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1.
Chem Sci ; 14(36): 9863-9871, 2023 Sep 20.
Article in English | MEDLINE | ID: mdl-37736642

ABSTRACT

Efficient access to the morphinan scaffold remains a major challenge in both synthetic chemistry and biotechnology. Here, a biomimetic chemo-enzymatic strategy to synthesize the natural promorphinan intermediate (+)-salutaridine is demonstrated. By combining early-stage organic synthesis with enzymatic asymmetric key step transformations, the prochiral natural intermediate 1,2-dehydroreticuline was prepared and subsequently stereoselectively reduced by the enzyme 1,2-dehydroreticuline reductase obtaining (R)-reticuline in high ee and yield (>99% ee, up to quant. conversion, 92% isol. yield). In the final step, membrane-bound salutaridine synthase was used to perform the selective ortho-para phenol coupling to give (+)-salutaridine. The synthetic route shows the potential of combining early-stage advanced organic chemistry to minimize protecting group techniques with late-stage multi-step biocatalysis to provide an unprecedented access to the medicinally important compound class of promorphinans.

2.
Angew Chem Int Ed Engl ; 62(46): e202312721, 2023 11 13.
Article in English | MEDLINE | ID: mdl-37743348

ABSTRACT

Identifying (bio)catalysts displaying high enantio-/stereoselectivity is a fundamental prerequisite for the advancement of asymmetric catalysis. Herein, a high-throughput, stereoselective screening assay is reported that gives information on enantioselectivity, stereopreference and activity as showcased for peroxygenase-catalyzed hydroxylation. The assay is based on spectrophotometric analysis of the simultaneous formation of NAD(P)H from the alcohol dehydrogenase catalyzed enantioselective oxidation of the sec-alcohol product formed in the peroxygenase reaction. The assay was applied to investigate a library comprising 44 unspecific peroxygenases (UPOs) containing 25 UPOs not reported yet. Thereby, previously non-described wild-type UPOs displaying (S)- as well as (R)-stereoselectivity for the hydroxylation of representative model substrates were identified, reaching up to 98 % ee for the (R)- and 94 % ee for the (S)-enantiomer. Homology models with concomitant docking studies indicated the structural reason for the observed complementary stereopreference.


Subject(s)
Mixed Function Oxygenases , Stereoisomerism , Mixed Function Oxygenases/metabolism , Oxidation-Reduction , Catalysis
3.
Chembiochem ; 24(11): e202300170, 2023 06 01.
Article in English | MEDLINE | ID: mdl-37057969

ABSTRACT

Mass spectrometry-based high-throughput screening methods combine the advantages of photometric or fluorometric assays and analytical chromatography, as they are reasonably fast (throughput ≥1 sample/min) and broadly applicable, with no need for labelled substrates or products. However, the established MS-based screening approaches require specialised and expensive hardware, which limits their broad use throughout the research community. We show that a more common instrumental platform, a single-quadrupole HPLC-MS, can be used to rapidly analyse diverse biotransformations by flow-injection mass spectrometry (FIA-MS), that is, by automated infusion of samples to the ESI-MS detector without prior chromatographic separation. Common organic buffers can be employed as internal standard for quantification, and the method provides readily validated activity and selectivity information with an analytical run time of one minute per sample. We report four application examples that cover a broad range of analyte structures and concentrations (0.1-50 mM before dilution) and diverse biocatalyst preparations (crude cell lysates and whole microbial cells). Our results establish FIA-MS as a versatile and reliable alternative to more traditional methods for screening enzymatic reactions.


