P450 BM3 Monooxygenase as an Efficient NAD(P)H‐Oxidase for Regeneration of Nicotinamide Cofactors in ADH‐Catalysed Preparative Scale Biotransformations

Enzymatic oxidations of primary and secondary alcohols catalysed by nicotinamide dependent alcohol dehydrogenases on the preparative scale require cofactor regeneration systems. Of critical value from an economic and ecological perspective is the application of NAD(P)H‐oxidases, which utilise molecular oxygen as a cost‐effective, atom‐efficient and environmentally benign oxidant to regenerate the cofactor NAD(P)⁺. Herein, the P450 BM3 monooxygenase from Bacillus megaterium is presented as an NAD(P)H‐oxidase for the successful regeneration of both NADP⁺ and NAD⁺ on the preparative scale. This enzyme was exemplarily applied for ADH‐catalysed oxidative kinetic resolutions of racemic secondary alcohols and the desymmetrisation of a meso‐diol leading to enantiomerically enriched secondary alcohols in both cases. Furthermore, the ADH‐catalysed oxidation of a primary alcohol targeting the corresponding aldehyde was performed. The obtained results significantly broaden the scope of feasible oxidative biotransformations, thereby increasing the number of synthetic reactions complying with key challenges of a modern and sustainable chemistry such as mild reaction conditions, environmentally benign solvents, and biodegradable non‐toxic catalysts.

     

Flavin‐grafted poly(vinyl alcohol): Preparation and properties

An NAD(P)H oxidase‐like activity was found in semisynthetic flavoadducts, prepared from an aldehyde derivative of riboflavin and commercial poly(vinylalcohol) (PVA), previously grafted with aminopropyl side chains. Both water‐soluble and water‐insoluble beaded preparations were obtained. The products showed a noticeable NAD(P)H oxidase‐like activity, converting the nucleotide substrate to its oxidized counterpart NAD(P)⁺ at the expense of molecular oxygen, the latter being reduced to hydrogen peroxide. In contrast to some “true” β‐NAD(P)H oxidases, the PVA adducts do not require the presence of additional flavin adenine dinucleotide (FAD) to work; some properties of the flavoadducts were studied that make these flavoadducts good candidates for technological applications. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 85: 2471–2477, 2002     

Evolution of Glucose Dehydrogenase for Cofactor Regeneration in Bioredox Processes with Denaturing Agents

Glucose dehydrogenase (GDH) is a general tool for driving nicotinamide (NAD(P)H) regeneration in synthetic biochemistry. An increasing number of synthetic bioreactions are carried out in media containing high amounts of organic cosolvents or hydrophobic substrates/products, which often denature native enzymes, including those for cofactor regeneration. In this work, we attempted to improve the chemical stability of Bacillus megaterium GDH (BmGDH_M0) in the presence of large amounts of 1-phenylethanol by directed evolution. Among the resulting mutants, BmGDH_M6 (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2-fold increase in tolerance against 10 % (v/v) 1-phenylethanol. Moreover, BmGDH_M6 was also more stable than BmGDH_M0 when exposed to hydrophobic and enzyme-inactivating compounds such as acetophenone, ethyl 2-oxo-4-phenylbutyrate, and ethyl (R)-2-hydroxy-4-phenylbutyrate. Coupled with a Candida glabrata carbonyl reductase, BmGDH_M6 was successfully used for the asymmetric reduction of deactivating ethyl 2-oxo-4-phenylbutyrate with total turnover number of 1800 for the nicotinamide cofactor, thus making it attractive for commercial application. Overall, the evolution of chemically robust GDH facilitates its wider use as a general tool for NAD(P)H regeneration in biocatalysis.     

Two‐Stage Oxidation of Glucose by an Enzymatic Bioanode

The present study reports the design of a novel bioanode to deeply oxidize glucose in an enzymatic biofuel cell (EFC). This enzymatic glucose cell utilizes three co‐immobilized enzymes: NAD‐dependent glucose dehydrogenase (GDH), NAD(P)⁺‐dependent gluconate‐5‐dehydrogenase (Ga5DH), and diaphorase (DI). Glucose is oxidized to gluconate by NAD‐dependent GDH, gaining two electrons per glucose; the gluconate obtained as a by‐product is oxidized at the C5 carbon to 5‐keto‐gluconate by Ga5DH. Operation of our bioanode enabled the oxidation of glucose in two stages, resulting in the gain of four electrons. The three‐enzyme EFC provides a maximum power density of 10.51 ± 1.72 μW cm^–2, which is about 1.6 times higher than the maximum power density of an EFC using a bioanode based on the co‐immobilization of two enzymes (GDH and DI). Our results hold promise for increasing the current density of EFCs, and for application in glucose biosensor.     

