聚脲多孔材料的简单制备及在酶固定和手性拆分中的应用

以甲苯二异氰酸酯(TDI)为单体,在水与丙酮混合溶剂中通过沉淀聚合一步法制备了富含胺基的聚脲多孔材料(PPU),通过扫描电镜和压汞法对其表面形貌和孔结构进行了表征.PPU经戊二醛(GA)活化后用于荧光假单胞菌脂肪酶(PFL)的固定,考察了GA活化过程中GA浓度对酶固定量及固定酶活性的影响.结果表明,PPU是一种粒子尺寸分布在30~50μm范围的形状不规则的多孔粒子,孔径在2 nm~100μm之间呈连续分布.在pH=8.0的缓冲溶液中用0.17 mol/L的GA对PPU进行改性,将改性后的PPU用于PFL的固定,当酶溶液浓度为2.56 mg/m L时,得到酶的最大固定量为95.2 mg/g,固定酶的活性为375 U/mg,相对活性为76%.将此固定酶作为催化剂,用于1-苯乙醇外消旋化合物的手性拆分,并与游离酶催化的结果相比较.结果表明,固定酶的反应活性和立体选择性都明显优于游离酶.通过沉淀聚合制备的聚脲多孔材料在酶固定及手性分子拆分方面具有应用前景.     

Enzyme activation and inactivation induced by low doses of irradiation

Activation phenomenon has been observed with two sets of enzymes under the conditions of low dosage irradiation. Activation was registered for angiotensin-converting enzyme under in vitro γ-irradiation (0.662 MeV, pulse duration approx 10s) at dose levels of 1–3 Gy and under X-ray irradiation (approx 9 keV, pulseduration approx 10^?9s) at dose levels of 2×10^?5 Gy. An activation effect has also occurred for native and recombinant horseradish peroxidase and tobacco peroxidase under γ-irradiation. The phenomenon observed is rationalized in terms of a kinetic model suggesting the existence of at least one activated enzyme conformation induced by radiolysis. The activity oscillations registered in dense plasma focus experiments were rationalized using the same model with the corresponding kinetic equation converted into the form describing the decaying oscillations caused by exciting force. The model analysis is presented.     

Inactivation of microbial arginine deiminases by L-canavanine

Arginine deiminase (ADI) catalyzes the hydrolytic conversion of L-arginine to ammonia and L-citrulline as part of the energy-producing L-arginine degradation pathway. The chemical mechanism for ADI catalysis involves initial formation and subsequent hydrolysis of a Cys-alkylthiouronium ion intermediate. The structure of the Pseudomonas aeruginosa ADI-(L-arginine) complex guided the design of arginine analogs that might react with the ADIs to form inactive covalent adducts during catalytic turnover. One such candidate is L-canavanine, in which an N-methylene of L-arginine is replaced by an N-O. This substance was shown to be a slow substrate-producing O-ureido-L-homoserine. An in depth kinetic and mass spectrometric analysis of P. aeruginosa ADI inhibition by L-canavanine showed that two competing pathways are followed that branch at the Cys-alkylthiouronium ion intermediate. One pathway leads to direct formation of O-ureido-L-homoserine via a reactive thiouronium intermediate. The other pathway leads to an inactive form of the enzyme, which was shown by chemical model and mass spectrometric studies to be a Cys-alkylisothiourea adduct. This adduct undergoes slow hydrolysis to form O-ureido-L-homoserine and regenerated enzyme. In contrast, kinetic and mass spectrometric investigations demonstrate that the Cys-alkylthiouronium ion intermediate formed in the reaction of L-canavanine with Bacillus cereus ADI partitions between the product forming pathway (O-ureido-L-homoserine and free enzyme) and an inactivation pathway that leads to a stable Cys-alkylthiocarbamate adduct. The ADIs from Escherichia coli, Burkholderia mallei, and Giardia intestinalis were examined in order to demonstrate the generality of the L-canavanine slow substrate inhibition and to distinguish the kinetic behavior that defines the irreversible inhibition observed with the B. cereus ADI from the time controlled inhibition observed with the P. aeruginosa, E. coli, B. mallei, and G. intestinalis ADIs.     

