离子液体修饰超顺磁性纳米颗粒用于青霉素G酰化酶固定化(英文)

Functionalized ionic liquids containing ethyoxyl groups were synthesized and immobilized on magnetic silica nanoparticles(MSNP) prepared by two steps,i.e.,Fe3O4 synthesis and silica shell growth on the surface.This magnetic nanoparticle supported ionic liquid(MNP-IL) were applied in the immobilization of penicillin G acylase(PGA).The MSNPs and MNP-ILs were characterized by the means of Fourier transform infrared spectroscopy(FTIR),scanning electron microscopy(SEM),transmission electron microscopy(TEM),and vibrating sample magnetometer(VSM).The results showed that the average size of magnetic Fe3O4 nanoparticles and MSNPs were ~10 and ~90 nm,respectively.The saturation magnetizations of magnetic Fe3O4 nanoparticles and MNP-ILs were 63.7 and 26.9 A?m2?kg?1,respectively.The MNP-IL was successfully applied in the immobilization of PGA.The maximum amount of loaded enzyme was about 209 mg?g?1(based on carrier),and the highest enzyme activity of immobilized PGA(based on ImPGA) was 261 U?g?1.Both the amount of loaded enzyme and the activity of ImPGA are at the same level of or higher than that in previous reports.After 10 consecutive operations,ImPGA still main-tained 62% of its initial activity,indicating the good recovery property of ImPGA activity.The ionic liquid modified magnetic particles integrate the magnetic properties of Fe3O4 and the structure-tunable properties of ionic liquids,and have extensive potential uses in protein immobilization and magnetic bioseparation.This work may open up a novel strategy to immobilize proteins by ionic liquids.     

Expansion of Access Tunnels and Active‐Site Cavities Influence Activity of Haloalkane Dehalogenases in Organic Cosolvents

The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein–solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady‐state kinetics assays, molecular dynamics simulations, and time‐resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme–solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K_0.5 and increasing k_cat, or caused enzyme inactivation by promoting substrate inhibition and decreasing k_cat. The induced activation and inhibition of the enzymes correlated with expansion of the active‐site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active‐sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein–solvent pair and effective cosolvent concentration.     

Stabilization of α‐amylase by chemical modification with carboxymethylcellulose

Carboxymethylcellulose activated by periodate oxidation was covalently linked to porcine pancreatic α‐amylase (EC 3.2.1.1). The specific activity of the conjugate prepared was 54% of the native enzyme. The carbohydrate content was estimated to be 62% by weight as a result of the modification of 67% of the amino groups from the protein. In comparison with the native enzyme, the thermostability and pH stability were improved for α‐amylase by this modification. The conjugate was also more resistant to the action of denaturant agents such as urea and sodium dodecylsulfate. We conclude that modification of enzymes by the anionic polysaccharide carboxymethylcellulose might be a useful method for improving enzyme stability under various denaturing conditions. © 1999 Society of Chemical Industry     

Recrystallization of Er3+:CaF2 in Transparent Fluorophosphate Glass‐Ceramics with the Co‐Firing Method

Transparent glass‐ceramics containing Er³⁺:CaF₂ crystallites were prepared with the co‐firing method. The formation process of the glass‐ceramics was investigated by means of SEM, XRD, and DSC. The results reveal that the Er³⁺:CaF₂ nanocrystals do not dissolve into the fluorophosphates (FP) glassmelt until the co‐firing temperature increase higher than about 920°C. Below this temperature, Er³⁺:CaF₂ survives the co‐firing process and the nanocrystals just grow to spherical crystals of micrometers in size. Co‐firing temperature higher than this temperature leads to the dissolution of Er³⁺:CaF₂ and the dissolved Er³⁺:CaF₂ recrystallized during quenching process and takes the shape of dendrite.     

Asymmetric Bio‐amination of Ketones in Organic Solvents

ω‐Transaminases, employed as a lyophilised crude cell‐free extract, were successfully employed in organic solvent for the asymmetric amination of ketones without the need for immobilisation. Best activity was found for methyl tert‐butyl ether (MTBE) at a water activity of 0.6. The ω‐transaminases (9 different enzymes) accepted efficiently 2‐propylamine as amine donor when used in the solvent, which is not the case when they are used in aqueous solution. The bio‐amination in organic solvent showed several advantages such as higher reaction rates (up to 17‐fold), general acceptance of 2‐propylamine as amine donor, simple work‐up procedure (i.e., no basification and extraction required), easy recycling of the catalyst and lack of substrate inhibition. The biocatalysts maintained their excellent stereoselectivity in MTBE allowing the preparation of optically pure amines (ee >99%) with up to >99% conversion.     

