Caulobacter segnis Dioxygenase CsO2: A Practical Biocatalyst for Stilbenoid Ozonolysis

Ozonolysis is a useful as well as dangerous reaction for performing alkene cleavage. On the other hand, enzymes are considered a more sustainable and safer alternative. Among them, Caulobacter segnis dioxygenase (CsO2) known so far for its ability to catalyze the coenzyme-free oxidation of vinylguaiacol into vanillin, was selected and its substrate scope evaluated towards diverse natural and synthetic stilbenoids. Under optimized conditions, CsO2 catalyzed the oxidative cleavage of the C=C double bonds of various trans-stilbenes, providing that a hydroxyl moiety was necessary in para-position of the phenyl group (e. g., resveratrol and its derivatives) for the reaction to take place, which was confirmed by modelling studies. The reactions occurred rapidly (0.5–3 h) with high conversions (95–99 %) and without formation of by-products. The resveratrol biotransformation was carried out on 50–mL scale thus confirming the feasibility of the biocatalytic system as a preparative method.     

Co-Oxidative Transformation of Piperine to Piperonal and 3,4-Methylenedioxycinnamaldehyde by a Lipoxygenase from Pleurotus sapidus

The valuable aroma compound piperonal with its vanilla-like olfactory properties is of high interest for the fragrance and flavor industry. A lipoxygenase (LOX_Psa1) of the basidiomycete Pleurotus sapidus was identified to convert piperine, the abundant pungent principle of black pepper (Piper nigrum), to piperonal and a second volatile product, 3,4-methylenedioxycinnamaldehyde, with a vanilla-like odor through an alkene cleavage. The reaction principle was co-oxidation, as proven by its dependence on the presence of linoleic or α-linolenic acid, common substrates of lipoxygenases. Optimization of the reaction conditions (substrate concentrations, reaction temperature and time) led to a 24-fold and 15-fold increase of the piperonal and 3,4-methylenedioxycinnamaldehyde concentration using the recombinant enzyme. Monokaryotic strains showed different concentrations of and ratios between the two reaction products.     

Oxidative Alkene Cleavage by Chemical and Enzymatic Methods

The cleavage of alkenes to the corresponding carbonyl products is a widely employed method in organic synthesis, especially to introduce oxygen functionalities into molecules, remove protecting groups and tailor large molecules. Chemical methods available for alkene cleavage include, for instance, ozonolysis, several metal‐based variants (KMnO₄, OsO₄, RuO₄, etc.), electrochemical alternatives, singlet oxygen, hypervalent iodine and organic molecules in combination with oxygen. Furthermore, several enzymatic methods for alkene cleavage have been described to establish safe, mild and selective oxidation methods. Various heme and non‐heme iron‐dependent enzymes catalyse the alkene cleavage at ambient temperature and atmospheric pressure in an aqueous buffer, showing good chemo‐ and regioselectivities in selected cases. Quite recently some Cu‐, Mn‐ and Ni‐dependent enzymes have been identified for this reaction. This review gives an overview of the different chemical and enzymatic methods available for the cleavage of alkenes.

     

Selective heterogeneous oxidation of light alkanes. What differentiates alkane from alkene feedstocks?

Some aspects of the reactivity of vanadyl pyrophosphate catalysts inn-butane andn-pentane oxidation and propane ammoxidation are discussed in order to illustrate differences and peculiarities of the catalysts for the selective oxidation of alkanes in comparison to the catalysts for the selective oxidation of alkenes. The formation of alkenes as reaction intermediates, the catalytic and mechanistic differences in alkane and alkene conversion, their different effects in changing the active surface configuration, the relationship between surface concentration of adspecies and catalytic behavior, and the problem of the surface isolation of the active intermediates are discussed. Some brief economic considerations about the advantages of the various processes of alkane oxidation as compared to alkene or aromatic oxidation are also given.     

