Enzyme column

The flow diagram of the enzyme reactor for continuous production of the L-amino add is given in Figure A85. The acetyl amino add is continuously charged into the enzyme column through a filter and a heat exchanger. The effluent is concentrated and the L-amino add is crystallised. The acyl-D-amino add contained in the mother liquor is racemised by heating in a racemisation tank, and reused. 

Blankenstein G., Preuschoff F., Spohn U., Mohr K.H., Kula M.R., Determination of L-glutamate and L-glutamine by flow-injection analysis and chemiluminescence detection comparison of an enzyme column and enzyme membrane sensor, Anal. Chim. Acta 1993 271 231-237. 

The same group reported in 1986 a sensitive and selective HPLC method employing CL detection utilizing immobilized enzymes for simultaneous determination of acetylcholine and choline [187], Both compounds were separated on a reversed-phase column, passed through an immobilized enzyme column (acetylcholine esterase and choline oxidase), and converted to hydrogen peroxide, which was subsequently detected by the PO-CL reaction. In this period, other advances in this area were carried out such as the combination of solid-state PO CL detection and postcolumn chemical reaction systems in LC [188] or the development of a new low-dispersion system for narrow-bore LC [189],... 

The techniques developed in enzyme immobilization have facilitated the development of enzyme electrodes and of novel enzyme -based, automated, analytical methods (l6,17,l8). Enzyme electrodes have resulted from the combination of an enzyme membrane and an ion-selective electrode they were used successfully to assay directly appropriate substrates. Enzyme columns or enzyme tubes, prepared in a conventional manner, were used as a specific auxiliary component in the indirect assay of substrates in many of the novel automated analytical procedures. 

Kurkijarvi et al. were the first to demonstrate the feasibility of seg-mented-flow bioluminescence assays by use of a bioreactor packed with bacterial bioluminescent enzymes immobilized on Sepharose 4B [60]. The packed glass colunrn used was placed in front of the photomultiplier tube of a luminometer. The luminescence signal obtained was linearly related to the NADH concentration from 1 pmol to 10 nmol for sample volumes of 2-20 pL. In the region of 400 NADH assays could be performed with a single enzyme column, with no appreciable change in sensitivity or accuracy. However, problems arising from packing or disruption of the matrix were encountered after 4 days of intensive use. 

The decomposition of polymer molecules in wastewater samples can also be achieved by a hybrid sensor, which consists of a consortium of microorganisms and hydrolases together. As an example, the BOD of lactose-containing wastewater was determined by a hybrid sensor consisting of cells of the yeast Lipomyces kononenkoae and of the enzyme /1-galactosidase [50, 69]. It is also possible to use a column containing immobilized enzymes, which is inserted in the measuring device (e.g., an FIA) before the biosensor [52, 70]. Such a combination of an enzyme column, which contains a-amylase and amy-... 

The modeling of real immobilized-enzyme column reactors, mainly the fluidized-bed type, has been described (Emeiy and Cardoso, 1978 Allen, Charles and Coughlin, 1979 Kobayashi and Moo-Young, 1971) by mathematical models based on the dispersion concept (Levenspiel, 1972), by incorporation of an additional term to account for back-mixing in the ideal plug-flow reactor. This term describes the non-ideal effects in terms of a dispersion coefficient. 

In 1989, to the best of our knowledge, the first report appeared on the use of an avidin-biotin system for the immobilization of enzymes in the preparation of biosensors. Walt et al. have immobilized biotin-modified enzymes (urease, esterase, and penicillinase) on the surface of biotin-modified optical fiber using avidin as a binder (Figure 5a). They have demonstrated the general use of this procedure in immobilizing several types of enzymes. At nearly the same time, Gunaratna and Wilson used an enzyme column in which choline esterase and choline oxidase were immobilized through avidin-biotin complexation for the determination of acetyl-... 

Fig. 12. Diagram of elution pattern of red cell acid phosphatase and various markers on Biogel P 60. The position of the various protein markers was determined both by optical density determination and by starch gel electrophoresis of the individual fractions (83). The experiment was carried out using a polyacrylamide gel (Biogel P 60, 50-150 mesh exclusion limit >60,000 Bio-Rad Laboratories, California) in 0.05 M tris buffer, pH 8.0, containing 0.08% (v/v) Tween 80 and 0.1% (v/v) 2-mercaptoethanol to stabilize the enzyme. Column 60 X 4 cm. Flow rate 20 ml/hr, 4 ml fractions. (A) OD at 280 nm, ( ) OD at 540 nm, ( ) LDH assay with p-nitrophenyl phosphate for AcP. From Hopkinson and Harris (85).

Puchades, R., Lemieux, L., and Simard, R. E. (1989). Sensitive, rapid and precise determination of L-glutamic acid in cheese using a flow injection system with immobilized enzyme column. /. Food Sci. 54, 423M26. 

Conventional calorimetric biosensors with thermistors as the transducer were invented early by proposing a thermal biosensor in a flow stream [10]. So far the design of enzyme thermistors does not entirely match the market demand well, but it seems well suited for special applications [10,11]. A number of devices have utilized discrete pairs of thermistors for differential measurements with immobilized enzymes or with separate enzyme columns [9,10,11],... 

