Ionic liquid-coated enzyme for biocatalysis in organic solvent

Ionic liquid-coated enzyme (ILCE) is described as a useful catalyst for biocatalysis in organic solvent. An ionic liquid, [PPMIM]-[PF(6)] (1, [PPMIM] = 1-(3'-phenylpropyl)-3-methylimidazolium), which is solid at room temperature and becomes liquid above 53 degrees C, was synthesized in two steps from N-methylimidazole. The coating of enzyme was done by simply mixing commercially available enzyme with 1 in the liquid phase above 53 degrees C and then allowing the mixture to cool. A representative ILCE, prepared with a lipase from Pseudomonas cepacia, showed markedly enhanced enantioselectivity without losing any significant activity.     

Activity of an enzyme immobilized on polyelectrolyte multilayers

The immobilization of α-chymotrypsin on the surface of boron silicate glass microspheres is conducted via the technique of multilayer adsorption of polyelectrolytes. It is shown that the enzyme is adsorbed on both positively and negatively charged surfaces and its activity is partially preserved relative to that in solution. The activity of the enzyme depends on the number of polyectrolyte layers preliminarily adsorbed on glass microspheres and on the charge of the surface. The activity of α-chymotrypsin adsorbed on the negatively charged surface is four times higher than the activity of this enzyme adsorbed on a positively charged surface.     

Site-specific functionalization on individual colloids: size control, stability, and multilayers

Individual colloidal particles are locally functionalized with nanoscale control. Here we use the particle lithography technique to mask one region of a silica or polystyrene particle (size 3.0 mum down to 170 nm), while the remaining 95% or more of the particle is coated with various sized nanocolloids. The images and data show precise and predictable control over the size of the region, with fine-tuned patch size control obtainable by changing the ionic strength of the solution. The coating on the particles remains stable even when subjected to sonication for 5 min. Both single regions and multilayer annuluses are readily formed. Particle lithography provides a general, reliable, stable, controllable, and scalable method for placing site-specific functionalizations on individual particles, opening the way to more complex particle patterning and the bottom-up assembly of more complex structures.     

Recent advances in biocatalysis by directed enzyme evolution

Naturally occurring enzymes are remarkable biocatalysts with numerous potential applications in industry and medicine. However, many of their catalyst properties often need to be further tailored to meet the specific requirements of a given application. Within this context, directed evolution has emerged over the past decade as a powerful tool for engineering enzymes with new or improved functions. This review summarizes recent advances in applying directed evolution approaches to alter various enzyme properties such as activity, selectivity (enantio- and regio-), substrate specificity, stability, and solubility. Special attention will be paid to the creation of novel enzyme activities and products by directed evolution.     

Preparation of organized protein multilayers

A novel technique of preparation of multilayer assemblies consisting of a controlled number of molecular layers of various proteins arranged according to a predetermined architecture is described. The multilayers were formed by consecutive adsorption of polyanions and positively charged proteins below their isoelectric point and covalently crosslinked via protein amino groups. Polyanions were washed out from the crosslinked protein assembly by rinsing with buffer in which the net charge of the proteins was inverted.     

