Immobilization of a Bacterial Cytochrome P450 Monooxygenase System on a Solid Support

Bacterial cytochrome P450s (P450s), which catalyze regio‐ and stereoselective oxidations of hydrocarbons with high turnover rates, are attractive biocatalysts for fine chemical production. Enzyme immobilization is needed for cost‐effective industrial manufacturing. However, immobilization of P450s is difficult because electron‐transfer proteins are involved in catalysis and anchoring these can prevent them from functioning as shuttle molecules for carrying electrons. We studied a heterotrimeric protein‐mediated co‐immobilization of a bacterial P450, and its electron‐transfer protein and reductase. Fusion with subunits of a heterotrimeric Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) enabled immobilization of the three proteins on a solid support. The co‐immobilized enzymes catalyzed monooxygenation because the electron‐transfer protein fused to PCNA via a single peptide linker retained its electron‐transport function.     

Synthesis and encapsulation properties of dendronized polymer with Fréchet‐type dendrons peripherally decorated by carboxylic acid functionalization

A new carboxylic acid‐terminated dendronized polymer (denpol), constructed with linear chain attaching Fréchet‐type dendrons at each repeat unit, has been designed and synthesized through a combination of macromonomer route and hydrolysis reaction. The resulting denpol exhibits excellent solubility in aqueous solution for pH ≥ 6, and significantly the denpol also encapsulates various aromatic molecules efficiently. The results of UV–vis and fluorescence spectra indicate that hydrophobic and π‐π interactions bring into effect between the water‐insoluble organic molecules and the denpol. Moreover, the photoisomerization of azobenzene solubilized in denpol aqueous solution was investigated, which indicated that trans‐to‐cis and cis‐to‐trans conversions were first‐order reactions. The enhancement of photoisomerization property may attribute to the proper microenvironment in the denpol. Therefore, the denpol is expected to be a potential candidate as amphiphilic unimolecular container. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 4564–4574, 2008     

Immobilization of liposomes on hydrophobically modified polymer gel particles in batch mode interaction

Immobilization of liposomal phospholipids onto Sephacryl S-1000 gels that were chemically conjugated with hydrophobic alkyl moieties, octyl, dodecyl and hexadecyl, was examined in batch mode interaction. Compared with the octyl gel, the dodecyl and the hexadecyl gels were found to immobilize the three to four times more phospholipids with the less hydrophobic moieties. The encapsulation of a water-soluble marker, with other evidences, suggests that the majority of the immobilized phospholipids maintained liposomal morphology. As the lipid of the interacting liposomes, egg yolk phosphatidylcholine (eggPC), 1,2-dimyristoylphosphatidylcholine (DMPC) and a mixture of DMPC and 1,2-dimyristamido-1,2-deoxyphosphatidylcholine were examined. At 22 °C, DMPC liposomes showed higher extent of immobilization than at 37 °C but not eggPC liposomes, suggesting that the phase of liposomal membrane could have influence on the immobilization. Exchange between the immobilized liposomes and free ones was found to be small, less than 3%. The gel that had been first interacted with liposomes to apparent saturation could further immobilize the newly added liposomes. The rate of this second immobilization was similar to that of the slow adsorption process; the both could be based on the same mechanism, possibly involving rearrangement of the immobilized liposomes on the gel as proposed by Lundahl. As had been observed in the flow mode, the immobilization had preference for smaller liposomes. In application of the system in batch mode, the size distributions of the immobilized liposomes and of those left in the supernatant may differ from that of the originally added liposomes.     

Effects of an avidin-biotin binding system on chondrocyte adhesion and growth on biodegradable polymers

Cell adhesion to a scaffold is a prerequisite for tissue engineering. Many studies have been focused on enhancing cell adhesion to synthetic materials that are used for scaffold fabrication. In this study, we applied an avidin-biotin binding system to enhance chondrocyte adhesion to biodegradable polymers. Biotin molecules were conjugated to the cell membrane of chondrocytes, and mediated cell adhesion to avidin-coated surfaces. We demonstrated that immobilization of biotin molecules to chondrocyte surfaces enhanced cell adhesion to avidin-coated biodegradable polymers such as poly(L-lactic acid), poly(D,L-lactic acid), and polycaprolactone, compared to the adhesion of normal chondrocytes to the same type of biodegradable polymer. The biotinylated chondrocytes still maintained their proliferation ability. This study showed the promise of applying the avidin-biotin system in cartilage tissue engineering. [diagram in text].     

