Fabrication, characterization, and enzymatic activity of fungal protease--nanogold membrane bioconjugate

This study describes the synthesis of a free-standing nanogold membrane by the spontaneous reduction of aqueous chloroaurate ions by the diamine molecule DAEE at a liquid-liquid interface. The free standing nanogold membrane, provides a biocompatible surface for the immobilization of proteins. F-Protease (F-Prot) was then bound to the nanogold membrane via interaction with the gold nanoparticles leading to a new class of biocatalyst. A highlight of the new biocatalyst wherein the enzyme is bound to the nanogold membrane is the ease with which separation from the reaction medium may be achieved by simple filtration. In relation to the free enzyme in solution, the F-Prot in the bioconjugate material exhibited a slightly higher biocatalytic activity and significantly enhanced pH and temperature stability. The F-Prot nanogold membrane bioconjugate material also exhibited excellent biocatalytic activity over ten successive reuse cycles.     

SP1 as a novel scaffold building block for self-assembly nanofabrication of submicron enzymatic structures

In this study, SP1, a ring-shaped highly stable homododecamer protein complex was utilized for the self-assembly of multiple domains in a predefined manner. Glucose oxidase (GOx) was fused in-frame to SP1 and expressed in Escherichia coli. Complexes where GOx encircled SP1 dodecamer were observed, and moreover, the enzymatic monomers self-assembled into active multienzyme nanotube particles containing hundreds of GOx molecules per tube. This work demonstrates the value of SP1 as a self-assembly scaffold.     

反胶团酶膜反应器研究进展

两亲分子溶入有机溶剂时可自组织形成球形聚集体，称作反胶团，酶能溶入反胶团的水池中以催化特定的反应文中对反胶团介质中的酶反应进行了介绍，并对膜反应器在反胶团酶反应中的应用进行了综述     

Microelectrode control of surface-bound enzymatic activity

Microelectrodes have been used to control the microscopically local reaction environment of surface-bound alcohol dehydrogenase (ADH). A surface comprised of agarose beads coated with immobilized ADH was prepared on a microscope slide and exhibited maximum activity toward the oxidation of ethanol, in the presence of nicotinamide adenine dinucleotide (NAD(+)), at a pH of 9.0. Microelectrode control of activity was achieved by submerging the slide in a solution of pH 6.0, well below the optimum value, and generating hydroxide at the microelectrode tip through the reduction of oxygen or water. An alkaline "sphere of influence" was set up around the microelectrode tip that, when positioned in close proximity to the enzyme surface, created a favorable reaction environment. The increased enzymatic activity was monitored by observing fluorescence of the reduced cofactor, NADH, using a fluorescence microscope equipped with an imaging camera. The fluorescent sphere diameter was characterized as a function of time, potential, and solution buffer strength. Optimum spatial resolution for enzymatic control was 7-12 μm.     

Effect of dialysis dose and membrane flux on hemoglobin cycling in hemodialysis patients

Many studies found that hemoglobin (Hb) fluctuation was closely related to the prognosis of the maintenance hemodialysis patients. We investigated the association of factors relating dialysis dose and dialyzer membrane with Hb levels. We undertook a randomized clinical trial in 140 patients undergoing thrice‐weekly dialysis and assigned patients randomly to a standard or high dose of dialysis; Hb level was measured every month for 12 months. In the standard‐dose group, the mean (±SD) urea reduction ratio was 65.1% ± 7.3%, the single‐pool Kt/V was 1.26 ± 0.11, and the equilibrated Kt/V was 1.05 ± 0.09; in the high‐dose group, the values were 73.5% ± 8.7%, 1.68 ± 0.15, and 1.47 ± 0.11, respectively. The standard deviation (SD) and residual SD (liner regression of Hb) values of Hb were significantly higher in the standard‐dose group and low‐flux group. The percentage achievement of target Hb in the high‐dose dialysis group and high‐flux dialyzer group was significantly higher than the standard‐dose group and low‐flux group, respectively. Patients undergoing hemodialysis thrice weekly appear to have benefit from a higher dialysis dose than that recommended by current KDQQI (Kidney Disease Qutcome Quality Initiative) guidelines or from the use of a high‐flux membrane, which is in favor of maintaining stable Hb levels.     