酶复合催化剂原位合成新方法及生物催化应用

工业生物催化面临两大重要挑战,一是可工业应用的酶催化反应类型仍然比较有限,远少于化学催化剂,因此需要拓展酶催化的反应类型;二是酶在苛刻的工业催化反应条件下尤其是在高温、有机溶剂、不适宜的pH等环境下稳定性较差,因此需要提高工业酶催化剂的稳定性.研究者已经开发了很多方法,以解决这两方面难题,例如酶的定向进化、定点突变、酶的计算机从头设计和构建人工金属酶等.本文系统介绍了本课题组开发的酶复合催化剂原位合成方法及其生物催化应用,期望为解决工业生物催化的上述挑战提供新思路.原位合成是构建酶-无机晶体复合催化剂的一种简便、高效、普适的方法.酶-无机晶体复合物中,限域包埋使酶具有高于常规固定化酶的催化活性和稳定性.该方法可以简便拓展至其它多种类型的无机晶体材料,显著提高酶的稳定性.无机晶体的限域包埋对酶分子结构和性能有着重要影响,通过理性设计复合催化剂的结构,可实现对酶的活性、稳定性以及多酶反应级联效率的有效调控.本课题组采用分子模拟和实验相结合的方法阐释了多酶-无机晶体复合催化剂所驱动的级联反应效率提高的关键因素.通过调控原位合成中金属离子和有机配体的浓度,实现了酶分子在缺陷型甚至无定形载体中的包埋.在此基础上,深入探讨了缺陷对酶分子结构和催化活性的调控机制,为酶复合催化剂的理性设计提供了依据.同样基于原位合成方法,本课题组构建了酶-金属团簇复合催化剂,实现了温和条件下酶催化和金属催化的高效耦合和协同.以脂肪酶-钯团簇复合催化剂为例,阐明了酶-金属团簇复合催化剂中二者相互作用对酶分子结构和活性以及金属催化活性的影响机制,为酶催化和金属催化相融合的研究提供了重要基础.我们对这一领域存在的挑战和未来重要的研究方向也进行了讨论,希望本文可以从催化剂工程角度为高效酶催化剂的设计以及生物催化应用领域的拓展提供新思路,推动该领域发展.     

Engineering proteinaceous colloidosomes as enzyme carriers for efficient and recyclable Pickering interfacial biocatalysis

Despite Pickering interfacial biocatalysis being a popular topic in biphasic biocatalysis, the development of water-in-oil (w/o) emulsion systems stabilized by single particles remains a challenge. For the first time, hydrophobized proteinaceous colloidosomes with magnetic-responsiveness are developed to function as both an enzyme carrier and emulsifier, achieving a breakthrough in protein-based w/o Pickering bioconversion. Enzyme-loaded protein colloidosomes are synthesized by a facile and mild method via emulsion templating. This system exhibits superior catalytic activity to other systems at the oil–water interface. Besides, feasible enzyme recovery and reusability ensure that this novel system can be employed as an efficient and eco-friendly recyclable platform.

Engineering proteinaceous colloidosomes with magnetic-responsiveness are designed as both enzyme carrier and emulsifier, achieving a breakthrough in protein-based w/o Pickering interfacial biocatalysis.     

Preparation of highly sulfonated polystyrene model colloids

Previous attempts to prepare monodisperse styrene/sodium styrene sulfonate copolymer latexes by batch, seeded, and semicontinuous emulsion polymerization were unsuccessful at high concentrations of the functional comonomer. Broad, and sometimes bimodal, size distributions, and large amounts of water soluble homopolymer were obtained. After removal of free monomer, solute and adsorbed homopolymer and copolymer, the overall incorporation of the functional comonomer was found to be low. To overcome these problems, a two stage “shot-growth” or in situ seeding technique was developed. A first stage copolymerization was carried out with a low concentration of sodium styrene sulfonate: the purpose of the functional comonomer was to enhance the stability and regulate the size of the seed particles. When this reaction had reached high conversion (> 90%), a second stage monomer mixture was added. The ratio of styrene to sodium styrene sulfonate in this mixture determined the final surface charge density. The mechanism by which the NaSS is incorporated in the polymer particles is considered to be by solution copolymerization with solute styrene monomer to form surface active oligoradicals. These radicals adsorb on the particle surface, initiate polymerization and become inextricably bound, preventing their transfer back to the aqueous phase. By this means, it was possible to vary independently the particle size and surface charge density. High concentrations of functional comonomer could be polymerized without undue wastage (incorporations were only slightly less than 100%) or loss of monodispersity. In extreme cases, the area per functional group fell below the theoretical minimum, indicating considerable hydration of the surface layers.     

