Automated standard addition for fluorimetric steady-state and reaction-rate measurements

Instrumentation and methodology are described in which fluorescence measurements are made on a sample before and after a microcomputer-controlled injection of a standard-addition aliquot. From the two measurements an dother information, the analyte concentration in the sample is calculated and reported. The automated standard-addition procedure is shown to reduce measurement errors of 25–40% due to the presence of matrix quenchers or absorbers to less than 2% for fluorimetric steady-state measurements of quinine sulfate and in a fluorimetric reaction-rate method for determination of aluminum.     

Real-time assay of immobilized tannase with a stopped-flow conductometric device

A stopped-flow manifold was developed to assay and characterize immobilized tannase (EC 3.1.1.20). The immobilized enzyme reactor was inserted within the tube-type electrode pair (cell constant=103.2 cm(-1)) for a real-time conductometric measurement. Tris buffer (2 mM, pH=7.0) was used as the carrier for sensitivity improvement. The activities and kinetic parameters (Km values) for propyl gallate, methyl gallate and tannic acid were investigated.     

Amperometric acetylcholine and choline sensors with immobilized enzymes

Acetylcholine and choline sensors are prepared by immbilizing enzymes on nylon net attached to a hydrogen peroxide snsor. Choline oxidase is used for the choline sensor; acetylcholinesterase choline oxidase are used for acetylcholine. The platinum/silver electrode pair is polarized at +0.6 V. The assembly is protected with an acetate cellulose membrane to enhance selectivity. The ranges measured are 1–10 μmol l^?1 in 0.1–1 ml of sample. The response times are 1–2 min.     

Flow-through microfluorometry of immobilized enzymes

The turnover of bioreactors is commonly determined from the total consumption or production rates of substrates and products. However, conventional techniques provide no information about the dispersion of catalytic particles such as cells or enzyme carrier beads. Flow-through microfluorometry enables the dispersive measurement on-line of static parameters (e.g. distribution of size and enzyme load), and of dynamic parameters of particle suspensions (e.g. distribution of individual turnover rates in bioreactors). This allows the influence of heterogeneity of the carrier matrix or cells on the kinetics of the system to be estimated.     

Thermal inactivation of immobilized enzymes: A kinetic study

The thermal inactivation kinetics of chymotrypsin, trypsin, and—-amylase bound to silica, polyacrylamide, and polystyrene were studied at different temperatures. The inactivation curves were analyzed by a kinetic model, assuming a first-order reaction of differently stable enzyme fractions on the matrix. In all cases the assumption of two enzyme fractions with distinctly different inactivation constants was sufficient for describing the inactivation progress (standard deviations between experimental and calculated inactivation curves, 1-4%). Both the inactivation constants as well as the relative concentrations of the enzyme fractions were found to change in dependence on temperature.     

Activity of enzymes immobilized on microspheres with thermosensitive hairs

Poly(N-isopropylacrylamide)s (PNIPAMs) carboxylated at one chain end or both ends were prepared by polymerization using 4,4-azobis(N,N,-cyanopentanoic acid) (V-501) as an initiator and β-mercaptopropionic acid (MPA) as a chain transfer reagent. One end group of PNIPAM carboxylated at both ends was conjugated with latex particles, and another with trypsin using carbodiimide. Differential scanning calorimetry (DSC) revealed that PNIPAM on the particles exhibited a drastic phase transition, and that the transition temperature was largely elevated when the enzyme was immobilized at the chain end. Therefore, PNIPAM on the particles showed two phase transitions because of the coexistence of the enzyme-conjugated and non-conjugated PNIPAMs. The activity of trypsin immobilized on the particles with the PNIPAM spacer showed significant temperature dependence. The apparent relative activity increased above the transition temperature of non enzyme-conjugated PNIPAM on the particles. One of the reasons for this is that the diffusion of the substrate changed discontinuously around the transition temperature. Therefore, the temperature dependence of the enzymatic activity was significantly affected by the molecular size of the substrates. The enzymatic activity was also influenced by the surface density of trypsin and PNIPAM on the particle, and the molecular weight of the PNIPAM spacer.     

Determination of phenol and catechol concentrations with oxygen probes coated with immobilized enzymes or immobilized cells

The enzyme phenol 2-hydroxylase was immobilized on Sepharose and used in conjunction with an O₂ electrode for quantitating phenol. Similarly, catechol 1,2-oxygenase was used for quantitating catechol. A third probe was prepared by immobilization ofTrichosporon cutaneum cells rather than purified phenol 2-hydroxylase for phenol quantitation. The whole cell system gave results comparable to the immobilized enzyme system.     

