Enrichment of Erucic and Gondoic Fatty Acids from Crambe and Camelina Oils Catalyzed by Geotrichum candidum Lipases I and II

Erucic (22:1, cisΔ13) and gondoic acids (20:1, cisΔ11) are building blocks obtained from renewable sources for the oleochemical industry. Different biocatalytic strategies for the enrichment of these compounds with high recovery yields were developed in our group. Geotrichum candidum lipases (GCL) strongly discriminate against fatty acids longer than 18 carbon atoms. Thus, GCL-I and -II were investigated using hydrolysis or ethanolysis reactions with Crambe and Camelina oils. Hydrolysis was also studied using fatty acid ethyl esters (FAEE) derived from the corresponding oil. Both isoforms were highly selective; however, interesting differences were observed. Although it has been reported that GCL-I displays a higher preference toward 18 cisΔ9, which is present in the studied oils at high levels, GCL-II showed higher enrichment values during hydrolysis independent of the substrate used. Hence, enrichments of 87% (Crambe oil) and 82% (Crambe FAEE) for erucic acid and 50% (Camelina oil) and 45% (Camelina FAEE) for gondoic acid, with recovery values between 89% and 99%, were achieved. On the contrary, the best enzyme for ethanolysis was GCL-I (82% and 41% for erucic and gondoic acid, respectively). In this case, although GCL-II also displayed good enrichment and recovery levels (77% and 28%, respectively), they were lower compared to the former reactions. In both ethanolysis reactions, the FAEE fraction contained between 92% and 97% of 18 unsaturated fatty acids.     

Characterization of enzymatically synthesized amylopectin analogs via asymmetrical flow field flow fractionation

Asymmetrical flow field flow fractionation (AF4), when coupled with multi-angle laser light scattering (MALLS), is a very powerful technique for determination of the macromolecular structure of high molar mass (branched) polysaccharides. AF4 is a size fractionation technique just as size exclusion chromatography (SEC), nevertheless can overcome some crucial problems found in SEC analysis especially in starch like structures. This paper describes a detailed investigation of the macromolecular structure of two groups of well-defined synthetic amylopectin analogs – synthesized via an in vitro enzyme-catalyzed reaction using the enzymes phosphorylase b from rabbit muscle and Deinococcus geothermalis glycogen branching enzyme (Dg GBE). Size, molar mass distributions and structural data were studied by AF4 coupled with online quasi-elastic light scattering (QELS) and multi-angle light scattering (MALLS).     

Multivalent Protein Assembly Using Monovalent Self-Assembling Building Blocks

Discotic molecules, which self-assemble in water into columnar supramolecular polymers, emerged as an alternative platform for the organization of proteins. Here, a monovalent discotic decorated with one single biotin was synthesized to study the self-assembling multivalency of this system in regard to streptavidin. Next to tetravalent streptavidin, monovalent streptavidin was used to study the protein assembly along the supramolecular polymer in detail without the interference of cross-linking. Upon self-assembly of the monovalent biotinylated discotics, multivalent proteins can be assembled along the supramolecular polymer. The concentration of discotics, which influences the length of the final polymers at the same time dictates the amount of assembled proteins.     

Application of the Thioacid-Azide Ligation (TAL) for the Preparation of Glycosylated and Fluorescently Labeled Amino Acids

The reaction of thiocarboxylic acids with azides (thioacid-azide ligation, TAL) is a chemoselective transformation, leading to the formation of amide bonds. Especially in the case of electron-deficient sulfonyl azides, the reaction occurs in excellent yields, within minutes at room temperature, in a broad range of solvents. In this article, the application of TAL for the synthesis of glycosylated and dansylated amino acids is reported. 2-Azidosulfonylethyl glycosides were prepared from peracetylated monosaccharides and reacted with thioacid-containing derivatives of aspartic acid and glutamic acid. Likewise, aromatic sulfonyl azides were employed. Ligation products were obtained in yields of up to 96 %. Different conditions for the generation of the thioacids were compared. Whereas reaction of succinimidyl esters with sodium hydrogen sulfide led to concomitant formation of diacyl disulfides, trityl thioesters turned out to be suitable precursors for the in situ preparation of thioacids by mild acidolysis.     

PCDD/F and other micropollutants in MSWI crude gas and ashes during plant start-up and shut-down processes

Nonstationary combustion conditions at municipal solid waste incineration (MSWI) plants cause increased crude gas concentrations of polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), and other products of incomplete combustion (PIC). Such transient conditions occur, e.g., during and after start-up processes in MSWI plants. The start-up and shut-down processes of a MSWI plant were investigated in detail. PCDD/F and other PIC concentrations were determined in the crude gas, in the boiler ash, and in the ash from the electrostatic precipitator (ESP ash), with the outcome that only the start-up procedure significantly affected the concentrations of the organic pollutants in the flue gas and in the ESP ash. The shut-down procedure was evaluated as less problematic for the concentration of the organic pollutants. Moreover the concentration of the PCDD/F and other PIC in the boiler ash was determined as not influenced by shut-down and start-up processes. The homologue profiles and the congener patterns as well as the PCDF/PCDD ratio in the flue gas and in the ESP ash change during MSWI start-up. The changing patterns point at a transition from dominant de novo synthesis to precursor synthesis.     