Subject(s)
High-Throughput Screening Assays , Mass Spectrometry/methods , Chromatography, High Pressure Liquid/methods , High-Throughput Screening Assays/methods
4.
Biotechnol Adv ; 60: 108016, 2022 11.
Article in English | MEDLINE | ID: mdl-35781046

ABSTRACT

Fusion proteins, understood as those created by joining two or more genes that originally encoded independent proteins, have numerous applications in biotechnology, from analytical methods to metabolic engineering. The use of fusion enzymes in biocatalysis may be even more interesting due to the physical connection of enzymes catalyzing successive reactions into covalently linked complexes. The proximity of the active sites of two enzymes in multi-enzyme complexes can make a significant contribution to the catalytic efficiency of the reaction. However, the physical proximity of the active sites does not guarantee this result. Other aspects, such as the nature and length of the linker used for the fusion or the order in which the enzymes are fused, must be considered and optimized to achieve the expected increase in catalytic efficiency. In this review, we will relate the new advances in the design, creation, and use of fused enzymes with those achieved in biocatalysis over the past 20 years. Thus, we will discuss some examples of genetically fused enzymes and their application in carbon­carbon bond formation and oxidative reactions, generation of chiral amines, synthesis of carbohydrates, biodegradation of plant biomass and plastics, and in the preparation of other high-value products.


Subject(s)
Multifunctional Enzymes , Protein Engineering , Amines/chemistry , Amines/metabolism , Biocatalysis , Carbohydrates , Carbon , Enzymes/chemistry , Multifunctional Enzymes/metabolism , Plastics/metabolism
5.
Adv Synth Catal ; 360(21): 4115-4131, 2018 11 05.
Article in English | MEDLINE | ID: mdl-30555288

ABSTRACT

Biotechnological strategies using renewable materials as starting substrates are a promising alternative to traditional oleochemical processes for the isolation of different fatty acids. Among them, long chain mono-unsaturated fatty acids are especially interesting in industrial lipid modification, since they are precursors of several economically relevant products, including detergents, plastics and lubricants. Therefore, the aim of this study was to develop an enzymatic method in order to increase the percentage of long chain mono-unsaturated fatty acids from Camelina and Crambe oil ethyl ester derivatives, by using selective lipases. Specifically, the focus was on the enrichment of gondoic (C20:1 cisΔ11) and erucic acid (C22:1 cisΔ13) from Camelina and Crambe oil derivatives, respectively. The pursuit of this goal entailed several steps, including: (i) the choice of a suitable lipase scaffold to serve as a protein engineering template (Candida antarctica lipase A); (ii) the identification of potential amino acid targets to disrupt the binding tunnel at the adequate location; (iii) the design, creation and high-throughput screening of lipase mutant libraries; (iv) the study of the selectivity towards different chain length p-nitrophenyl fatty acid esters of the best hits found, as well as the analysis of the contribution of each amino acid change and the outcome of combining several of the aforementioned residue alterations and, finally, (v) the selection and application of the most promising candidates for the fatty acid enrichment biocatalysis. As a result, enrichment of C22:1 from Crambe ethyl esters was achieved either, in the free fatty acid fraction (wt, 78%) or in the esterified fraction (variants V1, 77%; V9, 78% and V19, 74%). Concerning the enrichment of C20:1 when Camelina oil ethyl esters were used as substrate, the best variant was the single mutant V290W, which doubled its content in the esterified fraction from approximately 15% to 34%. A moderately lower increase was achieved by V9 and its two derived triple mutant variants V19 and V20 (27%).

6.
Prog Lipid Res ; 63: 153-64, 2016 07.
Article in English | MEDLINE | ID: mdl-27301784

ABSTRACT

This review first provides a brief introduction into the most important tools and strategies for protein engineering (i.e. directed evolution and rational protein design combined with high-throughput screening methods) followed by examples from literature, in which enzymes have been optimized for biocatalytic applications. This covers engineered lipases with altered fatty acid chain length selectivity, fatty acid specificity and improved performance in esterification reactions. Furthermore, recent achievements reported for phospholipases, lipoxygenases, P450 monooxygenases, decarboxylating enzymes, fatty acid hydratases and the use of enzymes in cascade reactions are treated.