Single‐Molecule Kinetics of an Enzyme in the Presence of Multiple Substrates

Herein, the catalytic activity of a single enzyme in the presence of multiple substrates is studied. Three different mechanisms of bisubstrate binding, namely, ordered sequential, random sequential and ping‐pong nonsequential pathway, are broadly discussed. By means of the chemical master equation approach, exact expressions for the waiting‐time distributions, the mean turnover time and the randomness parameter as a function of the substrate concentration, such that both concentrations are fixed, but one of them is changed quasi‐statically are obtained. The randomness parameter is not equal to unity at intermediate to high substrate concentrations, which indicates the presence of multiple rate‐limiting steps in the reaction pathway in all three modes of bisubstrate binding. This arises due to transitions between the free enzyme and the enzyme–substrate complexes that occur on comparable timescales. Such turnover statistics of the single enzyme can also distinguish between the different types of bisubstrate binding mechanisms.     

3,5‐Dimethylpyrazolium Fluorochromate(VI)‐Catalysed Oxidation of Organic Substrates by Hydrogen Peroxide under Solvent‐Free Conditions

3,5‐Dimethylpyrazolium fluorochromate (VI), C₅H₈N₂H[CrO₃F], DmpzHFC , serves as an efficient catalyst for oxidation of primary, secondary and allylic alcohols to the corresponding carbonyl compounds using H₂O₂ at room temperature under solvent‐free conditions. Oxidation of methyl phenyl sulfides and triphenylphosphine were also carried out successfully.     

Testing the Null of Co‐integration in the Presence of Variance Breaks

Abstract. We show that changes in the innovation covariance matrix of a vector of series can generate spurious rejections of the null hypothesis of co‐integration when applying standard residual‐based co‐integration tests. A bootstrap solution to the inference problem is suggested which is shown to perform well in practice, redressing the size problems associated with the standard test but not losing power relative to the standard test under the alternative.     

Purification and Characterization of a Lipase with High Thermostability and Polar Organic Solvent‐Tolerance from Aspergillus niger AN0512

An extracellular lipase (EC 3.1.1.3, AN0512Lip) from Aspergillus niger AN0512 was purified and its characteristics were investigated. After the process of ammonium sulfate precipitation followed by ion‐exchange chromatography and gel filtration, the purified lipase was achieved with 203.6‐fold purification and 22.1 % recovery. AN0512Lip exhibited the highest activity at 50 °C and pH 5.0. It was thermostable and pH‐stable, as indicated by that more than 50 % activity retained at 60 °C for 20 h and more than 90 % activity retained at pH 3.0 for 20 h, respectively. AN0512Lip activity was stimulated by some divalent metal ions (especially Cu²⁺, Ca²⁺), while greatly suppressed by EDTA, indicating that AN0512Lip was a metal‐activated enzyme. Moreover, AN0512Lip exhibited high tolerance for various polar organic solvents with log P < 0.8, and the highest lipase activity (476 % of its original activity) was achieved after addition of 90 % (V/V) isopropanol to the reaction mixture. AN0512Lip also displayed 3‐regiospecificity and great affinity for the long‐chain fatty ester. The preliminary test showed that AN0512Lip was a candidate for enriching EPA and DHA in fish oil. All the unique properties, such as thermostability, Cu²⁺‐dependent, 3‐regiospecificity, and polar organic solvent‐tolerance, indicated that AN0512Lip could have potential applications in the food industry, even in organic synthesis and the pharmaceutical industry.     

One‐Step C‐Terminal Deprotection and Activation of Peptides with Peptide Amidase from Stenotrophomonas maltophilia in Neat Organic Solvent

Chemoenzymatic peptide synthesis is a rapidly developing technology for cost effective peptide production on a large scale. As an alternative to the traditional C→N strategy, which employs expensive N‐protected building blocks in each step, we have investigated an N→C extension route that is based on activation of a peptide C‐terminal amide protecting group to the corresponding methyl ester. We found that this conversion is efficiently catalysed by Stenotrophomonas maltophilia peptide amidase in neat organic media. The system excludes the possibility of internal peptide cleavage as the enzyme lacks intrinsic protease activity. The produced peptide methyl ester was used for peptide chain extension in a kinetically controlled reaction by a thermostable protease.