Immobilization of chymotrypsin with interpolymer complexes of P(TM‐co‐AAm)/PAA

Chymotrypsin was immobilized with interpolymer complexes formed by the cationic polymer poly(allyltrimethyl ammonium chloride‐co‐acrylamide) [P(TM‐co‐AAm)] and poly(acrylic acid) (PAA). The introduction of a small amount of cationic groups led to a much stronger polymer–polymer interaction between P(TM‐co‐AAm) and PAA. The characteristic pH sensitivity of this kind of complex provided the possibilities of controlling the activity of the immobilized enzyme and separating the immobilized enzyme from the batch by changing the pH of the medium. Compared with the free enzyme, the immobilized chymotrypsin had higher thermal stability, acid–base stability, and stability in use. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 2013–2018, 2001     

Comparison of Selective Gas Phase‐ and Liquid Phase Hydrogenation of (Cyclo‐)Alkadienes towards Cycloalkenes on Pd/Alumina Egg‐Shell Catalysts

The hydrogenation of dienes such as 1,3‐butadiene, cyclooctadiene, and of acetylenic hydrocarbons on Pd catalysts shows high reaction rates and consequently, a strong influence of mass transfer on the selectivity of the intermediate alkene or cycloalkene product. 100 % selectivity towards (cyclo)‐alkene hydrogenation is achieved for the gas phase when the Thiele modulus is , where L is the thickness of the active layer and D_eff is the effective diffusion coefficient of the diene. The interdependencies expressed by this formula were studied in detail using model catalysts with regular pores of uniform length and diameter and perpendicular to the surface. These catalysts were prepared by anodic oxidation of aluminium wires and immobilization of the active Pd. For the liquid phase procedure of selective hydrogenation, a reaction mass transfer model has been derived in order to compare the gas phase and liquid phase procedures, in particular with respect to the selectivity. The hydrogenation of 1,3‐cyclooctadiene and of 1,3‐butadiene were studied for both procedures employing the same catalyst. The rate of hydrogenation can be represented for both cases by the identical kinetic equation r₁ = k₁ c_H2. This result is interpreted by assuming that the access of hydrogen to the surface through the dense layer of adsorbed diene is the rate determining step.     

Invertase immobilized on spacer‐arm attached poly(hydroxyethyl methacrylate) membrane: Preparation and properties

Microporous poly(2‐hydroxyethyl methacrylate) (pHEMA) membrane was prepared by UV‐initiated photopolymerization. The spacer arm (i.e., hexamethylene diamine) was attached covalently and then invertase was immobilized by the condensation reaction of the amino groups of the spacer arm with carboxyl groups of the enzyme in the presence of carbodiimides. The values of the Michael's constant K_m of invertase were significantly larger (ca. 2.5 times) upon immobilization, indicating decreased affinity by the enzyme for its substrate, whereas V_max was smaller for the immobilized invertase. Immobilization improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with immobilization and at 70°C the half times for the activity decay were 12 min for the free enzyme and 41 min for the immobilized enzyme. The immobilized enzyme activity was found to be quite stable in repeated experiments. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 75: 1685–1692, 2000     

Preparation of fatty epoxy alcohols using oat seed peroxygenase in nonaqueous media

Peroxygenase is an enzyme of higher plants that is capable of using hydroperoxide and hydrogen peroxide for oxidation of a double bond to an epoxide. A microsomal fraction was prepared from dry oat (Avena sativa) seeds. The peroxygenase activity of this fraction was tested using fatty acid hydroperoxide 2a [13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid] and its methyl ester 2b as sources of peroxygen. These were prepared by the action of soybean lipoxygenase on linoleic acid. A high-performance liquid chromatographic assay was used to differentiate between peroxygen cleavage and peroxygen cleavage with accompanying double-bond oxidation Higher activity was obtained with 2b compared to 2a, and peroxygen cleavage activity was observed in both aqueous and organic solvent media. Double-bond oxidation activity was high only in aqueous media and nonpolar organic solvents. Structural elucidation of the epoxidized product showed it to be the oxylipid, methyl cis-9,10-epoxy-13(S)-hydroxy-11(E)-octade-cenoate 4b, demonstrating specificity for epoxidation of the cis double bond. Trihydroxy product was not detected, demonstrating that the epoxide was not hydrolyzed.     