Preparation and characterization of polymer composite multilayers on SiO2

Poly(diallyldimethylammonium chloride) (PDADMAC) and poly(sodium 4-styrenesulfonate) (PSS) have been consecutively adsorbed onto 1.5-microm charged silica (SiO2) particles. Time-dependent adsorption studies indicate that, due to the strong ionic charge of the dissociated polycation in water, adsorption is complete in less than 30 min. Indications of the maximum adsorption density, changes in surface charge, and stability of the layered particles are demonstrated through adsorption isotherms and electrophoretic mobility (EPM) measurements. Further stability of the PDADMAC layer is demonstrated through multiwashing with ultra pure deionized water. Preliminary desorption studies of the PSS layer also illustrate a stabilized two-layer system. Due to the nature of the electrostatic charges on the surface of the SiO2 core particles and both polyelectrolytes in aqueous media, the use of polyelectrolytes as layering elements serves as a model for the assembly of time-released drug delivery particle systems.     

Preparation and catalytic properties of NR-stabilized palladium colloids

Palladium colloids revealing narrow particle size distributions can be obtained by chemical reduction using tetra–alkylammonium hydrotriorganoborates. Combining the stabilizing agent [NR] with the reducing agent [BEt₃H^?] provides a high concentration of the protecting group at the reduction centre. Alternatively, NR₄X (X = halogen) may be coupled to the metal salt prior to the reduction step: addition of N(octyl)₄Br to Pd(ac)₂ in THF, for example, evokes an active interaction between the stabilizing agent and the metal salt. Reduction of NR-stabilized palladium salts with simple reducing agents such as hydrogen at room temperature yields stable palladium organosols which may be isolated in the form of redispersible powders. The anion of the palladium salt is crucial for the success of the colloid synthesis. Electron microscopy shows that the mean particle size ranges between 1.8 and 4.0 nm. An X–ray–photoelectron spectrscopic examination demonstrated the presence of zerovalent palladium. These palladium colloids may serve as both homogeneous and heterogeneous hydrogenation catalysts. Adsorption of the colloids onto industrially important supports can be achieved without agglomeration of palladium particles. The standard activity of a charcoal catalyst containing 5% of colloidal palladium determined through the cinnamic acid standard test was found to exceed considerably the activity of the conventional technical catalysts. In addition, the lifespan of the catalyst containing a palladium colloid, isolated from the reduction of [N(octyl)₄]₂PdCl₂Br₂ with hydrogen, is superior to conventionally prepared palladium/charcoal (Pd/C) catalysts. For example, the activity of a conventional Pd/C catalyst is completely suppressed after 38×10³ catalytic cycles per Pd atom, whereas the colloidal Pd/C catalyst shows activity even after 96times;10³ catalytic cycles.     

Preparation and characterization of aqueous colloids of Pt-Ru nanoparticles

A synthetic method for platinum-ruthenium (PtRu) nanoparticles in aqueous media is proposed. This method employs citric acid as a capping agent and NaBH(4) as a reducing agent with the aid of pH control. The number-averaged size of the PtRu nanoparticles was ca. 2 nm. The crystal phase and chemical composition of the nanoparticles was investigated by X-ray diffraction measurement and scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy analysis, which indicated that the nanoparticles mainly consisted of an alloy of Pt and Ru. Electrochemical measurement showed that the PtRu nanoparticles had catalytic activity for methanol oxidation.     

An enzyme library approach to biocatalysis: development of nitrilases for enantioselective production of carboxylic acid derivatives

The discovery, from Nature, of a large and diverse set of nitrilases is reported. The utility of this nitrilase library for identifying enzymes that catalyze efficient production of valuable hydroxy carboxylic acid derivatives is demonstrated. Unprecedented enantioselectivity and substrate scope are highlighted for three newly discovered and distinct nitrilases. For example, a wide array of (R)-mandelic acid derivatives and analogues were produced with high rates, yields, and enantiomeric excesses (95-99% ee). We also have found nitrilases that provide direct access to (S)-phenyllactic acid and other aryllactic acid derivatives, again with high yields and enantioselectivities. Finally, different nitrilases have been discovered that catalyze enantiotopic hydrolysis of 3-hydroxyglutaronitrile to afford either enantiomer of 4-cyano-3-hydroxybutyric acid with high enantiomeric excesses (>95% ee). The first enzymes are reported that effect this transformation to furnish the (R)-4-cyano-3-hydroxybutyric acid which is a precursor to the blockbuster drug Lipitor.     