Deposition of cationic polymer micelles on planar SiO2 surfaces

The deposition of PS-PVPH+ polymer micelles from a pH 1 aqueous solution onto Si wafers has been studied using a simple dip-coating technique. It has been found that the rate of evaporation of the solvent and the rate of withdrawal have a considerable influence on the density and ordering of the adsorbed micelles. The highest density and degree of ordering (as judged by the 2D pair correlation function) is achieved when solvent evaporation dominates the deposition process, but a fairly homogeneous distribution of polymer micelles can be achieved over a distance of at least 3-4 mm by controlling the solvent evaporation rate and the rate of substrate withdrawal. We did not observe any significant effect of added KCl (up to 0.1 M) during the deposition process or soaking in 1 M KCl after deposition. The attachment of these micelles is quite robust, as they cannot be washed off in pH 1 water (with or without KCl) without significant mechanical assistance. However, we did find that the micelles are rather easily caused to dewet and partially aggregate under the influence of 65 degrees C water vapor.     

毛细管柱固定蛋白质方法的研究进展

在熔融石英毛细管中,固定化的蛋白质(酶)在手性拆分、蛋白质肽谱、药物筛选、样品预浓缩等方面具有重要应用.本文对用于毛细管中蛋白质固定的溶胶-凝胶法、物理吸附法、离子鳌合吸附法、基于脂质的蛋白固定和共价键合法进行了综述.     

Graphoepitaxial deposition of cationic polymer micelles on patterned SiO2 surfaces

We have studied the deposition of polymer micelles formed from poly(styrene)-block-poly(2-vinylpyridine) (PS-PVPH+) from room-temperature aqueous solutions at pH 1 onto a hydrophilic Si/SiO2 surface with a relief pattern 100 nm deep with variable widths. It has been found that the micelle density is substantially higher and the ordering of the micelles is improved for micelles that adsorb in the 100 nm depressions in the width range of ca. 500-5000 nm. We ascribe these effects to capillary forces that pull the aqueous solution into the canyons where the micelles can be trapped. While the ordering of the micelles can be substantial, they do not form a perfect hexagonal crystal. If the surface is chemically modified by a Au coating, the micelle-surface interaction is strengthened and the degree of ordering is diminished. These results demonstrate that a combination of graphoepitaxy and processing conditions (speed of substrate withdrawal or evaporation of solvent) can be used to make fairly ordered polymer micelle arrays over a space of (at least) several millimeters.     

Supramolecular Immobilization of Laccase on Carbon Nanotube Electrodes Functionalized with (Methylpyrenylaminomethyl)anthraquinone for Direct Electron Reduction of Oxygen

An efficient way of immobilizing and wiring a large amount of laccase on non‐covalently‐functionalized multi‐walled carbon nanotube (MWCNT) electrodes is reported. 1‐(2‐anthraquinonylaminomethyl)pyrene and 1‐[bis(2‐anthraquinonyl)aminomethyl]pyrene were synthesized and studied for their capability to non‐covalently functionalize MWCNT electrodes and immobilize and orientate laccase on the nanostructured electrodes. This led to high‐performance biocathodes for oxygen reduction by direct electron transfer with maximum current densities of (1±0.2) mA cm^?2. The performance of the resulting bioelectrodes could be doubled simply by using the bis‐anthraquinone compound. The bioelectrodes show excellent stability over weeks and can thus be envisioned in enzymatic biofuel cells.     

The Preparation and Enzyme Immobilization of Hydrophobic Polysiloxane Supports

Enzymes are versatile biocatalysts and find increasing applications in many areas. The major advantages of using enzymes in biocatalytic transformations are their chemo‐, regio‐, and stereospecificity, as well as the mild reaction conditions that can be used. However, even when an enzyme is identified as being useful for a given reaction, its application is often hampered by its lack of long‐term stability under process conditions, and also by difficulties in recovery and recycling. For ease of application and stabilization purposes, enzymes are often immobilized on solid supports. Among support matrices, hydrophobic biomaterials have been extensively used as supports for enzyme immobilization because the hydrophobic interactions not only can effectively increase the amount of enzyme immobilization, but also exhibit higher activity and retention of activity compared with hydrophilic supports. On the other hand, polysiloxane can evidently increase the amount of enzyme immobilization because of its hydrophobicity and strong affinity with enzyme. Therefore, this research details the first preparation and use of a hydrophobic polysiloxane support for enzyme immobilization in which the structural and functional characteristics of new supports have been investigated by using glucose oxidase (GOD) and a simple Fenton's assay method, and extremely interesting features were revealed. The results showed that the amount of GOD immobilization and the stability of GOD loaded, which are fundamental properties for enzyme separation and purification, can be significantly improved by adsorption. Moreover, the results indicated that hydrophobic polysiloxane supports can effectively increase the enzymatic affinity and durability of GOD, and decrease the rate of GOD desorbed.