     

Polyester Preparation in the Presence of Pristine and Phosphonic‐Acid‐Modified Zirconia Nanopowders

A novel zirconia polyester nanocomposite is prepared using an in situ approach. Surface‐functionalized zirconia nanoparticles are obtained by attaching 3‐phosphonopropionic acid to the metal oxide. Neat and surface‐covered metal oxide particles are incorporated at the beginning of the polyesterification reaction of isophthalic acid and neopentyl glycol resulting in zirconia/poly(neopentyl isophthalate) (PNI) nanocomposites. TEM shows that the dispersibility of the inorganic filler is improved by covering the zirconia surface with carboxylic acid groups. These results are verified by SAXS. Rheological measurements reveal that the viscosities are increasing compared to pristine PNI at particle loads of 10 wt% (neat zirconia) and 5 wt% (phosphonic‐acid‐capped zirconia), respectively.

     

Resolution of N‐hydroxymethyl vince lactam catalyzed by lipase in organic solvent

BACKGROUND: The enantiomers of N‐hydroxymethyl vince lactam are important intermediates during the synthesis of chiral drugs. The preparation of its single enantiomer can be performed through enzymatic resolution. The aim of this work is to obtain (1S, 4R)‐N‐hydroxymethyl vince lactam with high enantiomeric purity via lipase‐catalyzed enantioselective transesterification in organic solvents. To achieve this, effects of various reaction conditions (including lipase sources, acyl donor, substrate molar ratio, organic solvent, temperature, and water activity) on the enzyme activity as well as enantioselectivity were investigated. RESULTS: The results of the study showed that the enantiopreference for all the selected enzymes was (4S, 1R)‐N‐hydroxymethyl vince lactam in enantioselective transesterification of racemic N‐hydroxymethyl vince lactam. Under the selected optimum conditions, the highest enantioselectivity (E = 33.8) was obtained with a higher enzyme activity (20.3 µmol g^?1 min^?1) for Mucor miehei lipase (MML) when vinyl valerate was used as the acyl donor. Besides, the remained (1S, 4R)‐N‐hydroxymethyl vince lactam with high enantiomeric purity (ee > 99%) was obtained when the conversion was about 60%. CONCLUSION: The results obtained clearly demonstrated potential for industrial application of lipase in resolution of N‐hydroxymethyl vince lactam through enantioselective transesterification. © 2012 Society of Chemical Industry     

有机溶剂原位萃取法提高悬浮培养中国红豆杉细胞紫杉醇产量

The production of paclitaxel from suspension culture of Taxus chinensis var,mairei was improved by in situ extraction with organic solvents to avoid feedback repression and product degradation.Oleic acid and dibutyl phthalate were proved to be suitable solvents .The optimal volumetric percentage of organic solvents in the culture medium was found to be around 8%,and the favorable time for their introduction was at the exponential phase of cell growth,Paclitaxel production with the in situ extraction was ca 3-fold of that without extraction.     

Synergistic Silver Extraction from Hydrochloric Acid Solutions with Triisobutylphosphine Sulfide in the Presence of Organic Proton-Donor Additives

Silver chloride extraction from hydrochloric acid solutions with triisobutylphosphine sulfide (CYANEX 471, L) in the presence of organic proton-donor additives (HR) was studied. Both alkyl-, bromo-, nitro-substituted phenols, and organic acids of various structures were used as additives. A new synergistic effect (S) was found to take place in the presence of the additives. The degree of the synergistic effect in the systems containing phenols decreases in the series 4-nitrophenol > 4-bromophenol > 4-tert-butylphenol > 2,6-dimethylphenol > 2-methyl-6-cyclohexylphenol > 2,6-bis(tert-butyl) phenol. In the systems containing organic acids, the following series is observed: dinonylnaphthalenesulfonic acid (DNNSA) > 4-tert-butylphenol > caprylic acid > di(2-ethylhexyl)phosphoric acid > bis(2,4,4-trimethylpentyl)phosphinic acid. Based on the analysis of the extraction data and the IR and NMR spectra of the extracts, it was concluded that an increase in the silver extraction with CYANEX 471 in the presence of HR is due to the formation of the compound [AgCl?2L?HR] in the organic phase. It was shown that a mixture of CYANEX 471 and 4-tert-butylphenol can be used for the highly selective extraction of silver from hydrochloric acid solutions containing metal impurities (Ni, Cu, Co, Zn, Fe(III), and Na). The mixtures of CYANEX 471 with alkylphenols or DNNSA, in which the high synergistic effects (S ≥ 10 and ≥ 50, respectively) take place, can efficiently be used for processing different industrial silver chloride solutions.     