A Rapid and High‐Throughput Assay for the Estimation of Conversions of Ene‐Reductase‐Catalysed Reactions

A fast and sensitive colorimetric assay (FRED, fast and reliable ene‐reductases detection) that allows the estimation of levels of conversion of ene‐reductase (ER)‐catalysed reactions has been developed. The activated olefin is reduced by ER at the expense of NAD(P)H cofactor, whose regeneration is carried out in situ by the glucose/glucose dehydrogenase system. Subsequently, the consumption of the co‐substrate glucose is determined colorimetrically by a multienzymatic system. The FRED assay offers a wide range of possible applications, from enzyme fingerprinting and kinetic analysis, to primary screening of enzyme libraries and optimisation of ERs' performances under different reaction conditions.     

Cofactor Regeneration of NAD+ from NADH: Novel Water‐Forming NADH Oxidases

Dehydrogenases with their superb enantioselectivity can be employed advantageously to prepare enantiomerically pure alcohols, hydroxy acids, and amino acids. For economic syntheses, however, the co‐substrate of dehydrogenases, the NAD(P)(H) cofactor, has to be regenerated. Whereas the problem of regenerating NADH from NAD⁺ can be considered solved, the inverse problem of regenerating NAD⁺ from NADH still awaits a definitive and practical solution. A possible solution is the oxidation of NADH to NAD⁺ with concomitant reduction of oxygen catalyzed by NADH oxidase (E.C. 1.6.‐.‐) which can reduce O₂ either to undesirable H₂O₂ or to innocuous H₂O. We have found and cloned two novel genes from Borrelia burgdorferi and Lactobacillus sanfranciscensis with hitherto only machine‐annotated NADH oxidase function. We have overexpressed the corresponding proteins and could prove the annotated function to be correct. As demonstrated with a more sensitive assay than employed previously, the two novel NADH oxidases reduce O₂ to H₂O.     

Photochemically Induced Oxidative and Reductive Regeneration of NAD(P)+/NAD(P)H Cofactors: Applications in Biotransformations

Photosensitized regeneration of NAD(P)H cofactors is accomplished by biocatalyzed and artificially catalyzed transformations in photochemical assemblies. Photogenerated N,N'-dimethyl-4,4′-bipyridinium radical cation, MV^+., acts as electron carrier for the reduction of NADPH in the presence of the enzyme ferredoxin reductase and for the reduction of NADH in the presence of lipoamide dehydrogenase. For the photogeneration of MV^+. and subsequent NADPH formation, three different photosensitizers are applied: Ru(bpz)²₃⁺, Ru(bpy)²₃⁺, and Zn—TMPyP⁴⁺. The highest quantum yield for NADPH formation is observed with Ru(bpz)₃²⁺ and is ϕ = 1.7 × 10⁻¹. For NADH regeneration only Zn-TMPyP⁴⁺ can be applied. Ru(bpy)₃²⁺ and Ru(bpz)²⁺₃ interact with NADH in their excited or oxidized forms and therefore cannot be used as light-active compounds in the system. The NADPH regeneration cycle has been coupled to the biocatalyzed synthesis of glutamic acid. Although Ru(bpz)₃²⁺ is 42.5-fold more efficient than Ru(bpy)₃²⁺ in the regeneration of NADPH, the synthesis of glutamic acid is improved only by a factor of 2 in the presence of Ru(bpz)₃²⁺, implying that the coupled process is rate limiting. Oxidative regeneration of the NAD⁺ cofactor is accomplished in a photosystem that includes Ru(bpy)₃²⁺ as photosensitizer. The photoprocess is coupled to dehydrogenation of ethanol, propanol, lactic acid, and alanine with concomitant H₂ evolution. A photosystem that includes Ru(bpy)₃²⁺ as photosensitizer, ascorbate as electron donor, and chloro-tris-(3-diphenylphosphinobenzene sulfonate)Rh(I), RhCl(dpm)₃^3-, is catalytically active in the photoinduced regeneration of NAD(P)H cofactors. Mechanistic investigations show that photogenerated Ru(bpy)₃⁺ mediates the generation of a hydrido-rhodium complex that acts as a charge relay for the production of NAD(P)H.     

