1,720,994 research outputs found
Biosynthesis of the marine antibiotic pentabromopseudilin. 2. The pyrrole ring
No full textThe biosynthesis of the potent marine antibiotic, pentabromopseudilin (1), was investigated. Feeding studies with Alteromonas luteoviolaceus were performed on a defined medium. D,L-[5-C-13]proline was incorporated symmetrically, demonstrating that the pyrrole ring of pentabromopseudilin is derived from proline
Understanding Enzyme Immobilisation
No full textEnzymes are versatile catalysts in the laboratory and on an industrial scale. To broaden their applicability in the laboratory and to ensure their (re)use in manufacturing the stability of enzymes can often require improvement. Immobilisation can address the issue of enzymatic instability. Immobilisation can also help to enable the employment of enzymes in different solvents, at extremes of pH and temperature and exceptionally high substrate concentrations. At the same time substrate-specificity, enantioselectivity and reactivity can be modified. However, most often the molecular and physical–chemical bases of these phenomena have not been elucidated yet. This tutorial review focuses on the understanding of enzyme immobilisation
On the Michael addition of water to C = C bonds
No full text?-Hydroxy carbonyl compounds are an important class of compounds often found as a common structural motif in natural products. Although the molecules themselves look rather simple, their synthesis can be challenging. Water addition to conjugated C = C bonds opens up a straightforward route for the preparation of ?-hydroxy carbonyl compounds. Moreover, water addition to C = C bonds benefits a lot from its simplicity and excellent atom economy. However, the enantioselective addition of water to ?,?-unsaturated carbonyl (Michael) acceptors still represents a chemically very challenging reaction, due to the poor nucleophilicity of water and its small size, which make regio- and stereoinduction difficult. Equally, the often unfavorable equilibrium of water-addition reactions remains to be solved. In contrast, enzymes such as fumarase, malease, citraconase, aconitase, and enoyl-CoA hydratase have been successfully used on industrial scale, and their excellent (enantio-) selectivities are highly valued. Unfortunately, most hydratases are part of the primary metabolism where perfect substrate specificity is required. This very high substrate selectivity severely limits their practical applicability in organic synthesis. Thus, a straightforward approach with broad applicability still had not been described. The aim of the research presented in this thesis was to take up this challenge and dedicated to the search for a Michael hydratase with a more relaxed substrate specificity for the preparation of important ?-hydroxy carbonyl compounds. The stereospecificity of enzyme-catalysed reactions has been a fruitful source of information about the mechanisms of enzyme catalysis and vice versa; the application of stereospecifically labelled substrates allows for studying the course of the reaction. It offers a very promising opportunity to comprehensively understand the precise mechanistic and kinetic details of even the most complex enzymatic reactions. Thus Chapter 1 provides unifying ideas for stereochemistry of the enzymatic water addition to C = C bonds. This enhances our understanding of the chemistry of water addition to C = C bonds, and further allows us to find more hydratases from natural sources or obtained via protein engineering. In Chapter 2, a direct, enantioselective Michael addition of water in water to prepare important ?-hydroxy carbonyl compounds using whole cells of Rhodococcus strains is described. Good yields and excellent enantioselectivities were achieved with this method. This opens up an entirely new approach for the preparation of important ?-hydroxy carbonyl compounds. Deuterium labelling studies demonstrate that a Michael hydratase catalyzes the water addition exclusively with anti-stereochemistry, which belongs to the family members of hydratases: oleate hydratase, fumarase, malease, aconitase and type II dehydroquinase with a preference for the anti-addition; whereas, type I dehydroquinase, enoyl-CoA hydratase and artificial hydratase exclusive prefer for the syn-addition, as discussed in Chapter 1. The biocatalytic reaction system was carefully optimized for gram-scale synthesis, resulting in good conversions and excellent enantioselectivities. Under the optimized conditions, whole cells could be reused for 4 cycles without significant loss of activity while maintaining up to 90% ee. Since whole cells from Rhodococcus strains were used to catalyse the Michael addition of water in water to a series of ?,?