Cyclodextrin-based hyperbranched polymers by acyclic diene metathesis polymerization of an AB<Subscript>n</Subscript> monomer: molecule design,synthesis, and characterization

A novel cyclodextrin-based hyperbranched polymer (HBP) was synthetized via acyclic diene metathesis (ADMET) polymerization in homogeneous water/organic mixtures. A modified α-cyclodextrin (α-CD) molecule with one electron-rich terminal alkene and many electron-poor acrylates was first prepared through the esterification reaction, and then utilized as an AB_n-type monomer for subsequent ADMET polymerization between alkene and acrylate using the second generation Hoveyda-Grubbs catalyst, yielding HBP with the reaction time prolonged. The chemical structures of monomer and HBP were characterized by elemental analysis, IR, gel permeation chromatography with multiangle laser light scattering, and NMR measurements. The degree of branching was determined by using ¹H NMR spectroscopy and the values ranged from 0.51 to 0.42. Influence of the molecular weight on the properties (thermal stability and solubility) was also investigated. The resulting HBPs showed the good thermal stability, and higher molecular weight resulted in higher decomposition temperature from 361 °C to 383 °C. These thermally stable HBPs also displayed the excellent solubility in aprotic polar solvents.     

Cleavage of olefinic double bonds by mediated anodic oxidation

Seven alkenes, e.g. 1-decene, methyl oleate, cyclododecene, norbornene, are cleaved by indirect anodic oxidation with IO₄⁻/RuCl₃ as mediator to carboxylic acids. The best performance was achieved with two alternative ex cell-methods. Periodate is regenerated from iodate in a divided cell at a PbO₂/Ti-anode. In the chemical reactor alkene and the produced carboxylic acid are immobilized in a chromatography column on Chromosorb W and oxidized with IO₄⁻/RuO₄ in CH₃CN/water. In the alternative version the alkene is oxidized in an emulsion generated by sonication and the organic phase is retained in the reactor by a separator. Acids and diacids are obtained in 61-91% chemical yield and good current yields. The amount of consumed periodate can be reduced to less than 5% of the amount needed for the chemical oxidation. The mediated anodic cleavage of alkenes is altogether an interesting alternative to ozonolysis.     

Stabilization of the Reductase Domain in the Catalytically Self‐Sufficient Cytochrome P450BM3 by Consensus‐Guided Mutagenesis

The multidomain, catalytically self‐sufficient cytochrome P450 BM‐3 from Bacillus megaterium (P450_BM3) constitutes a versatile enzyme for the oxyfunctionalization of organic molecules and natural products. However, the limited stability of the diflavin reductase domain limits the utility of this enzyme for synthetic applications. In this work, a consensus‐guided mutagenesis approach was applied to enhance the thermal stability of the reductase domain of P450_BM3. Upon phylogenetic analysis of a set of distantly related P450s (>38 % identity), a total of 14 amino acid substitutions were identified and evaluated in terms of their stabilizing effects relative to the wild‐type reductase domain. Recombination of the six most stabilizing mutations generated two thermostable variants featuring up to tenfold longer half‐lives at 50 °C and increased catalytic performance at elevated temperatures. Further characterization of the engineered P450_BM3 variants indicated that the introduced mutations increased the thermal stability of the FAD‐binding domain and that the optimal temperature (T_opt) of the enzyme had shifted from 25 to 40 °C. This work demonstrates the effectiveness of consensus mutagenesis for enhancing the stability of the reductase component of a multidomain P450. The stabilized P450_BM3 variants developed here could potentially provide more robust scaffolds for the engineering of oxidation biocatalysts.     