     

2D MXenes as Perspective Immobilization Platforms for Design of Electrochemical Nanobiosensors

MXenes are a new group of 2D nanomaterials with fascinating properties including high electrical conductivity, hydrophilic nature, easily tunable structure and high surface area. This is why MXene modified interfaces are extremely promising for the preparation of sensitive electrochemical biosensors. While there are numerous reports on MXene‐based enzymatic biosensors for detection of a wide range of analytes, application of MXene for construction of affinity biosensors is in its infancy. The review article summarizes current state‐of the‐art in the field with a focus on MXene modifications needed for construction of robust and high performance MXene electrochemical biosensors.     

Kinetic analysis of semisynthetic peroxidase enzymes containing a covalent DNA-heme adduct as the cofactor

The reconstitution of apo enzymes with DNA oligonucleotide-modified heme (protoporphyrin IX) cofactors has been employed as a tool to produce artificial enzymes that can be specifically immobilized at the solid surfaces. To this end, covalent heme-DNA adducts were synthesized and subsequently used in the reconstitution of apo myoglobin (aMb) and apo horseradish peroxidase (aHRP). The reconstitution produced catalytically active enzymes that contained one or two DNA oligomers coupled to the enzyme in the close proximity to the active site. Kinetic studies of these DNA-enzyme conjugates, carried out with two substrates, ABTS and Amplex Red, showed a remarkable increase in peroxidase activity of the DNA-Mb enzymes while a decrease in enzymatic activity was observed for the DNA-HRP enzymes. All DNA-enzyme conjugates were capable of specific binding to a solid support containing complementary DNA oligomers as capture probes. Kinetic analysis of the enzymes immobilized by the DNA-directed immobilization method revealed that the enzymes remained active after hybridization to the capture oligomers. The programmable binding properties enabled by DNA hybridization make such semisynthetic enzyme conjugates useful for a broad range of applications, particularly in biocatalysis, electrochemical sensing, and as building blocks for biomaterials.     

Immobilization of glucoamylase onto novel porous polymer supports of vinylene carbonate and 2-hydroxyethyl methacrylate

Glucoamylase was immobilized onto novel porous polymer supports. The properties of immobilized glucoamylase and the relationship between the activity of immobilized enzyme and the properties of porous polymer supports were investigated. Compared with the native enzyme, the temperature profile of immobilized glucoamylase was widened, and the optimum pH was also changed. The optimum substrate concentration of immobilized glucoamylase was higher than that of native enzyme. After storage for 23 d, the immobilized glucoamylase still maintained about 84% of its initial activity, whereas the native enzyme only maintained about 58% of the initial activity. Moreover, after using repeatedly seven times, the immobilized enzyme maintained about 85% of its initial activity. Furthermore, the properties of porous polymer supports had an effect on the activity of the immobilized glucoamylase.     

Immobilization of polymer-protected metal colloid catalysts by the formation of polymer hydrogen bond complexes

poly(N-vinyl-2-pyrrolidone)-(PVP) and polyvinyl alcohol-(PVA) protected nanoscopic noble metal colloidal catalysts were immobilized to prepare the corresponding heterogeneous catalysts by forming polymer hydrogen bond complexes of polyacrylic acid (PAA) with PVP or PVA. A PVP–PAA–Pd catalyst was found to be very active and selective for the partial hydrogenation of cyclopentadiene to cyclopentene.     

Covalent Immobilization of Dehydrogenases on Carbon Felt for Reusable Anodes with Effective Electrochemical Cofactor Regeneration

This study presents the immobilization with aldehyde groups (glyoxyl carbon felt) of alcohol dehydrogenase (ADH) and formate dehydrogenase (FDH) on carbon‐felt‐based electrodes. The compatibility of the immobilization method with the electrochemical application was studied with the ADH bioelectrode. The electrochemical regeneration process of nicotinamide adenine dinucleotide in its oxidized form (NAD⁺), on a carbon felt surface, has been deeply studied with tests performed at different electrical potentials. By applying a potential of 0.4 V versus Ag/AgCl electrode, a good compromise between NAD⁺ regeneration and energy consumption was observed. The effectiveness of the regeneration of NAD⁺ was confirmed by electrochemical oxidation of ethanol catalyzed by ADH in the presence of NADH, which is the no active form of the cofactor for this reaction. Good reusability was observed by using ADH immobilized on glyoxyl functionalized carbon felt with a residual activity higher than 60 % after 3 batches.     