-unsaturated carbonyl compounds, and when the work presented in Chapter 2 started, no genomic information of Rhodococcus strains was publically available, we sequenced and annotated the strain R. rhodochrous ATCC 17895. This is described in Chapter 3 together with features of the R. rhodochrous ATCC 17895. It is a Gram-positive aerobic bacterium with a rod-like morphology. The 6,869,887 bp long genome contains 6,609 protein-coding genes and 53 RNA genes. Our study suggests the Michael hydratase has not been described before. In the work presented in Chapter 2, we found that most ?-hydroxy ketones are not commercially available or commercially expensive as we mentioned in the first paragraph, which made the stereoselectivity determination of Michael addition products difficult. Indeed, many seemingly simple molecules have to be prepared via multi-step syntheses, in particular so if they are optically active. Therefore a straightforward approach to enantiomerically enriched (R)- and (S)-3-hydroxycyclopentanone was established by kinetic resolution in Chapter 4. This methodology allows us to prepare more ?-hydroxy carbonyl compounds structurally closely related to 3-hydroxycyclopentanone. The isolated chiral alcohols were used to determine the stereochemistry of the Michael addition of water in Chapter 2, saving us a lot of laboratory work. Moreover, unexpected stereoselective reduction of conjugated C = C bonds was discovered during studies on the enantioselective Michael addition of water. As mentioned in Chapter 2, the whole cells of R. rhodochrous ATCC 17895 reduced ?,?-unsaturated cyclic ketones into the corresponding ketones as initially undesired side reaction for the addition of water to C = C bonds. Therefore, ene-reductase activity was also investigated in Chapter 5. A series of substrates, including activated ketones, aldehydes, amines and nitro-compounds were screened for ene-reductase activity using whole cells of R. rhodochrous ATCC 17895. This showed that R. rhodochrous is a very promising catalyst for the reduction of C = C bonds and harbours ene-reductases. Indeed, looking for the annotated ene reductase from the genome of R. rhodochrous ATCC 17895 as described in Chapter 3, three candidates were observed and were classified as ene-reductases by amino acid sequence alignment with the known Old Yellow Enzymes (OYEs). Thus, the putative ene-reductase genes from R. rhodochrous ATCC 17895 were heterologously overexpressed in Escherichia coli and one of the encoded proteins was purified and characterized for their biocatalytic and biochemical properties. Based on these accomplishments it can be concluded that we have discovered a new Michael hydratase and three new ene reductases from Rhodococcus strains. Genome sequence and annotation of strain R. rhodochrous ATCC 17895 has been done, offering an excellent opportunity for the discovering novel enzymes, for instance, the Michael hydratase and S-selective ene reductase. The important chiral ?-hydroxy carbonyl compounds can be prepared by kinetic resolution of racemic alcohols using lipases or the direct enantioselective Michael addition of water using whole cells of Rhodococcus strains. The isolated products from kinetic resolution were readily used for the stereochemistry determination of Michael addition of water in water, completes the story of water addition to C = C bonds.BiotechnologyApplied Science
Combination of Asymmetric Organo- and Biocatalysis in Flow Processes and Comparison with their Analogous Batch Syntheses
No full textSchober L, Tonin F, Hanefeld U, Gröger H. Combination of Asymmetric Organo- and Biocatalysis in Flow Processes and Comparison with their Analogous Batch Syntheses. European Journal of Organic Chemistry . 2022;(7): e202101035.A sequential-type as well as a tandem-type chemoenzymatic flow cascade combining an organocatalytic aldol reaction and a biocatalytic reduction to form stereoselectively a 1,3-diol with two stereogenic centers were developed. Initially, a comprehensive screening of 24 alcohol dehydrogenases was carried out and the identified candidates were applied in different multistep flow cascades. All four stereoisomers of the desired 1,3-diol product are accessible via a sequential flow approach with product formation-related conversions of up to 76% over two steps, isolated yields of up to 64% and enantiomeric excess of >99% in all cases. In addition, a tandem-type flow process, performing both reaction steps simultaneously, was established leading to 51 % conversion with >99% ee and 8:1 d.r. and representing a combination of the fields of asymmetric chemo-catalysis, biocatalysis and flow chemistry
Hydroxynitrile Lyase-Catalyzed Enantioselective Conversion of Ketones into Cyanohydrins