Sequential injection Lab-at-valve (SI-LAV) segmented flow system for kinetic study of an enzyme

A sequential injection-Lab-at-valve (SI-LAV) segmented flow system for kinetic study of an enzyme was developed. Air segments were introduced for separation of enzyme and substrate zones and separation of the stacked zones from the carrier solution which ensure the measurement of the initial rate and minimize the dilution/dispersion effect. The open- ended mixing chamber makes it possible to use air segments in the flow system without the need for additional air segment discarding steps. The enzyme horseradish peroxidase (HRP) kinetic parameters based on initial rate was used as a model study. The operation of the system is virtually the same as that of the conventional batch-wise process. The kinetic parameters (i.e. K(m) and V(max)) of HRP obtained using the proposed system agree well with those obtained using the batch-wise process as well. The proposed system offers additional benefits of volume down scaling, improved rapidity and automatic features that does not require a skillful operator.     

Templating Irreversible Covalent Macrocyclization by Using Anions

Inorganic anions were used as templates in the reaction between a diamine and an activated diacid to form macrocyclic amides. The reaction conditions were found to perform the macrocyclization sufficiently slow to observe a template effect. A number of analytical methods were used to clarify the reaction mechanisms and to show that the structure of the intermediate plays a decisive role in determining the product distribution. For the macrocyclization under kinetic control, it was shown that the amount of a template, the conformational rigidity of building blocks, and the anion affinities of reaction components and intermediates are important parameters that one should take into consideration to achieve high yields.     

Spectroscopic and kinetic evidence for an accumulating intermediate in an SNV reaction with amine nucleophiles. Reaction of methyl beta-methylthio-alpha-nitrocinnamate with piperidine and morpholine

A spectroscopic and kinetic study of the reaction of methyl beta-methylthio-alpha-nitrocinnamate (4-SMe) with morpholine, piperidine, and hydroxide ion in 50% DMSO/50% water (v/v) at 20 degrees C is reported. The reactions of 4-SMe with piperidine in a pH range from 10.12 to 11.66 and those with morpholine at pH 12.0 are characterized by two kinetic processes when monitored at lambdamax (364 nm) of the substrate, but by only one process when monitored at lambdamax (388) nm of the product. The rate constants obtained at 388 nm were the same as those determined for the slower of the two processes at 364 nm. These rate constants refer to product formation, whereas the faster process observed at 364 nm is associated with the loss of reactant to form an intermediate. In contrast, for the reaction of 4-SMe with morpholine at pH 8.62 the rates of product formation and disappearance of the substrate were the same, i.e., there is no accumulation of an intermediate. Likewise, the reaction of 4-SMe with OH- did not yield a detectable intermediate. The factors that allow the accumulation of intermediates in certain SNV reactions but not in others are discussed in detail, and structure-reactivity comparisons are made with reactions of piperidine and morpholine with other highly activated vinylic substrates.     

Microwave‐synthesized Pepper Peduncle Carbon,Characterization, and Its Adsorption Studies

Adsorption on activated carbon is an efficient method for the removal of toxic dyes. However, since commercially available charcoal is quite expensive, activated carbon obtained from agricultural by‐products may serve as a good replacement. In this study, activated carbon was prepared from pepper peduncle, an agricultural waste product, by microwave activation. The synthesized carbon was characterized by X‐ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy, and thermal gravimetric analysis techniques. It was then used for the adsorption of methylene blue dye from an aqueous solution, which was studied as a function of the dye concentration, contact time, and temperature. The adsorption data were fitted to Freundlich and Langmuir isotherm models. The adsorption kinetics was studied by employing first‐ and second‐order kinetic models, and it was found that the adsorption of methylene blue on the synthesized activated carbon follows a second‐order kinetic model. Effect of temperature on the adsorption process was studied, and the thermodynamic parameters such as activation energy, change in enthalpy, entropy, and free energy of adsorption were calculated on the basis of the absolute theory of reaction rate expressions. About 99.5–91.8% of the dye was removed for an initial dye concentration in the range 20–100 mg/g in 1 h. Thus the synthesized activated carbon was found to be very efficient in adsorbing the dye.     