The anomalous behaviour of isoquinoline relative to quinoline and its significance in relation to structure and reactivity

Vapour phase catalytic oxidation of quinoline over a vanadia base catalyst yielded nicotinic acid, while vapour phase catalytic ammoxidation gave 3-cyano-pyridine, the benzenoid ring undergoing cleavage in both cases. The isomeric molecule, isoquinoline, was however found to behave in sharp contrast, undergoing an unexpected reaction in its transformation into phthalimide during identical conditions of oxidation and into ortho-phthalonitrile during ammoxidation, the pyridine ring undergoing cleavage in both cases, with the appearance of the heterocyclic ring nitrogen in a reactive functional group. The significance of these observations is discussed in relation to structure and reactivity and possible reaction mechanisms are suggested.     

Elucidating mechanism of ethylbenzene oxidation catalyzed by carbon-based catalysts with kinetic modeling

Ethylbenzene (EB) oxidation to oxygenated products catalyzed by carbon nanotubes (CNTs) and CNTs with confined cobalt nanoparticles (Co@C/CNTs) are conducted. Co@C/CNTs with enhanced interfacial charge transfer displays a unique activity. A kinetic model with the simplified network is established, in which seven elementary reactions with prominent features of EB oxidation are selected. The main parameters, reaction rate constant (k) and active energy (E_a) are obtained. Co@C/CNTs could speed up most elementary steps and reduce energy barriers, thus improving the overall activity. The homolytic cleavage of peroxide is confirmed to be the rate-determining step (RDS). The k ratio (1.55) of acetophenone (AcPO)/1-phenylethyl alcohol (PEA) is found to be close to the experimental result (r_AcPO/r_PEA, 1.30). The prediction of EB oxidation is simulated, showing that the synthesis of AcPO dominates the reaction route, while the addition of PEA would prohibit the reaction.     

Transition metal catalyzed oxidations. 13

The secondary N-aryl-N-alkyl amines 1 – 3 and 5 are oxidized by the Zr(O-t-Bu)₄/TBHP system to nitrobenzene ( 6 ) and the corresponding carbonyl compounds with cleavage of the C–N bond. Nitrones are postulated as reaction intermediates as demonstrated by the cleavage of 9 – 11 to nitrobenzene ( 6 ) and the benzaldehydes 12 – 14 by the same catalytic oxidation system.     

Trametes versicolor laccase immobilized poly(glycidyl methacrylate) based cryogels for phenol degradation from aqueous media

This study aims removal of phenols in wastewater by enzymatic oxidation method. In this study, Trametes versicolor laccase was covalently immobilized onto a cryogel matrix by the nucleophilic attack of amino groups of laccase to epoxy groups of matrix. Glycidyl methacrylate was chosen as functional monomer to prepare poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) [p(HEMA‐co‐GMA)] cryogels. The enzyme immobilized matrix was characterized by FTIR, SEM, and swelling tests. The effect of pH, reaction time, temperature, substrate concentration, enzyme concentration, and storage period on immobilized enzyme activity was determined and compared with those of free enzyme. The model substrate was 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid (ABTS). Lineweaver‐Burk plots were used to calculate K_m and V_m values. K_m values were 165.1 and 156.0 µM while V_m values were 55.2 µM min^?1 and 1.57 µM min^?1 for free and immobilized laccase, respectively. Immobilized enzyme was determined to retain 82.5% and 72.0% of the original activity, respectively, after 6 consecutive use and storage period of 4 weeks. The free enzyme retained only 24.0% of its original activity following the same storage period. Lastly, decomposition products resulting from enzymatic oxidation of a model phenolic compound (3,5‐dinitrosalicylic acid) in aqueous solution were identified by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41981.     

Photocatalytic Oxidative Cleavage of Alkenes Followed by Carbonyl Stereoselective Bioreduction for the Synthesis of Enantioenriched Secondary Alcohols

Oxidative alkene cleavage of a series of (hetero)aryl alkyl styrenes in aqueous medium has been developed using either 9-mesityl-10-methylacridinium perchlorate ([AcrMes]ClO₄) and sodium anthraquinone-2-sulfonate (SAS) as photosensitizers under blue LED irradiation. Reaction conditions were studied to find a suitable media for the development of a linear cascade after subsequent stereoselective reduction of the corresponding ketone intermediate. The use of cesium carbonate provided an adequate pH to the reaction medium for the alcohol dehydrogenase action. [AcrMes]ClO₄ was found to be the best photocatalyst, allowing the development of concurrent or sequential cascades depending on the ability of the photosensitizer to oxidize back the chiral alcohol to the ketone. Overall, the photobiocatalytic approach has allowed the synthesis of a wide number of alcohol compounds, the formation of (S)- or (R)-enantiomers being attained with excellent stereoselectivity and moderate to good yields.     