An Amine‐, Copper‐ and Phosphine‐Free Sonogashira Coupling Reaction Catalyzed by Immobilization of Palladium in Organic–Inorganic Hybrid Materials

An immobilization of palladium in organic‐inorganic hybrid materials‐catalyzed Sonogashira coupling reaction has been described. Terminal alkynes reacted with aryl iodides and aryl bromides only in the presence of a 3‐(2‐aminoethylamino)propyl‐functionalized, silica gel‐immobilized palladium catalyst under amine‐, copper‐ and phosphine‐free reaction conditions. The reaction generates the corresponding cross‐coupling products in excellent yields. Furthermore, the silica‐supported palladium can be recovered and recycled by a simple filtration of the reaction solution and used for 30 consecutive trials without any decreases in activity.     

Orientation control of regioregular‐poly(3‐dodecylthiophene) films formed by the friction‐transfer method and the performance of organic photovoltaic devices based on these films

Control of the molecular orientation of regioregular poly(3‐alkylthiophene)s (RR‐P3ATs) improves the performance of field‐effect transistors and organic photovoltaic devices (OPVs). However, most thiophene ring planes of the RR‐P3AT molecules (except RR‐poly(3‐butylthiophene)) in films formed by the conventional spin‐coating method stand on the substrate, that is, edge‐on orientation. Orientation control of RR‐poly(3‐dodecylthiophene) (RR‐P3DDT) molecules in films formed by the friction transfer method is reported and the performance of OPVs based on friction‐transferred RR‐P3DDT films is compared to that of OPVs based on spin‐coated films. The films are investigated by polarized ultraviolet–visible light absorption spectroscopy, Fourier transform infrared spectroscopy, and grazing‐incidence X‐ray diffraction measurement. For friction‐transferred films, the RR‐P3DDT molecular chain is uniaxially aligned parallel to the substrate plane. In addition, the thiophene ring planes of the RR‐P3DDT molecules are also oriented parallel to the substrate plane, that is, face‐on orientation. The power conversion efficiency (PCE) and fill factor of the RR‐P3DDT/C₆₀ bilayer OPVs based on the friction‐transferred RR‐P3DDT films are higher than those of devices based on spin‐coated films. The PCE and photocurrent of the device based on the friction‐transferred film are larger under irradiation with polarized light parallel to the RR‐P3DDT molecular chain direction than with polarized light orthogonal to the chain direction. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40136.     

Polyamides obtained by direct polycondesation of 4‐[4‐[9‐[4‐(4‐aminophenoxy)‐3‐methyl‐phenyl] fluoren‐9‐YL]‐2‐methyl‐phenoxy]aniline with dicarboxylic acids based on a diphenyl‐silane moiety

Polyamides (PAs) containing fluorene, oxyether, and diphenyl‐silane moieties in the repeating unit were synthesized in > 85% yield by direct polycondesation between a diamine and four dicarboxylic acids. Alternatively, one PA was synthesized from an acid dichloride. The diamine 4‐[4‐[9‐[4‐(4‐aminophenoxy)‐3‐methyl‐phenyl]fluoren‐9‐yl]‐2‐methyl‐phenoxy]aniline ( 3 ) was obtained from the corresponding dinitro compound, which was synthesized by nucleophilic aromatic halogen displacement from p‐chloronitrobenzene and 9,9‐bis (4‐hydroxy‐3‐methyl‐phenyl)fluorene ( 1 ). Monomers and polymers were characterized by FTIR and ¹H, ¹³C, and ²⁹Si‐NMR spectroscopy and the results were in agreement with the proposed structures. PAs showed inherent viscosity values between 0.14 and 0.43 dL/g, indicative of low molecular weight species, probably of oligomeric nature. The glass transition temperature (T_g) values were observed in the 188–211°C range by DSC analysis. Thermal decomposition temperature (TDT_10%) values were above 400°C due to the presence of the aromatic rings in the diamine. All PAs showed good transparency in the visible region (>88% at 400 nm) due to the incorporation of the fluorene moiety. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011