Kinetic pathway investigations of three‐component photoinitiator systems for visible‐light activated free radical polymerizations

Three‐component photoinitiator systems generally include a light‐absorbing photosensitizer (PS), an electron donor, and an electron acceptor. To investigate the key factors involved with visible‐light activated free radical polymerizations involving three‐component photoinitiators and 2‐hydroxyethyl methacrylate, we used thermodynamic feasibility and kinetic considerations to study photopolymerizations initiated with either rose bengal or fluorescein as the PS. The Rehm–Weller equation was used to verify the thermodynamic feasibility for the photo‐induced electron transfer reaction. It was concluded that key kinetic factors for efficient visible‐light activated initiation process are summarized in two ways: (1) to retard back electron transfer and recombination reaction steps and (2) to use a secondary reaction step for consuming dye‐based radical and regenerating the original PS (dye). Using the thermodynamic feasibility and kinetic data, we suggest three different kinetic mechanisms, which are (i) photo‐reducible series mechanism, (ii) photo‐oxidizable series mechanism, and (iii) parallel‐series mechanism. Because the photo‐oxidizable series mechanisms most efficiently allow the key kinetic factors, this kinetic pathway showed the highest conversion and rate of polymerization. The kinetic data measured by near‐IR and photo‐differential scanning calorimeter verified that the photo‐oxidizable series mechanism provides the most efficient kinetic pathway in the visible‐light activated free radical polymerizations. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 887–898, 2009     

Sorption of atrazine on conventional and surface modified activated carbons

The sorption of atrazine from water has been studied using a conventional activated carbon, F400, an annealed carbon sample, F400AN, and an aminated carbon sample, F400NH(2). Characterisation of the carbon samples showed that sample F400NH(2) had the highest proportion of micropores, but had the lowest values of point of zero charge (PZC) and iso-electric point (IEP). This was attributed to the existence of a high proportion of oxygen containing functional groups. Sorption data showed that sample F400AN was superior in the sorption of atrazine to samples F400 and F400NH(2). It was noted that pore size distribution alone was not the only contributing factor for the uptake of atrazine onto the activated carbons. The sorption data were fitted well using the Freundlich isotherm. The free energy change showed that sorption of atrazine on activated carbons is a spontaneous process. A pseudo-second order kinetic model was used for analysing the kinetic data, and it was concluded that adsorption of atrazine was controlled by a film diffusion mechanism.     

Kinetic models applied to erbium adsorption on activated charcoal from aqueous solutions

The adsorption of erbium (Er) ions on activated charcoal (AC) is investigated at temperatures 10–40 °C from aqueous solutions to understand the kinetics behavior. The intra-particle diffusion, the pseudo-first order kinetic and pseudo-second order kinetic models were used to describe the kinetic data. Results shows that the adsorption of Er ions on AC occurs in two stages and the surface adsorption and diffusion phenomena are operative in the adsorption process. The result also reveals that intra-particle diffusion is not only the main rate determining step through out the adsorption process, but the boundary layer diffusion also play significant role in rate determination. Values of the intra-particle diffusion rate constant and the extent of the boundary layer diffusion were calculated. A comparison of the kinetics models on the overall adsorption rate indicates that the Er/AC system is best described by the pseudo-second order kinetic model than the pseudo-first order model, and the overall rate of the Er ions adsorption on AC appears to be controlled by more than one step, i.e., external mass transfer and diffusion mechanism.     

Kinetics and modeling of (R,S)‐1‐phenylethanol acylation over lipase

The kinetics of the acylation of (R,S)‐1‐phenylethanol was investigated using lipase as a catalyst. The main parameters were temperature, reaction atmosphere, different acyl donors, and different amounts of acyl donor as well as the presence of some additives in the reaction mixture. The initial reaction rate increased with increasing temperature and with a decreasing amount of an acyl donor. The activated esters, such as isopropenyl‐ and vinyl acetate, exhibited very high acylation rates for R‐1‐phenylethanol, whereas low rates were obtained with ethyl acetate and 2‐methoxyethyl acetate. The addition of water and acetophenone decreased the acylation rate. A kinetic model was developed based on a sequential step mechanism, in which enzyme was reacting in the first step with an acyl donor followed by the reaction of a modified enzyme complex with the reactant, R‐1‐phenylethanol. Comparison with experimental data obtained at different temperatures allowed simplification of this model, leading to a kinetic equation with just one apparent parameter. The influence of the amount of acyl donor, ethyl acetate, could be quantitatively described by taking into account the competitive inhibition of the ethanol produced. The rate constants and apparent activation energy for experiments performed under different temperatures and the amounts of acylation agent were determined. The apparent activation energy was 24.5 kJ/mol. © 2010 Wiley Periodicals, Inc. Int J Chem Kinet 42: 629–639, 2010     

Biomolecular Release from Alginate‐modified Electrode Triggered by Chemical Inputs Processed through a Biocatalytic Cascade – Integration of Biomolecular Computing and Actuation