Statistical analysis of intramembranous particles using freeze fracture specimens

We studied the point processes of intramembranous particles of mitochondrial membranes from HeLa cells using the freeze fracture technique. Three groups – under normal conditions, after exposition with rotenone, and after exposition with sodium acid – were compared. First, we used several summary statistics in order to study the two‐dimensional point patterns of intramembranous particles within each group. Then, we compared the patterns in different groups by bootstrap tests using the K‐function and the nearest neighbour distance function G(r). Estimation of the G‐function provided significant results but no significant differences between groups were found using the classical K‐function; estimation of G(r) should therefore not be omitted when studying observed planar point patterns.     

Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases

Stereoselective carbon-carbon bond forming reactions are quintessential transformations in organic synthesis. One example is the Diels-Alder reaction, a [4+2] cycloaddition between a conjugated diene and a dienophile to form cyclohexenes. The development of biocatalysts for this reaction is paramount for unlocking sustainable routes to a plethora of important molecules. To obtain a comprehensive understanding of naturally evolved [4+2] cyclases, and to identify hitherto uncharacterised biocatalysts for this reaction, we constructed a library comprising forty-five enzymes with reported or predicted [4+2] cycloaddition activity. Thirty-one library members were successfully produced in recombinant form. In vitro assays employing a synthetic substrate incorporating a diene and a dienophile revealed broad-ranging cycloaddition activity amongst these polypeptides. The hypothetical protein Cyc15 was found to catalyse an intramolecular cycloaddition to generate a novel spirotetronate. The crystal structure of this enzyme, along with docking studies, establishes the basis for stereoselectivity in Cyc15, as compared to other spirotetronate cyclases.     

Maximizing batch fermentation efficiency by constrained model-based optimization and predictive control of adenosine triphosphate turnover

We present a constrained model-based optimization and predictive control framework to maximize the production efficiency of batch fermentations based on the core idea of manipulating adenosine triphosphate (ATP) wasting. In many bioprocesses, enforced ATP wasting —rerouting ATP use towards an energetically possibly suboptimal path— allows increasing the metabolic flux towards the product, thereby enhancing product yields and specific productivities. However, this often comes at the expense of lower biomass yields and reduced volumetric productivities. To maximize the overall efficiency, we formulate ATP wasting as a model-based optimal control problem. This allows for balancing trade-offs between different objectives such as product yield and volumetric productivity for batch fermentations. Unlike static metabolic control, one obtains a higher degree of flexibility, adaptability, and competitiveness. This can be advantageous towards achieving a sustainable and economically efficient biotechnology industry. To compensate for model-plant mismatch, disturbances, and uncertainties, we propose not only solving the optimal control problem once. Instead, we exploit the concept of moving horizon model predictive control combined with constraint-based dynamic modeling to capture the fermentation dynamics. The approach is underlined considering the industrially relevant bioproduction of lactate by Escherichia coli. We discuss practical challenges for the described control strategy and provide an outlook towards future developments.     

Additive Benefits of Radium-223 Dichloride and Bortezomib Combination in a Systemic Multiple Myeloma Mouse Model

Osteolytic bone disease is a hallmark of multiple myeloma (MM) mediated by MM cell proliferation, increased osteoclast activity, and suppressed osteoblast function. The proteasome inhibitor bortezomib targets MM cells and improves bone health in MM patients. Radium-223 dichloride (radium-223), the first targeted alpha therapy approved, specifically targets bone metastases, where it disrupts the activity of both tumor cells and tumor-supporting bone cells in mouse models of breast and prostate cancer bone metastasis. We hypothesized that radium-223 and bortezomib combination treatment would have additive effects on MM. In vitro experiments revealed that the combination treatment inhibited MM cell proliferation and demonstrated additive efficacy. In the systemic, syngeneic 5TGM1 mouse MM model, both bortezomib and radium-223 decreased the osteolytic lesion area, and their combination was more effective than either monotherapy alone. Bortezomib decreased the number of osteoclasts at the tumor–bone interface, and the combination therapy resulted in almost complete eradication of osteoclasts. Furthermore, the combination therapy improved the incorporation of radium-223 into MM-bearing bone. Importantly, the combination therapy decreased tumor burden and restored body weights in MM mice. These results suggest that the combination of radium-223 with bortezomib could constitute a novel, effective therapy for MM and, in particular, myeloma bone disease.