Self‐Immolative Bioluminogenic Quinone Luciferins for NAD(P)H Assays and Reducing Capacity‐Based Cell Viability Assays

Highly sensitive self‐cleavable trimethyl lock quinone‐luciferin substrates for diaphorase were designed and synthesized to measure NAD(P)H in biological samples and monitor viable cells via NAD(P)H‐dependent cellular oxidoreductase enzymes and their NAD(P)H cofactors.     

Extended Catalytic Scope of a Well‐Known Enzyme: Asymmetric Reduction of Iminium Substrates by Glucose Dehydrogenase

NADP(H)‐dependent imine reductases (IREDs) are of interest in biocatalytic research due to their ability to generate chiral amines from imine/iminium substrates. In reaction protocols involving IREDs, glucose dehydrogenase (GDH) is generally used to regenerate the expensive cofactor NADPH by oxidation of d ‐glucose to gluconolactone. We have characterized different IREDs with regard to reduction of a set of bicyclic iminium compounds and have utilized ¹H NMR and GC analyses to determine degree of substrate conversion and product enantiomeric excess (ee). All IREDs reduced the tested iminium compounds to the corresponding chiral amines. Blank experiments without IREDs also showed substrate conversion, however, thus suggesting an iminium reductase activity of GDH. This unexpected observation was confirmed by additional experiments with GDHs of different origin. The reduction of C=N bonds with good levels of conversion (>50 %) and excellent enantioselectivity (up to >99 % ee) by GDH represents a promiscuous catalytic activity of this enzyme.     

Enzymatic production of l-alanine from malic acid with malic enzyme and alanine dehydrogenase with coenzyme regeneration

An enzyme reaction system with coenzyme regeneration was investigated for L-alanine production. Alanine dehyrogenase L-alanine: NAD⁺ oxidoreductase (EC 1.4.1.1)] from Corynebacterium flaccumfaciens AHU-1622 was used as the catalyst for reductive amination of pyruvate to L-alanine. NAD- and NADP-linked malic enzyme [L-malate: NAD(P)⁺ oxidoreductase (EC 1.1.1.39)] from Pseudomonas diminuta IFO-13182 was used for the regeneration of NADH. Optimum conditions for L-alanine production were determined, including L-malic acid concentration, MgCl₂ concentration and pH. Under suitable conditions, the conversion of L-malic acid to L-alanine reached 95% after 72 h of incubation at 30°C, yielding 106 mol/m³ of L-alanine. The L-alanine produced was purified in crystal form; its purity was 99.4%, based on HPLC analysis.     

Synthesis of copolymer from 1,3,5‐trioxane and 1,3‐dioxolane catalyzed by Maghnite‐H+

Copolymers (polyoxymethylene) were prepared by cationic copolymerization of 1,3,5‐trioxane (TOX) with 1,3‐dioxolane (DOX) in the presence of Maghnite‐H⁺ (Mag‐H⁺) in solution. Maghnite is a Montmorillonite sheet silicate clay, with exchanged protons to produce Mag‐H⁺. Various techniques, including ¹H‐NMR, ¹³C‐NMR, FT‐IR spectroscopy, and Ubbelohde viscometer were used to elucidate structural characteristics properties of the resulting copolymers. The influence of the amount of catalyst, of dioxolane (DOX), temperature, solvent, and time of copolymerization on yield and on intrinsic viscosity of copolymers was studied. The yield of copolymerization depends on the amount of Mag‐H⁺ used and the reaction time. We also propose mechanisms involved in the synthesis of copolymer (polyoxymethylene). © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

A New Strategy to Prepare Poly(2‐methyl‐2‐oxazoline)‐Based Antifouling Coatings: Using UV‐Crosslinked Copolymer Anchors

A series of the copolymer, poly(styrene‐random‐glycidyl methacrylate) (P(St‐r‐GMA)), is synthesized by free radical polymerization, and characterized by ¹H NMR spectroscopy and gel permeation chromatography. The various substrates are then modified by P(St‐r‐GMA) under ultraviolet (UV) irradiation. Subsequently, the poly(2‐methyl‐2‐oxazoline) (PMOXA) based coatings are prepared by anchoring amino‐terminated PMOXA onto the P(St‐r‐GMA) modified surfaces through the reaction between the amino group of PMOXA and epoxy group of P(St‐r‐GMA). The results of ellipsometry, X‐ray photoelectron spectroscopy, atomic force microscopy, and water contact angle reveal that PMOXA‐based coatings can be prepared successfully on the substrates through UV‐crosslinked P(St‐r‐GMA) as anchoring coatings. Besides, the PMOXA‐based coatings display not only a superior antifouling property but long‐term stability as well. Furthermore, the location of the coating formed on the substrate can be well controlled through selecting the site of UV irradiation, which can be utilized for the selectivity of protein adsorption (or resistance) on special devices.     