Biocatalytic cascades involving more than one or two enzyme‐catalyzed step are inefficient inside alginate hydrogel prepared on an electrode surface. The problem originates from slow diffusion of intermediate products through the hydrogel from one enzyme to another. However, enzyme activity can be improved by surface immobilization. We demonstrate that a complex cascade of four consecutive biocatalytic reactions can be designed, with the enzymes immobilized in an LBL‐assembled polymeric layer at the alginate‐modified electrode surface. The product, hydrogen peroxide, then induces dissolution of iron‐cross‐linked alginate, which results in release process of entrapped biomolecular species, here fluorescently marked oligonucleotides, denoted F‐DNA. The enzymatic cascade can be viewed as a biocomputing network of concatenated AND gates, activated by combinations of four chemical input signals, which trigger the release of F‐DNA. The reactions, and diffusion/release processes were investigated by means of theoretical modeling. A bottleneck reaction step associated with one of the enzymes was observed. The developed system provides a model for biochemical actuation triggered by a biocomputing network of reactions.     

The Mechanism of Nonenzymatic Template Copying with Imidazole‐Activated Nucleotides

The emergence of the replication of RNA oligonucleotides was a critical step in the origin of life. An important model for the study of nonenzymatic template copying, which would be a key part of any such pathway, involves the reaction of ribonucleoside‐5′‐phosphorimidazolides with an RNA primer/template complex. The mechanism by which the primer becomes extended by one nucleotide was assumed to be a classical in‐line nucleophilic‐substitution reaction in which the 3′‐hydroxyl of the primer attacks the phosphate of the incoming activated monomer with displacement of the imidazole leaving group. Surprisingly, this simple model has turned out to be incorrect, and the dominant pathway has now been shown to involve the reaction of two activated nucleotides with each other to form a 5′–5′‐imidazolium bridged dinucleotide intermediate. Here we review the discovery of this unexpected intermediate, and the chemical, kinetic, and structural evidence for its role in template copying chemistry.     

Additive perturbed molecular assembly in two-dimensional crystals: differentiating kinetic and thermodynamic pathways

During attempts to produce novel two-dimensional cocrystals by coadsorbing components in a binary mixture, the formation of a metastable form was observed in analogy to the phenomenon of additive-induced polymorph formation reported in three-dimensional crystallization. Mechanistic insights into this phenomenon were gained through the use of scanning tunneling microscopy and several adsorbate/additive combinations. One additive plays a critical role in forming a disordered assembly through a process that is primarily kinetic whereas another additive thermodynamically stabilized an intermediate form, resulting in interrupting a phase transformation to a more stable form. These additive effects elucidate one of the potential pathways to kinetically isolate a metastable polymorph formed during cocrystallization in three-dimensional crystallization.     

Nickel-Impregnated Silica Nanoparticle Synthesis and Their Evaluation for Biocatalyst Immobilization

In the present investigation, impact of nickel-impregnated silica paramagnetic particles (NSP) as biocatalyst immobilization matrices was investigated. These nanoparticles were synthesized by sol–gel route using a nonionic surfactant block co polymer [poly (ethylene glycol)-block-poly-(propylene glycol)-block-poly (ethylene glycol)]. Diastase enzyme was immobilized on these particles (enzyme-impregnated NSP) as model enzyme and characterized using Fourier-transform infrared spectroscopy and X-ray crystallography. Analysis of enzyme-binding nature with these nanoparticles at different physiological conditions revealed that binding pattern and activity profile varied with the pH of the reaction mixture. The immobilized enzyme was further characterized for its biocatalytic activity with respect to kinetic properties such as Km and Vmax and compared with free enzyme. Paramagnetic nanoparticle-immobilized enzyme showed more affinity for substrate compared to free one. The nature of silica and nickel varied from amorphous to crystalline nature and vice versa upon immobilization of enzyme. To the best of our knowledge, this is the first report of its kind for change of nature from one form to other under normal temperatures upon diastase interaction with NSP.     