A new simple instrument for thermomagnetometry. Applications to simultaneous thermomechanical measurements

A new instrument has been set up to study magnetic materials. Curie points measured by means of this simple apparatus are in perfect agreement with those given by a conventional magnetic balance. The apparatus may be used alone, but also combined with an electronic thermodilatometer. Curie points are emphasized simultaneously by the thermodilatometric and impedance curves. It appears that the sensitivity and reliability of the apparatus demonstrate magnetic phenomena of very low intensity such as the order point of α-Fe₂O₃.     

A new instrument for time-resolved reduction of scattered radiation in fluorescence measurements

An opto-electronic cross-correlation system was employed to reduce the scattering influence in fluorescence measurements. A stable optical delay line incorporated into the instrument was positioned to yield detection at a fixed time after excitation; the optimal delay time was determined simply from the ratio of the fluorescence decay curve to a similar curve portraying scattering response. Signal-to-scattering background enhancements greater than two were measured for the very short-lived (τ = 0.7 ns) fluorophores whereas a six-fold increase was measured for fluorophores with longer lifetimes. The shortest lifetime which would benefit from time-resolution in this system is limited by the time-response of the photomultiplier tube (1.1 ns FWHM); the excitation pulses are on the order of 6 ps.     

Modification of milk using immobilized enzymes

The preparation and use of immobilized enzyme systems for the modification of the principle components of milk—casein, lactose, and butterfat—are discussed. Design of a pilot-scale bioreactor is described. Also the use of tea polyphenols as a crosslinking agent is reported. A review of methods for hydrolyzing lactose and characteristics of a concentrated liquid sweetener derived from dairy byproducts is presented. Further, the application of immobilized esterases to modify milk, cream, and butterfat is reported.     

Enzymatic protein digest in chip-based nanovials with immobilized proteolytic enzymes

In the present work, protein digest reactions in silicon-based microchips, coated with immobilized proteolytic enzymes, have been carried out. The performance of such vials, modified with trypsin or chymotrypsin, was tested with myoglobin as a substrate. Capillary electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry were utilized for analysis of the digests, and the influence of different instrumentation setups, immobilization procedures and reaction conditions are discussed.     

Stopped-flow kinetic determination of glucose and lactate with immobilized enzymes

Kinetic procedures are described for the measurement of lactate and glucose with enzymes immobilized in the observation cellof a stopped-flow spectrophotometer. Kinetic data are obtained by two-point or multipoint methods with data acquisition over any time range desired. Glucose was quantified in a fixed-time mode with a linear range of 0–10 mM. Lactate was quantified in the range 0–50 μM by obtaining the slope of the absorbance/time profile between 10 and 20 s after each reaction was initiated. Results for these two substrates in control sera are presented.     

A control over accessibility of immobilized enzymes through porous coating layer

We report immobilization of an enzyme by layer-by-layer (LbL) film deposition technique. All the enzyme layers, including the inner ones, contributed to the activity. We put-forwarded additional coating layers to protect the enzymes. To control the accessibility of the enzymes beneath the coating layer, pores have been introduced. Our results show controlled accessibility of immobilized enzymes in solid-state matrices.     

Use of the clark oxygen sensor with immobilized enzymes for determinations in flow systems

A small-volume cell has been constructed for amperometric flow measurements with a Clark oxygen sensor and its performance was tested. The Clark sensor can be combined with immobilized enzymes for determination of substances after enzymatic conversion during which oxygen is consumed or released. Two enzymes, glucose oxidase and tyrosinase, were used and two measuring techniques, employing the enzyme immobilized on the Clark sensor membrane and with the enzyme bound on a support in a preceding reactor, were tested and compared. It was found that, in the given system, measurement with the enzyme immobilized on the sensor membrane has better sensitivity, precision and response rate.     

Determination of intrinsic properties of immobilized enzymes

Staphylococcal nuclease has been insolubilized, directly through its amino groups, on CNBr-activated Sepharose 2B. For kinetic studies, a small substrate (thymidine 5′-(p-nitrophenyl phosphate) 3′-phosphate) has been used to measure the hydrolytic activity. With this system the absence of diffusional limitation has been proven. Eadie-Hofstee analysis of the data has been employed to determine the intrinsic kinetic constants of the insolubilized enzyme. Thek _cat-pH andK _M−pH profiles and the activation energies are similar for the soluble and for the insolubilized nuclease. At the same time conditions are established in which a stirred batch reactor containing particles of insolubilized nuclease behaves as an open system.     