Immobilization of manganese peroxidase fromLentinula edodes on alkylaminated emphazeTM AB 1 polymer for generation of Mn3+ as an oxidizing agent

Manganese peroxidase (MnP) is secreted by white-rot fungi and participates in the degradation of lignin by these organisms. MnP uses H₂O₂ as an oxidant to oxidize Mn^II to Mn^III as the manganic ion Mn³⁺. The Mn³⁺ stabilized by chelation, is a highly reactive nonspecific oxidant capable of oxidizing a variety of toxic organic compounds. Previous attempts at immobilization of MnP, purified fromLentinula edodes through reactive amino groups, have been hindered by the protein’s low lysine content of only 1% and its instability above pH 6.0. As an alternative to amine coupling, the enzyme has now been covalently immobilized through its carboxyl groups, using an azlactonefunctional copolymer derivatized with ethylenediamine and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) as a coupling reagent. The immobilization reaction was performed under acidic (pH 5.25) conditions, and 90% coupling efficiency was achieved within 2 h. Net immobilization efficiencies, expressed as the product of protein coupling efficiency and enzyme activity, have been measured at > 95% within 4 h. The MnP-NH-polymer and the free soluble protein were characterized and compared for their pH, temperature, and storage stabilities, as well as their H₂O₂ dependence and kinetics. The tethered MnP, employed in an immobilized enzyme bioreactor for generation of chelated Mn₃₊ may have industrial applications as a nonspecific oxidant of organopollutants.     

Adsorption and inactivation behavior of horseradish peroxidase on cellulosic fiber surfaces

The physical immobilization behavior of horseradish peroxidase (HRP) on cellulosic fiber surfaces was characterized using adsorption and inactivation isotherms measured by the depletion method followed by fitting of Langmuir’s and Freundlich’s models to the experimental data. The adsorption and inactivation behavior of simpler and relatively non-porous high and low crystalline cellulosic substrates (microcrystalline cellulose and regenerated cellulose) as well as more complex and porous cellulosic pulp fibers (bleached kraft softwood fibers) were investigated. The effect of the sorbent surface energy on HRP adsorption was demonstrated by increasing the hydrophobicity of the cellulosic fibers using an internal sizing agent. The influence of the fiber surface charge density on HRP adsorption was studied via modification of the cellulosic fibers using TEMPO (2,2,6,6-tetramethyl-1-piperidiniloxy radical)-mediated oxidation methods. Results showed that hydrophobic interactions had a much larger effect on HRP adsorption than electrostatic interactions. More hydrophobic fiber surfaces (lower polar surface energy) result in larger enzyme-fiber binding affinity constants and higher binding heterogeneity. It was also found that oxidation of the cellulosic fiber substrate reduces enzyme adsorption affinity but significantly increases the loading capacity per unit weight of the surface.     

Immobilization of Penicillin G Acylase on Vinyl Sulfone-Agarose: An Unexpected Effect of the Ionic Strength on the Performance of the Immobilization Process

Penicillin G acylase (PGA) from Escherichia coli was immobilized on vinyl sulfone (VS) agarose. The immobilization of the enzyme failed at all pH values using 50 mM of buffer, while the progressive increase of ionic strength permitted its rapid immobilization under all studied pH values. This suggests that the moderate hydrophobicity of VS groups is enough to transform the VS-agarose in a heterofunctional support, that is, a support bearing hydrophobic features (able to adsorb the proteins) and chemical reactivity (able to give covalent bonds). Once PGA was immobilized on this support, the PGA immobilization on VS-agarose was optimized with the purpose of obtaining a stable and active biocatalyst, optimizing the immobilization, incubation and blocking steps characteristics of this immobilization protocol. Optimal conditions were immobilization in 1 M of sodium sulfate at pH 7.0, incubation at pH 10.0 for 3 h in the presence of glycerol and phenyl acetic acid, and final blocking with glycine or ethanolamine. This produced biocatalysts with stabilities similar to that of the glyoxyl-PGA (the most stable biocatalyst of this enzyme described in literature), although presenting just over 55% of the initially offered enzyme activity versus the 80% that is recovered using the glyoxyl-PGA. This heterofuncionality of agarose VS beads opens new possibilities for enzyme immobilization on this support.     

磁性SiO2载体的表面改性与纳米TiO2光催化剂的固载

以Fe3O4米粒子为磁响应成分,制备了SiO2/Fe3O4磁性载体,采用回流热处理的方式对载体进行了表而改性.利用傅里叶变换红外和X-射线光电子能谱等手段对改性前后的载体进行分析.结果表明,载体改性后表面的Si-O-Si键数量明显减少,Si-OH的数量有所增加.以TiO2为探针催化剂,Procion染料为探针分子考察了载体的表面改性对催化剂固载量和光降解催化活性的影响,结果表明,改性后的载体明显提高了TiO2催化剂的固载量和降解Procion的催化活性.