Preparation and characterisation of polypyrrole colloids in non-aqueous media

We have prepared polypyrrole both as a bulk powder and as a sterically stabilised colloidal dispersion in a noved non-aqueous solvent, 2-merhoxyethanol. The steric stabiliser utilised in the latter system was a poly(vinyl acetate) homopolymer. The electroactivity, morphology, thermal stability and chemical composition of these materials were characterised by d.c. conductivity measurememts, electron microscopy, thermogravimetry, FTIR and visible absorption spectroscopy and clemental microanalyses. The mass ratio of poly(vinyl acetate) to polypyrrole in the colloidal particles was determined indirectly by spectroscopic assay of the post-reaction supernatant solution for non-adsorbed stabiliser.     

Preparation of magnetic polymer colloids with Brownian magnetic relaxation

Magnetic polymer colloids (MPCs) consisting of CoFe₂O₄ nanoparticles (NPs) embedded in a poly(methyl methacrylate) (PMMA) matrix were synthesized by magnetic miniemulsion polymerization. CoFe₂O₄ NPs were modified with 3-trimethoxysilylpropylmethacrylate and directly emulsified with different concentrations of sodium dodecyl sulfate under ultrasonication for subsequent miniemulsion polymerization. The average diameter of the CoFe₂O₄/PMMA spheres (about 200 nm) was controlled by varying the amount of surfactant. Thermogravimetric analysis indicated that the magnetic content was in the range of 44 to 73 %. Magnetic properties of the dispersions were investigated by measuring equilibrium magnetization curves and the dynamic magnetic susceptibility as a function of frequency. The MPCs were found to follow the Debye model for the dynamic magnetic susceptibility, with a characteristic time given by the rotational hydrodynamic resistance and thermal energy through the Stokes-Einstein relation. This demonstrates that the MPCs respond to applied magnetic fields by rotating. Due to their uniform size and high magnetic loading, these colloids may be suitable in a variety of applications, including nanoscale mechanical probes and actuators in complex fluids and biological systems.     

Interfacial biocatalysis on charged and immobilized substrates: the roles of enzyme and substrate surface charge

An enzyme charge ladder was used to examine the role of electrostatic interactions involved in biocatalysis at the solid-liquid interface. The reactive substrate consisted of an immobilized bovine serum albumin (BSA) multilayer prepared using a layer-by-layer technique. The zeta potential of the BSA substrate and each enzyme variant was measured to determine the absolute charge in solution. Enzyme adsorption and the rate of substrate surface hydrolysis were monitored for the enzyme charge ladder series to provide information regarding the strength of the enzyme-substrate interaction and the rate of interfacial biocatalysis. First, each variant of the charge ladder was examined at pH 8 for various solution ionic strengths. We found that for positively charged variants the adsorption increased with the magnitude of the charge until the surface became saturated. For higher ionic strength solutions, a greater positive enzyme charge was required to induce adsorption. Interestingly, the maximum catalytic rate was not achieved at enzyme saturation but at an invariable intermediate level of adsorption for each ionic strength value. Furthermore, the maximum achievable reaction rate for the charge ladder was larger for higher ionic strength values. We propose that diffusion plays an important role in interfacial biocatalysis, and for strong enzyme-substrate interaction, the rate of diffusion is reduced, leading to a decrease in the overall reaction rate. We investigated the effect of substrate charge by varying the solution pH from 6.1 to 8.7 and by examining multiple ionic strength values for each pH. The same intermediate level of adsorption was found to maximize the overall reaction rate. However, the ionic strength response of the maximum achievable rate was clearly dependent on the pH of the experiment. We propose that this observation is not a direct effect of pH but is caused by the change in substrate surface charge induced by changing the pH. To prove this hypothesis, BSA substrates were chemically modified to reduce the magnitude of the negative charge at pH 8. Chemical modification was accomplished by the amidation of aspartic and glutamic acids to asparagine and glutamine. The ionic strength response of the chemically modified substrate was considerably different than that for the native BSA substrate at an identical pH, consistent with the trend based on substrate surface charge. Consequently, for substrates with a low net surface charge, the maximum achievable catalytic rate of the charge ladder was relatively independent of the solution ionic strength over the range examined; however, at high net substrate surface charge, the maximum rate showed a considerable ionic strength dependence.     