Immobilization of Lecitase? Ultra onto a Novel Polystyrene DA-201 Resin: Characterization and Biochemical Properties

A simple, rapid, and economic method of enzyme immobilization was developed for phospholipase Lecitase? ultra (LU) via interfacial adsorption. The effect of nature of the polystyrene supports and the kinetic behavior and stability of immobilized lecitase? ultra (IM-LU) were evaluated. Six macroporous resins (AB-8, X-5, DA-201, NKA-9, D101, D4006) and two anion resins (D318 and D201) were studied as the supports. DA-201 resin was selected because of its best immobilization effect for LU. Immobilization conditions were investigated, including immobilization time, pH, and enzyme concentration. IM-LU with a lipase activity of 1,652.4?±?8.6?U/g was obtained. The adsorption process was modeled by Langmuir and Freundlich equations, and the experimental data were better fit for the former one. The kinetic constant (K _m) values were found to be 192.7?±?2.2?mM for the free LU and 249.3?±?5.4?mM for the IM-LU, respectively. The V _max value of free LU (169.5?±?4.3?mM/min) was higher than that of the IM-LU (53.8?±?1.5?mM/min). Combined strategies of scanning electron micrograph, thermogravimetric analysis, and Fourier transform infrared (FTIR) spectroscopy were employed to characterize the IM-LU. FTIR spectroscopy showed that the secondary conformation of the enzyme had changed after immobilization, through which a decrease of ??-helix content and an increase of ??-sheet content were observed. The IM-LU possessed an improved thermal stability as well as metal ionic tolerance when compared with its free form. The reusability of IM-LU was also evaluated through catalyzing esterification reaction between oleic acid and glycerol. It exhibited approximately 70?% of relative esterification efficiency after six successive cycles. This immobilized enzyme on hydrophobic support may well be used for the synthesis of structural lipids in lipid area.     

(S)-selective kinetic resolution and chemoenzymatic dynamic kinetic resolution of secondary alcohols

(S)-Selective kinetic resolution was achieved through the use of a commercially available protease, which was activated with a combination of two different surfactants. The kinetic resolution (KR) process was optimized with respect to activation of the protease and to the acyl donor. The KR proved to be compatible with a range of functionalized sec-alcohols, giving good to high enantiomeric ratio values (up to >200). The enzymatic resolution was combined with a ruthenium-catalyzed racemization to give an (S)-selective dynamic kinetic resolution (DKR) of sec-alcohols. The DKR process works under very mild reaction conditions to give the corresponding esters in high yields and with excellent enantioselectivities.     

One electron transfer mechanism in the enzymatic oxygenation of sulfoxide to sulfone promoted by a reconstituted system with purified cytochrome p-450

A kinetic study on enzymatic S-oxygenation of sulfoxides to sulfones was carried out by a reconstited system with purified cytochrome P-450. A linear correlation observed between log(Vmax)'s and the one-electron oxidation potentials of sulfoxides suggests that the oxygenation of sulfoxides proceeds via one electron transfer process to the active “oxenoid” intermediate of the enzyme.     

EPDM accelerated sulfur vulcanization: a kinetic model based on a genetic algorithm

A simple closed form equation for the prediction of crosslinking of EPDM during accelerated sulfur vulcanization is presented. Such a closed form solution is derived from a second order non homogeneous differential equation, deduced from a kinetic model. The kinetic model is based on the assumption that, during vulcanization, a number of partial reactions occurs, both in series and in parallel, which determine the formation of intermediate compounds, including activated and matured polymer. Once written standard first order differential equations for each partial reaction, the differential equation system so obtained is rearranged and, after few considerations, a single second order non homogeneous differential equation with constant coefficients is derived, for which a solution may be found in closed form, provided that the non-homogeneous term is approximated with an exponential function. To estimate numerically the degree of crosslinking, kinetic model constants are evaluated through a simple data fitting, performed on experimental rheometer cure curves. The fitting procedure is a new one, and is achieved using an ad-hoc genetic algorithm, provided that a few points, strictly necessary to estimate model unknown constants with sufficient accuracy, are selected from the whole experimental cure curve. To assess the results obtained with the model proposed, a number of different compounds are analyzed, for which experimental or numerical data are available from the literature. The important cases of moderate and strong reversions are also considered, experiencing a convincing convergence of the analytical model proposed. For the single cases analyzed, partial reaction kinetic constants are also provided.     

固定化葡萄糖氧化酶活性的X射线微区分析

利用X射线微区分析方法,对固定化活性葡萄糖氧化酶进行了定位分析;葡萄糖作为底物,FeSO4和KI作为捕捉剂,底物经固定化葡萄糖氧化酶催化产生H2O2,后者和捕捉剂反应生成沉淀,可以确定固定化葡萄糖氧化酶的催化活性部位。结果表明：颗粒越小,酶活越高,活性葡萄糖氧化酶在凝胶内分布均匀,且绝大多数葡萄糖氧化酶固定在凝胶的内部。作者还研究了固定化活性葡萄糖氧化酶定位的最佳条件。