Fig-i- Micro-thermistorarray with immobilized enzymes for calorimetric analysis T,... Thermistors, E ... Enzyme columns... 

Fig. 19. Schematic design of a flow injection analysis (FIA) system. A selection valve (top) allows a selection between sample stream and standard(s). The selected specimen is pumped through an injection loop. Repeatedly, the injection valve is switched for a short while so that the contents of the loop are transported by the carrier stream into the dispersion/reaction manifold. In this manifold, any type of chemical or physical reaction can be implemented (e.g. by addition of other chemicals, passing through an enzyme column, dilution by another injection, diffusion through a membrane, liquid-liquid extraction, etc. not shown). On its way through the manifold, the original plug undergoes axial dispersion which results in the typical shape of the finally detected signal peak...

Eicompak AC-GEL 0.07 M-Phosphate buffer containing 60 ppm of Na2 EDTA, 0.065% of tetramethyl ammonium chloride and 0.3 of sodium octane-sulfonate Electrochemical Mouse brain tissues. An immobilized enzyme column with ChO and AChE was used. [172]... 

The thermostability of the immobilized lipase is most easily demonstrated in continuous interesterification. A general test system is shewn in Figure 6. The immobilized enzyme is placed in the enzyme column. The precolumn is used to saturate the reactants with water as they are pumped through the system. 

The most widely used immobilized enzyme process involves the use of the enzyme glucose isomerase for the conversion of glucose to fructose in com syrup (Carasik and Carroll 1983). The organism Bacillus coagu-lans has been selected for the production of glucose isomerase. The development of the immobilized cell slurry has not proceeded to the point where half-lives of the enzyme are more than 75 days. A half-life is defined as the time taken for a 50 percent decrease in activity. Such immobilized enzyme columns can be operated for periods of over three half-lives. 

Figure 9.61 HPLC for the determination of protoporphyrinogen oxidase activity in human leukocytes, (a) Enzyme incubation mixture, (b) Blank incubation with boiled enzyme. Column ODS-Hypersil (250 mm X 5.0 mm i.d.) eluent, 88% (v/v) methanol in 1 M ammonium acetate, pH 5.16. Flow rate, 1.5 mL/min fluorescence detection, excitation at 400 nm and emission at 618 nm. Peaks 1, mesoporphyrin (internal standard) 2, protoporphyrin IX. (From Guo et al., 1991.)...

The activity of the immobilized enzyme bioreactor plays an important role in the quality of the spectra. Immobilized enzyme activity is dependent on pH, temperature, solvents and buffers used for the hydrolysis. Extremes in any one condition can irreversibly destroy the bioreactor activity. Hydrolysis at conditions far from optimal can lead to no-hydrolysis or partial hydrolysis of a peptide, providing limited information on the peptide. In order to successfully employ an immobilized enzyme column on-line with HPLC/thermospray MS, organic modifiers must be kept minimal (less than 30-50%), pH must be between 6.5 and 8.5 and buffer (ammonium acetate) concentration around 0.05-0.1M [12]. 

The combination of immobilized enzyme columns with HPLC/thermospray MS can be very useful in peptide identification and sequencing [6,7], There are a number of ways of combining the immobilized enzyme column, HPLC and MS detection for peptide analysis. Use of an endopeptidase column prior to HPI.C separation and MS detection will enable separation of each hydrolysis product for Identification. Figure 2 shows the trypsin column/HPLC/thermospray MS of Y-endorphin. The selected ion chromatograms show the retention time for each tryptic hydrolysis product Tj and T. Typically, this column configuration can only be used on purified samples since no separation or column clean-up is performed before hydrolysis. 

Combination of two immobilized enzyme columns with HPLC/thermospray MS can be useful for amino acid sequencing and identification. The use of an endopeptidase bioreactor followed by HPLC separation then an exopeptidase column and MS detection can enable sequencing of 3-5 amino acids of each endopeptidase hydrolysis product. The trypsin, hydrolysis/HPLC/ carboxypeptidase A, B, and Y (1 1 1) hydrolysis/ thermospray MS analysis assist in the sequencing of Y-endorphin (Figure 2C,C ). 

The earlier investigations employed several different types of plexiglas constructions containing the immobilized enzyme column. These devices were thermostated in a water bath, and the temperature at the point of exit from the column was monitored with a thermistor connected to a commercial Wheatstone bridge. The latter was constructed for general temperature measurements and osmometry. Later, we developed more sensitive instruments for temperature monitoring indigenously the water bath was replaced by a carefully temperature-controlled metal block, which contained the enzyme column. The enzyme thermistor concept has been patented in several major countries. 

A compact sensor of greatly reduced dimensions (outer diameter x length 36 x 46 mm) has been constructed and is shown in Fig. 2. In order to conveniently accommodate enzyme columns and to ensure isolation from ambient temperature fluctuations, a cylindrical copper heat sink was included. An outer Delrin jacket further improved the insulation. The enzyme column (inner diameter x length 3x4 mm), constructed of Delrin, was held tightly against the inner terminals of the copper core. Short pieces of well-insulated gold capillaries (outer diameter/inner diameter 0.3/0.2 mm) were placed next to the enzyme column as temperature-sensitive elements. Microbead thermistors were mounted on the capillaries with a heat-conducting epoxy. Two types of mini system has been constructed as discussed below. 

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