Preparation of ZnO colloids by aggregation of the nanocrystal subunits

Colloidal ZnO particles with narrow size distribution were prepared via a sol-gel process by base-catalyzed hydrolysis of zinc acetate. The morphology of ordered arrays of the particles was recorded by SEM. SEM also reveals that these uniform particles were composed of tiny ZnO subunits (singlets) sized of several nanometers. The size of the singlets, which is confirmed by X-ray diffraction and UV-vis absorption spectra, increases as the aging time is prolonged. The size-selective formation of colloids by aggregation of nanosized subunits is proposed to consist of two-stage growth by nucleation of nanosized crystalline primary particles and their subsequent aggregation into polycrystalline secondary colloids. The aggregates are all spherical because the internal rearrangement processes are fast enough. The ZnO colloids, i.e., the aggregates, tend to self-assemble into well-ordered hexagonal close-packed structures. Room-temperature photoluminescence was characterized for green and aged ZnO.     

Preparation of monodisperse, fluorescent PMMA-latex colloids by dispersion polymerization

We report a single step procedure to prepare monodisperse colloidal poly(methyl)methacrylate (PMMA) particles where fluorescent dyes are incorporated into the polymer network. The particles are sterically stabilized against flocculation by poly(12-hydroxystearic acid). The fluorescent dyes are RITC (rhodamine isothiocyanate)-aminostyrene (RAS) and 4-methylaminoethylmethacrylate-7-nitrobenzo-2-oxa-1,3-diazol (NBD-MAEM), which are prepared from (commercially available) RITC and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), respectively, as starting materials. The particles can be grown larger with nonfluorescent PMMA. Examples of the usefulness of these model particles in colloid science are given.     

Preparation and optical properties of silica@Ag-Cu alloy core-shell composite colloids

The silica@Ag-Cu alloy core-shell composite colloids have been successfully synthesized by an electroless plating approach to explore the possibility of modifying the plasmon resonance at the nanoshell surface by varying the metal nanoshell composition for the first time. The surface plasmon resonance of the composite colloids increases in intensity and shifts towards longer, then shorter wavelengths as the Cu/Ag ratio in the alloy shell is increased. The variations in intensity of the surface plasmon resonance with the Cu/Ag ratio obviously affect the Raman bands of the silica colloid core. The report here may supply a new technique to effectively modify the surface plasmon resonance.     

Smart nanotubes for bioseparations and biocatalysis

Tube-shaped nanostructures (nanotubes) have a number of attributes that make them potentially useful for biomedical applications such as drug delivery/detoxification and enzyme immobilization. Template synthesis provides a route for preparing monodisperse nanotubes of nearly any size and composed of nearly any material. We show here that template-synthesized silica nanotubes can be biochemically functionalized such that they act as biocatalysts and highly selective nano-phase extraction agents for bioseparations. For example, nanotubes containing an enantioselective antibody selectively extract the enantiomer of a drug molecule that binds to the antibody, relative to the enantiomer that has no specific interaction with the antibody. Nanotubes containing the enzyme glucose oxidase function as nanophase bioreactors to catalyze the oxidation of glucose.