Alkaline protease production by immobilized growing cells of Serratia marcescens with interpolymer complexes of P(TM‐co‐AAm)/PAA

The growing cells of Serratia marcescens (SM) were immobilized with the interpolymer complex carrier, which is formed by the cationic polymer, poly(allyltrimethyl ammonium chloride‐co‐acrylamide) [P(TM‐co‐AAm)], and poly(acrylic acid) (PAA). When the association degree of PAA is suitable to the cationic degree of P(TM‐co‐AAm), the effective crosslinking network provides the most favorable circumstances for the cell immobilization. The alkaline protease can be produced by the immobilized SM with high activity. Compared with the free cells, the immobilized SM has higher thermal stability, acid‐base stability, operational stability, and storage stability. Under the optimum immobilizing conditions, not only the living cells of SM but also thermophilic Bacillus firmus (TBF) were immobilized with the complex of P(TM‐co‐AAm)/PAA. The results show the carrier of P(TM‐co‐AAm)/PAA complex to be superior in properties to the usual carriers, such as Na‐alginate and carrageenan. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 84: 178–183, 2002; DOI 10.1002/app.10293     

Phenol Removal by Modified Peroxidases

Horseradish peroxidase (HRP) catalyses the oxidation of toxic aromatic compounds, especially phenols, in the presence of hydrogen peroxide. Reaction products polymerise to form insoluble precipitates which readily separate from aqueous solution, unlike their monomeric precursors. High-temperature phenol-containing gas liquors (produced from coal conversion processes) or effluent from bleach plants of kraft mills can substantially affect the stability of enzymes such as HRP and thus their oxidation capabilities. Apparent inactivation of peroxidase during high temperature polymerisation reactions is mainly due to unfolding of the protein backbone. The catalytic lifetime of HRP at high temperatures can be extended by chemical modification of lysine ε-amino groups using succinimides. The bifunctional, ethylene glycol bis-succinimidyl succinate (EG-NHS) and the monofunctional, acetic acid N-hydroxysuccinimide ester (AA-NHS) were used. The extent of stabilisation is dependent on the nature and concentration of the reagent used. The optimum pH for phenol removal is 9·0 (8·0 for 4-chlorophenol) for both native and modified forms of the enzyme; the optimum molar ratio of hydrogen peroxide and phenolic substrate is around 1·0. The effects of peroxide and enzyme concentration on the polymerisation reaction were investigated. HRP derivatives significantly reduced the oxidation reaction time at 70°C.     

Insight into β-Carotene Thermal Degradation in Oils with Multiresponse Modeling

The aim of this study was to gain further insight into β-carotene thermal degradation in oils. Multiresponse modeling was applied to experimental high-performance liquid chromatography–diode array detection (HPLC–DAD) data (trans-, 13-cis-, and 9-cis-β-carotene concentrations) during the heat treatments (120–180 °C) of two β-carotene-enriched oils, i.e., palm olein and copra. The test of different reaction schemes showed that β-carotene isomerization reactions were dominant and reversible. The resulting cis isomers and trans-β-carotene simultaneously underwent oxidation and cleavage reactions at the same rate constant. From the kinetic analysis, it appeared that—contrary to oxidation and cleavage reactions—isomerization rate constants did not follow the Arrhenius law. However, the isomerization equilibrium constant increased with temperature, favoring isomer production, particularly 9-cis-β-carotene. Its production was shown to be concomitant with oxidation and cleavage reactions, indicating that 9-cis-β-carotene could be a good degradation indicator during oil storage or processing.