No full textIn this thesis I have addressed several issues related to the HNL-catalyzed preparation of cyanohydrins. I first demonstrated in Chapter 2 that immobilized HNL as sol-gels and as commercially available Cross Linked Enzyme Aggregates (CLEA®) improved several features of the biocatalyst such as solvent stability, and substrate or product inhibition/deactivation. In particular, MeCLEA was remarkably stable towards the deleterious effect of organic solvent and the enzymatic reaction could be carried out in organic media. The CLEA immobilization strategy is nonetheless enzyme-dependent and I successfully developed the biocatalyst LuCLEA for optimum catalytic performances in organic media as described in Chapter 3. This enantioselective and recyclable biocatalyst appeared to be particularly effective for the preparation of 2-butanone cyanohydrin. In Chapter 4, I used benzaldehyde as a model substrate to develop multistep strategies towards cyanohydrin derivatives based on HNL-CLEA catalysis in organic solvents. The reaction could be carried out in one pot or with limited downstream processing/purification of the cyanohydrin intermediate. In the case of ketones such as acetophenone where unfavourable thermodynamics limit the practical conversion, all attempts to derivatize the cyanohydrin in situ in order to shift the equilibrium were not successful. Cyanohydrins from ketones can indeed be considered as tertiary alcohols which require relatively reactive reagents for derivatization. Under these conditions the biocatalyst was rendered inactive. Since no in situ derivatization method could de designed to enable complete conversion of unreactive ketones, kinetic resolution as a means to produce chiral cyanohydrin was explored in Chapter 5. I established enzymatic activity for a previously unreported ?,?-unsaturated ketone and showed that kinetic resolution was more suitable than the direct synthetic route for the preparation of the corresponding chiral cyanohydrin. As an extension of this work I also described the rearrangement of a similar ?,?-unsatuared cyanohydrin acetate into the corresponding tetronic acid derivative. Chapter 6 concludes this thesis with straightforward synthetic procedures towards racemic cyanohydrins from unreactive ketones in order to improve the overall cost efficiency of the kinetic resolution approach.Biocatalysis and Orgnanic ChemistryApplied Science
2-Deoxy-d-ribose-5-phosphate aldolase (DERA): applications and modifications
No full text© 2018, The Author(s). 2-Deoxy-d-ribose-5-phosphate aldolase (DERA) is a class I aldolase that offers access to several building blocks for organic synthesis. It catalyzes the stereoselective C–C bond formation between acetaldehyde and numerous other aldehydes. However, the practical application of DERA as a biocatalyst is limited by its poor tolerance towards industrially relevant concentrations of aldehydes, in particular acetaldehyde. Therefore, the development of proper experimental conditions, including protein engineering and/or immobilization on appropriate supports, is required. The present review is aimed to provide a brief overview of DERA, its history, and progress made in understanding the functioning of the enzyme. Furthermore, the current understanding regarding aldehyde resistance of DERA and the various optimizations carried out to modify this property are discussed.ChemE/Product and Process EngineeringBT/BiocatalysisBT/Biotechnologi
Immobilisation of hydroxynitrile lyases
No full textHydroxynitrile lyases are a versatile group of enzymes that are applied both in the laboratory and on an industrial scale. What makes them particularly interesting is that to date five structurally unrelated categories of hydroxynitrile lyases have been discovered. Given their great importance they have often been immobilised utilising many different methodologies. Therefore the hydroxynitrile lyases are ideally suited to compare different immobilisation methods and their dependence on the structural features of the enzyme in question, since the activity is the same in all cases. This review examines all the different immobilisation methods applied to hydroxynitrile lyases and draws conclusions on the effect of the approach.BiotechnologyApplied Science
The selective addition of water
No full textWater is omnipresent and essential. Yet at the same time it is a rather unreactive molecule. The direct addition of water to C[double bond, length as m-dash]C double bonds is therefore a challenge not answered convincingly. In this perspective we critically evaluate the selectivity and the applicability of the different catalytic approaches for water addition reactions: homogeneous, heterogeneous and bio-catalytic. Here we would like to discuss how to speed up water addition and even make it selective.BiotechnologyApplied Science
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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