [nM]/s reaching nearly background levels in liposomes (13 [nM]/s). A first B-factor analysis based on MD simulations confirmed that position K556 is the least fluctuating lysine among the six in the channel interior of FhuA Δ1-160 and therefore well suited for controlling compound fluxes through steric hindrance.</p> <p>Conclusions</p> <p>A FhuA Δ1-160 based reduction triggered release system has been shown to control the compound flux by the presence of only one inner channel sterical hindrance based on 3-(2-pyridyldithio)propionic-acid labeling (amino acid position K556). As a consequence, the release kinetic can be modulated by introducing an opportune number of hindrances. The FhuA Δ1-160 channel embedded in liposomes can be advanced to a universal and compound independent release system which allows a size selective compound release through rationally re-engineered channels.</p

In Silico and Experimental ADAM17 Kinetic Modeling as Basis for Future Screening System for Modulators

Understanding the mechanisms of modulators’ action on enzymes is crucial for optimizing and designing pharmaceutical substances. The acute inflammatory response, in particular, is regu lated mainly by a disintegrin and metalloproteinase (ADAM) 17. ADAM17 processes several disease mediators such as TNFα and APP, releasing their soluble ectodomains (shedding). A malfunction of this process leads to a disturbed inflammatory response. Chemical protease inhibitors such as TAPI-1 were used in the past to inhibit ADAM17 proteolytic activity. However, due to ADAM170 s broad expression and activity profile, the development of active-site-directed ADAM17 inhibitor was discontinued. New ‘exosite’ (secondary substrate binding site) inhibitors with substrate selectivity raised the hope of a substrate-selective modulation as a promising approach for inflammatory disease therapy. This work aimed to develop a high-throughput screen for potential ADAM17 modula tors as therapeutic drugs. By combining experimental and in silico methods (structural modeling and docking), we modeled the kinetics of ADAM17 inhibitor. The results explain ADAM17 inhibi tion mechanisms and give a methodology for studying selective inhibition towards the design of pharmaceutical substances with higher selectivity

CaLB Catalyzed Conversion of ε-Caprolactone in Aqueous Medium. Part 1: Immobilization of CaLB to Microgels

The enzymatic ring-opening polymerization of lactones is a method of increasing interest for the synthesis of biodegradable and biocompatible polymers. In the past it was shown that immobilization of Candida antarctica lipase B (CaLB) and the reaction medium play an important role in the polymerization ability especially of medium ring size lactones like ε-caprolactone (ε-CL). We investigated a route for the preparation of compartmentalized microgels based on poly(glycidol) in which CaLB was immobilized to increase its esterification ability. To find the ideal environment for CaLB, we investigated the acceptable water concentration and the accessibility for the monomer in model polymerizations in toluene and analyzed the obtained oligomers/polymers by NMR and SEC. We observed a sufficient accessibility for ε-CL to a toluene like hydrophobic phase imitating a hydrophobic microgel. Comparing free CaLB and Novozym® 435 we found that not the monomer concentration but rather the solubility of the enzyme, as well as the water concentration, strongly influences the equilibrium of esterification and hydrolysis. On the basis of these investigations, microgels of different polarity were prepared and successfully loaded with CaLB by physical entrapment. By comparison of immobilized and free CaLB, we demonstrated an effect of the hydrophobicity of the microenvironment of CaLB on its enzymatic activity