Structure and enzymatic activity of laser immobilized ribonuclease A

Ribonuclease A (RNase A) enzyme was immobilized on solid holders by matrix-assisted pulsed laser evaporation (MAPLE) technique. The experiments were performed inside a stainless steel irradiation chamber. A UV KrF* (λ = 248 nm, τ_FWHM ? 25 ns, ν = 10 Hz) excimer laser source was used for irradiations. Surface morphology, molecular structure, and enzymatic activity of laser transferred RNase A samples were investigated as a function of RNase A concentration in the frozen composite MAPLE targets. Surface morphology and thickness of the immobilized enzyme were investigated by atomic force microscopy, scanning electron microscopy, and surface profilometry. The molecular structure of the laser transferred RNase A was determined by Fourier transform infrared spectroscopy. The enzymatic activity of RNase A after immobilization was tested through ribonucleic acid removal from deoxyribonucleic acid (DNA) extract solutions isolated from plant and animal tissues. A molecular method based on polymerase chain reaction was used to investigate the functional properties of DNA extracts treated with laser immobilized RNase A.     

同时具有光敏感性和酶催化功能的复合敏感膜

采用溶液浇铸法成膜,把荧光指示剂和葡萄糖氧化物酶(GOD)同时固定于醋酸纤维素膜上,得到同时具有光敏感性和酶催化能力的复合敏感膜.利用SEM和紫外-可见光分光光度计对复合敏感膜表面形貌和酶活性进行了分析.荧光指示剂没有泄漏,固定化酶的稳定性高于游离酶,表明此醋酸纤维素膜可作为一种优良的荧光指示剂和酶固定化载体.     

Nanoreactors for pH controlled sequential activity switching in multistep enzymatic processes

This theoretical model predicts that the activity of multiple enzymes may be controlled simultaneously with superior efficiency in nanosized reactors by adjusting pH. Multistep enzymatic processes employed for various purposes including organic biotransformation may require application of multiple reactions and isolation of intermediates. Sequential activity switching would offer substantial advantages. Nanoreactors would provide better option to fully appreciate the pH switching approach.     

A hydrogen peroxide biosensor based on peroxidase activity of hemoglobin in polymeric film

A Hydrogen peroxide (H2O2) biosensor, based on hemoglobin (Hb) and ortho-phenylenediamine (o-PD) gold electrode, was fabricated. Hb was immobilized onto the electrode surface by electrochemical polymerize method with o-PD. The designed biosensor showed a well defined redox peak which was attributed to the direct electrochemical response of Hb. The immobilized Hb exhibited an excellent electrocatalytical response to the reduction of hydrogen peroxide, enabling the sensitivity determination of H2O2. Factors and performances such as pH, potential, influencing the designed biosensor, were studied carefully. The amperometric detection of H2O2 was carried out at -300 mV in phosphate buffer solution (PBS) (0.1 M) with pH 6.0. This biosensor showed a fast amperometric response (less then 5 s) to H2O2. The levels of the (Relative standard deviation) RSDs (< 3.5%) for the entire analyses reflected a highly reproducible sensor performance. Using the optimized conditions, the detection limit of the biosensor was 1 x 10(-7) M and linear range was from 5 x 10(-6) to 1.25 x 10(-4) M. In addition, this sensor showed long-term stability and good sensitivity.     

Electron-transfer studies with a new flavin adenine dinucleotide dependent glucose dehydrogenase and osmium polymers of different redox potentials

A new extracellular flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase from Glomerella cingulata (GcGDH) was electrochemically studied as a recognition element in glucose biosensors. The redox enzyme was recombinantly produced in Pichia pastoris and homogeneously purified, and its glucose-oxidizing properties on spectrographic graphite electrodes were investigated. Six different Os polymers, the redox potentials of which ranged in a broad potential window between +15 and +489 mV versus the normal hydrogen electrode (NHE), were used to immobilize and "wire" GcGDH to the spectrographic graphite electrode's surface. The GcGDH/Os polymer modified electrodes were evaluated by chronoamperometry using flow injection analysis. The current response was investigated using a stepwisely increased applied potential. It was observed that the ratio of GcGDH/Os polymer and the overall loading of the enzyme electrode significantly affect the performance of the enzyme electrode for glucose oxidation. The best-suited Os polymer [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(PVI)Cl](+) had a potential of +309 mV versus NHE, and the optimum GcGDH/Os polymer ratio was 1:2 yielding a maximum current density of 493 μA·cm(-2) at a 30 mM glucose concentration.     