Subject(s)
Enzymes/metabolism , Protein Engineering , Biocatalysis , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Enzymes/genetics , Fatty Acids/metabolism , Humans , Lipase/genetics , Lipase/metabolism , Lipoxygenase/genetics , Lipoxygenase/metabolism , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Phospholipases/genetics , Phospholipases/metabolism
7.
Appl Microbiol Biotechnol ; 99(7): 3057-68, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25324130

ABSTRACT

The TM1072 gene from Thermotoga maritima codifies for a putative form of a rhamnulose-1-phosphate aldolase (Rha-1PA Tm). To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified enzyme was activated by Co(2+) as a divalent metal ion cofactor, instead of Zn(2+) as its E. coli homologue, and exhibited a maximum of activity at 95 °C. Furthermore, the enzyme displayed a high stability against extreme reaction conditions, retaining 90 % of its activity in the presence of 40 % of acetonitrile and showing a half-life greater than 3 h at 115 °C. The kinetic parameters at room temperature (R/T) were also studied; the K M was calculated to be 3.6 ± 0.33 mM, while k cat/K M was found to be 0.7 × 10(3) s(-1) M(-1). Given these characteristics, Rha-1PA Tm is an attractive enzyme for use as a biocatalyst for industrial applications, offering intriguing possibilities for practical biocatalysis.


Subject(s)
Aldehyde-Lyases/genetics , Aldehyde-Lyases/metabolism , Thermotoga maritima/enzymology , Aldehyde-Lyases/chemistry , Amino Acid Sequence , Catalysis , Cloning, Molecular , Enzyme Stability , Escherichia coli/genetics , Half-Life , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Temperature , Thermotoga maritima/genetics
8.
Curr Opin Chem Biol ; 17(2): 236-49, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23490810

ABSTRACT

To transfer to the laboratory, the excellent efficiency shown by enzymes in Nature, biocatalysis, had to mimic several synthetic strategies used by the living organisms. Biosynthetic pathways are examples of tandem catalysis and may be assimilated in the biocatalysis field for the use of isolated multi-enzyme systems in the homogeneous phase. The concurrent action of several enzymes that work sequentially presents extraordinary advantages from the synthetic point of view, since it permits a reversible process to become irreversible, to shift the equilibrium reaction in such a way that enantiopure compounds can be obtained from prochiral or racemic substrates, reduce or eliminate problems due to product inhibition or prevent the shortage of substrates by dilution or degradation in the bulk media, etc. In this review we want to illustrate the developments of recent studies involving in vitro multi-enzyme reactions for the synthesis of different classes of organic compounds.


Subject(s)
Enzymes/chemistry , Enzymes/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Biocatalysis , Biotechnology , Metabolic Networks and Pathways , Plant Proteins/chemistry , Plant Proteins/metabolism , Stereoisomerism
9.
Chemistry ; 16(13): 4018-30, 2010 Apr 06.
Article in English | MEDLINE | ID: mdl-20198665

ABSTRACT

A bifunctional aldolase/kinase enzyme named DLF has been constructed by gene fusion through overlap extension. This fusion enzyme consists of monomeric fructose-1,6-bisphosphate aldolase (FBPA) from Staphylococcus carnosus and the homodimeric dihydroxyacetone kinase (DHAK) from Citrobacter freundii CECT 4626 with an intervening linker of five amino acid residues. The fusion protein was expressed soluble and retained both kinase and aldolase activities. The secondary structures of the bifunctional enzyme and the parental enzymes were analyzed by circular dichroism (CD) spectroscopy to study the effect of the covalent coupling of the two parent proteins on the structure of the fused enzyme. Because S. carnosus FBPA is a thermostable protein, the effect of the fusion on the thermal stability of the bifunctional enzyme has also been studied. The proximity of the active centers in the fused enzyme promotes a kinetic advantage as the 20-fold increment in the initial velocity of the overall aldol reaction indicates. Experimental evidence supports that this increase in the reaction rate can be explained in terms of substrate channeling.


Subject(s)
Aldehyde-Lyases/chemistry , Phosphotransferases/chemistry , Staphylococcus/chemistry , Aldehyde-Lyases/metabolism , Amino Acid Sequence , Binding Sites , Biocatalysis , Circular Dichroism , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Phosphotransferases/metabolism , Protein Engineering , Stereoisomerism
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