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- ItemAre Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents?(Basel : MDPI, 2017) Markel, Ulrich; Zhu, Leilei; Frauenkron-Machedjou, Victorine; Zhao, Jing; Bocola, Marco; Davari, Mehdi; Jaeger, Karl-Erich; Schwaneberg, UlrichDespite the significant advances in the field of protein engineering, general design principles to improve organic cosolvent resistance of enzymes still remain undiscovered. Previous studies drew conclusions to engineer enzymes for their use in water-miscible organic solvents based on few amino acid substitutions. In this study, we conduct a comparison of a Bacillus subtilis lipase A (BSLA) library—covering the full natural diversity of single amino acid substitutions at all 181 positions of BSLA—with three state of the art random mutagenesis methods: error-prone PCR (epPCR) with low and high mutagenesis frequency (epPCR-low and high) as well as a transversion-enriched Sequence Saturation Mutagenesis (SeSaM-Tv P/P) method. Libraries were searched for amino acid substitutions that increase the enzyme’s resistance to the water-miscible organic cosolvents 1,4-dioxane (DOX), 2,2,2-trifluoroethanol (TFE), and dimethyl sulfoxide (DMSO). Our analysis revealed that 5%–11% of all possible single substitutions (BSLA site-saturation mutagenesis (SSM) library) contribute to improved cosolvent resistance. However, only a fraction of these substitutions (7%–12%) could be detected in the three random mutagenesis libraries. To our knowledge, this is the first study that quantifies the capability of these diversity generation methods generally employed in directed evolution campaigns and compares them to the entire natural diversity with a single substitution. Additionally, the investigation of the BSLA SSM library revealed only few common beneficial substitutions for all three cosolvents as well as the importance of introducing surface charges for organic cosolvent resistance—most likely due to a stronger attraction of water molecules. © 2017 by the authors.
- ItemCaLB Catalyzed Conversion of ε-Caprolactone in Aqueous Medium. Part 1: Immobilization of CaLB to Microgels(Basel : MDPI, 2016) Engel, Stefan; Höck, Heidi; Bocola, Marco; Keul, Helmut; Schwaneberg, Ulrich; Möller, MartinThe 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.
- ItemKnowVolution of the Polymer-Binding Peptide LCI for Improved Polypropylene Binding(Basel : MDPI, 2018) Rübsam, Kristin; Davari, Mehdi D.; Jakob, Felix; Schwaneberg, UlrichThe 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.
- ItemSortase-Mediated Ligation of Purely Artificial Building Blocks(Basel : MDPI, 2018) Dai, Xiaolin; Mate, Diana M.; Glebe, Ulrich; Mirzaei Garakani, Tayebeh; Körner, Andrea; Schwaneberg, Ulrich; Böker, AlexanderSortase A (SrtA) from Staphylococcus aureus has been often used for ligating a protein with other natural or synthetic compounds in recent years. Here we show that SrtA-mediated ligation (SML) is universally applicable for the linkage of two purely artificial building blocks. Silica nanoparticles (NPs), poly(ethylene glycol) and poly(N-isopropyl acrylamide) are chosen as synthetic building blocks. As a proof of concept, NP–polymer, NP–NP, and polymer–polymer structures are formed by SrtA catalysis. Therefore, the building blocks are equipped with the recognition sequence needed for SrtA reaction—the conserved peptide LPETG—and a pentaglycine motif. The successful formation of the reaction products is shown by means of transmission electron microscopy (TEM), matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS), and dynamic light scattering (DLS). The sortase catalyzed linkage of artificial building blocks sets the stage for the development of a new approach to link synthetic structures in cases where their synthesis by established chemical methods is complicated.
- ItemComparison of Candida antarctica Lipase B Variants for Conversion of ε-Caprolactone in Aqueous Medium-Part 2(Basel : MDPI, 2018) Höck, Heidi; Engel, Stefan; Weingarten, Simone; Keul, Helmut; Schwaneberg, Ulrich; Möller, Martin; Bocola, MarcoEnzyme-catalyzed ring-opening polymerization of lactones is a method of increasing interest for the synthesis of polyesters. In the present work, we investigated which changes in the structure of Candida antarctica lipase B (CaLB) shift the catalytic equilibrium between esterification and hydrolysis towards polymerization. Therefore, we present two concepts: (i) removing the glycosylation of CaLB to increase the surface hydrophobicity; and (ii) introducing a hydrophobic lid adapted from Pseudomonas cepacia lipase (PsCL) to enhance the interaction of a growing polymer chain to the elongated lid helix. The deglycosylated CaLB (CaLB-degl) was successfully generated by site-saturation mutagenesis of asparagine 74. Furthermore, computational modeling showed that the introduction of a lid helix at position Ala148 was structurally feasible and the geometry of the active site remained intact. Via overlap extension PCR the lid was successfully inserted, and the variant was produced in large scale in Pichia pastoris with glycosylation (CaLB-lid) and without (CaLB-degl-lid). While the lid variants show a minor positive effect on the polymerization activity, CaLB-degl showed a clearly reduced hydrolytic and enhanced polymerization activity. Immobilization in a hydrophobic polyglycidol-based microgel intensified this effect such that a higher polymerization activity was achieved, compared to the “gold standard” Novozym® 435.