Peptide synthesis by means of immobilized enzymes

-Chymotrypsin covalently bound to silica, enzacryl AA, and enzacryl AH catalyzes peptide bond formation between N-protected dipeptide methyl esters and H-Leu-NH₂ with results similar to those with the free enzyme. The influence of water-miscible and water-immiscible cosolvents, of the supports, and of the structure of the substrates is shown to be of importance for the ease of the chymotrypsin-medicated coupling reactions. The best yields were obtained using biphasic aqueous-organic solvent mixtures, silica-bound chymotrypsin, and substrates with leucine in the P₂-position. The yields of the syntheses are discussed in terms of the reactivity of substrates with similar structure in enzymatic hydrolyses. All the immobilized chymotrypsin preparations could be re-utilized successfully for further couplings. Abbreviations: IUPAC/IUB rules for peptides are followed, see Eur. J. Biochem.27, 201 (1972); Glt=4-carboxybutyrul (glutaryl),-Nan=4-nitroanilide. All amino acids except glycine are of L-configuration.     

Analytical applications of reactors containing immobilized enzymes

The theory for analytical packed-enzyme reactors is discussed and it is shown that a 100% conversion efficiency gives many advantages. This concept has been applied to methods for substrate determinations of urea, amino acids, and glucose. Enzyme reactors have also been used in the effluent from a Chromatographic column to enhance selectivity and sensitivity for cholesterol. Enzyme reactors for the determination of inhibitors, e.g. mercury ions, should be designed differently. A low conversion efficiency gives high sensitivity and a linear response.     

Use of immobilized enzymes in chemical analysis

Immobilized enzymes are becoming increasingly popular as analytical reagents because of their reusability, stability, and sensitivity to many inhibitors that would seriously interfere in assays using soluble enzymes. In this article, some of the kinetic and catalytic effects of immobilized enzymes in analysis will be discussed. The shift of the activity-pH profile curves on immobilization, the changes in temperature dependence, the inhibitor constants (K_i), Michaelis constants (K_m), and the maximum velocity ( V_max), plus others, will be discussed. Finally, the use of these immobilized enzymes in fluorometric and electrochemical monitoring systems will be shown, and the future of these reagents in various areas will be discussed. A survey of enzyme electrodes will be presented as an example of the use of immobilized enzymes. Application of immobilized enzyme technology to the assay of BUN, glucose, uric acid, amino acids, ethanol, and other metabolites will be discussed.     

纳米载体固定化酶的最新研究进展

催化剂是化学工业的重要基础,其中酶是重要的高效天然催化剂。近年来,酶被越来越多地应用于工业领域,如精细化工、食品工业、制药工业、纺织业和制浆造纸。然而,由于游离酶存在价格昂贵及操作稳定性(特别是回收与重复使用性能)低等缺点,其在工业上的进一步应用受到一定限制。对酶进行固定化是解决上述问题的有效途径。一个理想的酶固定化技术需要载体具有良好的生物相容性和高比表面积,能够负载适量的酶并且具有很好的重复使用性能,固定化酶的过程简单温和,所得到的固定化酶制剂具有良好的催化性能、稳定性以及工业应用价值。尽管固定化酶技术经过了多年的发展,但仍需进一步研究。近几年,人们研究了基于纤维素纳米晶类、聚多巴胺类纳米载体以及生物相容性合成有机物纳米胶等新型载体对酶的固定化,取得了较好的成果。本文综述了这些新型纳米载体的制备以及酶的固定化过程,阐述了纳米载体固定化酶的结构和催化性能,并展望了发展前景。纤维素是全球产量最高、来源最广的生物聚合物。纤维素经过一定的酸(常用硫酸和盐酸)水解处理后,剩下的是具有高结晶度的纤维素纳米晶。它具有高比表面积、高机械强度和高长径比等优异性能。因此,研究者利用纤维素纳米晶作为载体进行酶固定化,获得了高负载量、高催化性能的固定化酶制剂。基于仿生矿化法制备的聚多巴胺类材料近年来获得研究者越来越多的关注。多巴胺具有良好的自聚合能力,可以对无机、有机等各种材料进行表面修饰。同时,聚多巴胺中含有的活性官能团可以与酶发生交联,从而达到固定化酶的效果。基于合成性聚合物纳米胶载体的固定化酶技术同样是一个新兴的、有意义的研究领域。相关的固定化过程可分为两大类：(1)在酶分子表面通过原位聚合生成纳米胶(growing-from过程);(2)将酶与预先合成的纳米胶进行交联(grafting-to过程)。其中, growing-from过程是先将酶分子丙烯酰化,再进行原位聚合。而原位聚合又可分为自由基聚合、原子转移自由基聚合(ATRP)和可逆加成-断裂链转移聚合(RAFT)。其中, ATRP和 RAFT主要用于制备环境响应型的酶-聚合物纳米凝胶。