News from HPCE '99: Correlating enzymatic activity and cell cycle

Britt Erickson reports from Palm Springs, CA.     

Discovery of enzymatic activity using stable isotope metabolite labeling and liquid chromatography-mass spectrometry

Stable isotope labeling of an intracellular chemical precursor or metabolite allows direct detection of downstream metabolites of that precursor, arising from novel enzymatic activity of interest, using metabolite profiling liquid chromatography-mass spectrometry. This approach allows the discrimination of downstream metabolites produced from novel enzymatic activity from the unlabeled form of the metabolite arising from native metabolic processes within the cell. Even for the case in which a given product of novel enzymatic activity is a transient, the novel enzymatic activity that produced it can be demonstrated to exist indirectly by identification of product-specific downstream metabolites. Therefore, direct or indirect discovery of novel enzymatic machinery engineered within a cell can be accomplished without a requirement for direct product purification or identification. The application of this approach to confirm the presence of a novel metabolic activity, alanine 2,3-aminomutase, obtained by mutagenesis and selection are discussed. The advantages of metabolite profiling approaches to metabolic engineering in terms of accelerating enzyme discovery and development of intellectual property will also be highlighted.     

Fabrication, characterization, and enzymatic activity of encapsulated fungal protease--fatty lipid biocomposite films

Encapsulation of an aspartic protease from the fungus Aspergillus saitoi (F-prot) in thermally evaporated fatty acid films by a simple beaker-based immersion technique under enzyme-friendly conditions is described. The approach is based on diffusion of the enzyme from aqueous solution, driven primarily by attractive electrostatic interaction between charged groups on the enzyme surface and ionized lipid molecules in the film. The encapsulated enzyme molecules could be "pumped out" of the biocomposite film into solution by modulating the electrostatic interaction between the enzyme and fatty acid molecules via solution pH variation. The kinetics of F-prot diffusion into the acid films was followed using quartz crystal microgravimetry measurements while the secondary and tertiary structure of the enzyme in the lipid matrix was studied using Fourier transform infrared (FT-IR) and fluorescence spectroscopies. FT-IR and fluorescence measurements indicated little perturbation to the native structure of the enzyme. A chemical analysis of the F-prot-fatty acid biocomposite film was also performed using X-ray photoelectron spectroscopy. The encapsulated F-prot molecules showed catalytic activity (as estimated by reaction with hemoglobin) comparable to free enzyme molecules in solution, indicating facile access of biological analytes/reactants in solution to the enzyme molecules. The advantages/disadvantages of this approach vis-à-vis methods currently used for encapsulation of biomolecules are briefly discussed.     

制备多孔壳聚糖膜的新方法:选择性酶解原位成孔

利用溶菌酶对中等脱乙酰度壳聚糖的选择性降解,将其与高脱乙酰度壳聚糖以不同比例混合浇铸成膜.考察了降解过程中材料的失重和溶液还原糖含量的变化,用傅立叶红外光谱分析了混合膜的成分改变和降解过程中膜的变化,并用扫描电镜观察了混合膜降解后的形态变化.结果表明,壳聚糖混合膜中的中等脱乙酰度壳聚糖被溶菌酶选择性的降解,而且当混合膜中该组分含量为0.5时,降解后膜表面及内部均产生了互相贯穿的纳米尺寸的孔,剩余的高脱乙酰度成分表现为纳米尺寸的纤维.该方法通过壳聚糖的选择性酶解原位成孔,为制备纳米结构的多孔壳聚糖膜提供了一个新颖的途径.     