Engineering a Formate Dehydrogenase for NADPH Regeneration**

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) constitute major hydrogen donors for oxidative/reductive bio-transformations. NAD(P)H regeneration systems coupled with formate dehydrogenases (FDHs) represent a dreamful method. However, most of the native FDHs are NAD⁺-dependent and suffer from insufficient reactivity compared to other enzymatic tools, such as glucose dehydrogenase. An efficient and competitive NADP⁺-utilizing FDH necessitates the availability and robustness of NADPH regeneration systems. Herein, we report the engineering of a new FDH from Candida dubliniensis (CdFDH), which showed no strict NAD⁺ preference by a structure-guided rational/semi-rational design. A combinatorial mutant CdFDH-M4 (D197Q/Y198R/Q199N/A372S/K371T/▵Q375/K167R/H16L/K159R) exhibited 75-fold intensification of catalytic efficiency (k_cat/K_m). Moreover, CdFDH-M4 has been successfully employed in diverse asymmetric oxidative/reductive processes with cofactor total turnover numbers (TTNs) ranging from 135 to 986, making it potentially useful for NADPH-required biocatalytic transformations.     

Hydroxyalkylation of barbituric acid. I. Hydroxymethyl derivatives of barbituric acid—Precursors of polyetherols with a pyrimidine ring

The reaction of barbituric acid with formaldehyde at variable molar ratios was studied. The structure of the products was elucidated using ¹H‐NMR and IR spectroscopy as well as acidic number and formaldehyde percentage analysis. The products were potential substrates for the synthesis of linear and multifunctional polyetherols with a pyrimidine ring by reaction with oxiranes. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101:3468–3478, 2006     

Esterification of Renewable Levulinic Acid to n‐Butyl Levulinate over Modified H‐ZSM‐5

The synthesis of n‐butyl levulinate, one of the most important biodiesel additives, by catalytic esterification of biomass‐derived levulinic acid (LA) with n‐butanol over modified H‐ZSM‐5 (micro/meso‐HZ‐5) in a closed‐batch system is reported for the first time. The optimization of the reaction conditions such as the reactant molar ratio, the catalyst loading, the reaction time and the temperature was performed in view to maximize the yield of n‐butyl levulinate. Micro/meso‐HZ‐5 was found to be the most efficient catalyst, with 98 % yield of n‐butyl levulinate and a reusability for six cycles, which is higher than reported in the literature. A possible catalytic mechanism for the esterification reaction is also proposed. A second‐order pseudo‐homogeneous model with R² > 0.97 confirmed that the esterification reaction is performed in the kinetic regime due to the high activation energy of 23.84 kJ mol^?1.     

Thermal regeneration of spent coal‐based activated carbon using carbon dioxide: process optimisation,Methylene Blue decolorisation isotherms and kinetics

Spent coal‐based activated carbon from the silicon industry has been used as raw material for the regeneration of activated carbon, with carbon dioxide as the regenerating agent. The regeneration process was optimised using response surface methodology and the optimum regeneration conditions were: regeneration temperature 985 °C; regeneration time 120 min; and carbon dioxide flow rate of 600 ml/min. The iodine number and yield of the activated carbon obtained under the optimum regeneration conditions were 1071 mg/g and 67%, with a Brunauer–Emmet–Teller surface area of 1270 m²/g and pore volume of 0.91 cm³/g. The regenerated carbon was tested for the removal of Methylene Blue dyes. The maximum adsorption capacity was found to be 395 mg/g and the equilibrium data fitted to the Langmuir isotherm model. The kinetic data indicated that the best fit corresponds to the pseudo‐second‐order kinetic model.     

