Enzymen op maat

Applied Science

Latest development in the synthesis of ursodeoxycholic acid (UDCA): A critical review

Ursodeoxycholic acid (UDCA) is a pharmaceutical ingredient widely used in clinics. As bile acid it solubilizes cholesterol gallstones and improves the liver function in case of cholestatic diseases. UDCA can be obtained from cholic acid (CA), which is the most abundant and least expensive bile acid available. The now available chemical routes for the obtainment of UDCA yield about 30% of final product. For these syntheses several protection and deprotection steps requiring toxic and dangerous reagents have to be performed, leading to the production of a series of waste products. In many cases the cholic acid itself first needs to be prepared from its taurinated and glycilated derivatives in the bile, thus adding to the complexity and multitude of steps involved of the synthetic process. For these reasons, several studies have been performed towards the development of microbial transformations or chemoenzymatic procedures for the synthesis of UDCA starting from CA or chenodeoxycholic acid (CDCA). This promising approach led several research groups to focus their attention on the development of biotransformations with non-pathogenic, easy-to-manage microorganisms, and their enzymes. In particular, the enzymatic reactions involved are selective hydrolysis, epimerization of the hydroxy functions (by oxidation and subsequent reduction) and the specific hydroxylation and dehydroxylation of suitable positions in the steroid rings. In this minireview, we critically analyze the state of the art of the production of UDCA by several chemical, chemoenzymatic and enzymatic routes reported, highlighting the bottlenecks of each production step. Particular attention is placed on the precursors availability as well as the substrate loading in the process. Potential new routes and recent developments are discussed, in particular on the employment of flow-reactors. The latter technology allows to develop processes with shorter reaction times and lower costs for the chemical and enzymatic reactions involved.BT/BiocatalysisBT/Biotechnolog

Discovery of Putative Halogenases in Environmental Samples Using Metagenomics

BACKGROUND: Halogenases are involved in the halogenation of secondary metabolites with pharmaceutical relevance, such as the antibiotics chloramphenicol and vancomycin. Hereby flavin- or FADH2-dependent halogenases represents the major class of halogenases. Previous investigations on flavin-dependent halogenases have focused on Actinomycetes and sponge-associated microorganisms. In order to extend our knowledge on the diversity and distribution of halogenases we have applied a PCR-based screening approach to show the wide distribution of flavin-dependent halogenases in metagenomes of microbial communities from different environments, such as soils and sediments. METHODS: For the screening of new flavin-dependent halogenases a metagenomic approach was used. Metagenomic libraries were constructed and screened by homology-driven PCR based on degenerate primers designed from known flavin-dependent halogenases. RESULTS: We screened 55 environmental samples that we collected from different habitats by a PCRbased screening for novel flavin-dependent halogenases. The phylogenetic analysis of the putative halogenases together with the 16S Amplicon sequencing results of the environmental samples demonstrates the wide distribution of the halogenases and the large sequence diversity. In addition, metagenomic libraries were constructed to screen for flavin-dependent halogenases. CONCLUSION: The metagenomic approach described in this paper is a promising method to explore novel putative halogenases from different environments

Production of fatty acid estolides

It has been found that esterification of a hydroxy-fatty acid by a lipase can be coupled with oleate hydratase (OHase) generation of that hydroxy-FA from an unsaturated FA with a cis C9-C10 double bond, e.g. oleic acid, in a single aqueous buffered reaction medium at low temperature,e.g. 30° C. A simple one-pot enzymatic method to produce fatty acid estolides from one or more triglycerides, e.g. starting from a natural plant oil, is thereby enabled in which the same lipase catalyses both the initial hydrolysis of triglyceride and the final esterification step

Heterogeneous Catalysts For Liquid-phase Oxidations: Philosophers' Stones Or Trojan Horses?

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Identification of catalytically important residues of the carotenoid 1,2-hydratases from Rubrivivax gelatinosus and Thiocapsa roseopersicina

Carotenoid 1,2-hydratases (CrtC) catalyze the selective addition of water to an isolated carbon–carbon double bond. Although their involvement in the carotenoid biosynthetic pathway is well understood, little is known about the mechanism by which these hydratases transform carotenoids such as lycopene into the corresponding hydroxyl compounds. Key residues were identified at positions His239, Trp241, Tyr266, and Asp268 in CrtC from Rubrivivax gelatinosus (and corresponding positions in Thiocapsa roseopersicina). Alanine mutants at these positions were found to be completely inactive, suggesting their direct involvement in the catalytic reaction. Our resulting mechanistic hypothesis is in analogy with the recently studied class of terpenoid cyclase enzymes containing a highly acidic aspartic residue in their active site. We propose that a similar aspartic acid residue, which is conserved through all putative CrtCs, is involved in initial protonation of the double bond in lycopene.BT/BiotechnologyApplied Science

Peroxygenase-Catalysed Epoxidation of Styrene Derivatives in Neat Reaction Media

Biocatalytic oxyfunctionalisation reactions are traditionally conducted in aqueous media limiting their production yield. Here we report the application of a peroxygenase in neat reaction conditions reaching product concentrations of up to 360 mM.Financial support by the European Research Council (ERC Consolidator Grant No. 648026) is gratefully acknowledged