PS-Ti超滤膜反应器进行酶水解预处理的蔗渣和酶回收的研究

研究了膜式糖化反应器及酶回收的工艺过程首次提出了ＰＳ－Ｔｉ复合管式膜糖化器水解蔗渣喷爆物及循环使用纤维素酶的新工艺研究了水解酶失活的机理为非降解底物的吸附作用，即水解酶大部分失活是由于吸附于非降解的蔗渣上所致超滤与丹宁沉淀法结合，使水解酶回收率均大于９０％膜式糖化反应器的半连续操作的最高总转化率可达９７％，在２０ｈ内总转化率水平比间歇提高７倍，还原糖产率提高６倍水解液中葡萄糖与木糖的质量比为３∶１     

Preparation of an enzyme/inorganic nanosheet/magnetic bead complex and its enzymatic activity

A complex composed of enzyme (horseradish peroxidase, HRP), layered titanate (TiO_x), and magnetic beads (MB) was prepared, and the enzymatic activity of the complex and the repeatable use for the enzyme in the complex were investigated. The HRP/TiO_x/MB complex was formed in acetate buffer (pH 4) by electrostatic interaction among the components where HRP and MB have positive charge, whereas TiO_x is negatively charged. The resultant complex was sufficiently stable in an aqueous solution, and HRP immobilized on the layered titanate in the complex kept a certain enzymatic activity in which typical enzymatic relationship was observed between substrate concentration and initial reaction rate. By giving a magnetic field using commercial magnets, the HRP/TiO_x/MB complex was easily and rapidly recovered, and the recovered complex was able to be used for the next enzymatic reactions. The enzyme/inorganic nanosheet/MB complex proposed in this study is expected to be applied as a new tool in the bioengineering and nanobiotechnological fields.     

8-hydroxyquinolinyl azo dyes: a class of surface-enhanced resonance Raman scattering-based probes for ultrasensitive monitoring of enzymatic activity

A series of surface-enhanced resonance Raman scattering (SERRS) based probes for the detection of lipase activity are reported. A number of novel SERRS-active 8-hydroxylquinolinyl azo dyes have been prepared and via synthetic esterification or subsequent enzymatic hydrolysis at the 8-hydroxyl position the SERRS signal can be "switched" on or off. In the first instance, the technique has been demonstrated for the successful detection of lipase from Pseudomonas cepacia, and these new compounds offer a limit of detection of 0.2 ng mL-1 enzyme, up to a 100-fold lower limit than observed for benzotriazolyl dyes used in previous studies. The chemical synthesis is straightforward and allows for facile introduction of a wide range of different masking groups, using commonly known synthetic methodologies. The potential for multiplexing analysis of enzyme activity using this technology is presented within.     

Luminescent quantum dots fluorescence resonance energy transfer-based probes for enzymatic activity and enzyme inhibitors

The paper describes the development and characterization of analytical properties of quantum dot-based probes for enzymatic activity and for screening enzyme inhibitors. The luminescent probes are based on fluorescence resonance energy transfer (FRET) between luminescent quantum dots that serve as donors and rhodamine acceptors that are immobilized to the surface of the quantum dots through peptide linkers. Peptide-coated CdSe/ZnS quantum dots were prepared using a one-step ligand exchange process in which RGDC peptide molecules replace trioctylphosphine oxide (TOPO) molecules as the capping ligands of the quantum dots. The peptide molecules were bound to the surface of the CdSe/ZnS quantum dots through the thiol group of the peptide cysteine residue. The peptide-coated quantum dots were labeled with rhodamine to form the FRET probes. The emission quantum yield of the quantum dot FRET probes was 4-fold lower than the emission quantum yield of TOPO-capped quantum dots. However, the quantum dot FRET probes were sufficiently bright to enable quantitative enzyme and enzyme inhibition assays. The probes were used first to test the enzymatic activity of trypsin in solution based on FRET signal changes of the quantum dot-based enzymatic probes in the presence of proteolytic enzymes. For example, exposure of the quantum dot FRET probes to 500 microg/mL trypsin for 15 min resulted in 60% increase in the photoluminescence of the quantum dots and a corresponding decrease in the emission of the rhodamine molecules. These changes resulted from the release of rhodamine molecules from the surface of the quantum dots due to enzymatic cleavage of the peptide molecules. The quantum dot FRET-based probes were used to monitor the enzymatic activity of trypsin and to screen trypsin inhibitors for their inhibition efficiency.