Homogeneous complex solution formed through interactions of poly(acrylamide‐co‐acrylic acid)/poly(acrylamide‐co‐dimethyldiallylammonium chloride) complex with M n+ hydration metal ions in aqueous media

A homogeneous complex solution, formed through inter‐polyelectrolyte complexation of poly(acrylamide‐co‐acrylic acid) (P(AM‐AA)) with poly(acrylamide‐co‐dimethyldiallylammonium chloride) (P(AM‐DMDAAC)) and interaction of the P(AM‐AA)/P(AM‐DMDAAC) complex with M ⁿ⁺ hydrated metal ion, was prepared and the structure and properties of the P(AM‐AA)/P(AM‐DMDAAC)/M ⁿ⁺ homogeneous complex solution were studied by UV spectrometry, dynamic light scattering and viscometry. The experimental results show that the homogeneous complex solution can be obtained by controlling the composition of the P(AM‐AA)/P(AM‐DMDAAC) complex and the M ⁿ⁺ metal ion content. Compared to the constituents, ie the P(AM‐AA) solution, the P(AM‐DMDAAC) solution and the P(AM‐AA)/P(AM‐DMDAAC) complex solution, the P(AM‐AA)/P(AM‐DMDAAC)/M ⁿ⁺ complex solution has a new peak at 270 nm in its UV spectrum, a larger hydrodynamic radius, and hence a higher solution viscosity, all of which indicate that there exist specific interactions between polymers and M ⁿ⁺ metal ions. These interactions lead to the formation of a network structure and hence an obvious increase not only in solution viscosity but also in resistance of the polymer solution to simple salts, to temperature changes and to shearing. © 2001 Society of Chemical Industry     

Structural development Of TMMA and SSQXN‐8 as porous chelating resins

Superfast kinetics of Nd⁺³, Eu⁺³, and Gd⁺³ ions were studied on the surfaces of N,N,N^\,N^\‐tetramethylmalonamide (TMMA) and silsesquioxane (SSQXN‐8) resins. TMMA and SSQXN‐8 were prepared by suspension polymerization and sol–gel routes, respectively. They were identified using elemental analysis, FTIR, H‐NMR, ¹³C‐NMR, MIP, and BET surface area. Kinetic investigations were performed in batch conditions and different models were used to fit the data; Boyd and Helfferich models were found the best. The diffusion of the ions through the resins were very fast and found to be in the order of 10^?16 m²/S. Effective diffusions of the studied ions were found to be 10^?15 order of magnitude and directly proportional to the kinetic energy of the transition state. ΔS* values from ?166.044 to ?179.297 J mol^?1 K^?1 were estimated as entropy stability factors of the system. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Efficient Syntheses of (Thio)phosphonylated Isobenzofurans by Tandem Nucleophilic Addition and Regioselective 5‐exo‐dig Addition to Carbon‐Carbon Triple Bond: Cooperative Effect to 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU)

The tandem nucleophilic addition‐cyclization reaction of o‐alkynylbenzaldehydes or o‐alkynylacetophenones 2 with dialkyl phosphites or dialkyl phosphonothioates 1 took place very smoothly in the presence of 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) in THF at room temperature. In all cases, the reaction proceeded in a regioselective manner leading to the 5‐exo‐dig products 3 in excellent yields. The phenomenon of a 1,5‐sigmatropic hydrogen shift or a 1,5‐sigmatropic methyl shift was observed in this reaction depending on the different substituent groups such as R³ in the o‐alkynylbenzaldehyde or o‐alkynylacetophenone 2 substrates.     

Preparation and release behavior of temperature‐ and pH‐responsive chitosan material

BACKGROUND: Chitosan is a polymer with good biocompatibility which makes it promising for potential applications in the field of drug delivery. A novel kind of copolymer, P(CS‐Ma‐graft‐NIPAm), was synthesized with chitosan (CS), maleic anhydride (Ma) and N‐isopropylacrylamide (NIPAm) by grafting and copolymerization. RESULTS: The copolymers were characterized using Fourier transform infrared, ¹H NMR and ultraviolet spectroscopies, and the molecular weight and polydispersity were determined using gel permeation chromatography. The aqueous solution properties of the copolymer and the controlled delivery of coenzyme A from it were also studied. The results showed that the copolymer had temperature and pH sensitivities, and that the release of coenzyme A from the copolymer was dependent on the release medium, namely the concentration of the copolymer, pH and temperature. Higher concentrations of the copolymer absorbed more coenzyme A than lower ones. Increasing temperature accelerated coenzyme A release from the copolymer. Also, the pH of the solution had a significant impact on the release of coenzyme A. CONCLUSION: These results suggest that the novel copolymer could be used in drug delivery systems. Copyright © 2007 Society of Chemical Industry