Immobilized enzyme system

RP-HPLC has also been used for the analysis of flavan-3-ols and theaflavins during the study of the oxidation of flavan-3-ols in an immobilized enzyme system. Powdered tea leaves (20Qmg) were extracted with 3 X 5 ml of 70 per cent aqueous methanol at 70°C for lQmin. The combined supernatants were filtered and used for HPLC analysis. Flavan-3-ols were separated in a phenyl hexyl column (250 X 4.6 mm i.d. particle size 5 /im) at 30°C. Solvents A and B were 2 per cent acetic acid in ACN and 2 per cent acetic acid in water, respectively. Gradient elution was 0-lQmin, 95 per cent B 10-4Qmin, to 82 per cent B to 40-5Qmin 82 per cent B. The flow rate was 1 ml/min. Theaflavins were determined in an ODS column (100 X 4.6 mm i.d. particle size 3pm) at 30°C. The flow rate was 1.8 ml/min and solvent B was the isocratic mobile phase. The data demonstrated that flavan-3-ols disappear during the oxidation process while the amount of theaflavins with different chemical structures increases [177],... 

D. Thomas and J. P. Kernevez, Eds.. Analysis and Control of Immobilized Enzyme Systems. North-Holland, New York. 1976. 

A renewed interest in this research field may lead to the construction of functional immobilized biocatalysts that surpass the conventional definition, or usually credited advantages, of immobilized biocatalysts with regard to their capabilities as catalysts [22-24], i.e. immobilized enzyme systems in which, for example, an enzymatic process can be controlled by externally applied stimuli such as light, electric fields, pH, temperature, and mechanical force. In such cases, what is crucial in system construction is not to rely on a possible... 

As described above, volume-phase transitions in gels with immobilized enzymes are available for the biochemical creation of mechanical energies when coupled with enzymatic changes within the gel phase. In the design of such immobilized enzyme systems, the concept of controlling the phase transition threshold by... 

The authors experience is that the enzymic phosphorylation and diphosphorylation of nucleoside monophosphates is very efficient the yields are nearly quantitative and the immobilized enzyme system appears reusable for at least three months. 

The Immobilized Enzyme System. The glucose isomerases used are immobilized and granulated to a particle size between 0.3 and 1.0 mm. The enzyme granulates must be rigid enough to withstand compaction when they are packed into the column. Ca2+ acts as an inhibitor in the system, and therefore calcium salts need to be removed from the feed syrup. Conversely, Mg2+ acts as an activator, and magnesium salts are added to the feed syrup. 

Immobilized enzyme systems can be differentiated according to mode of immobilization, carrier properties, and rate-determining step. 

Affinity chromatography combines the analytical and chemical capacities of chemically bonded stationary phases and immobilized enzymes. Technology and methodology of both techniques are joined in the development of affinity stationary phases. Since steric requirements are even more determining than in simple immobilized enzyme systems, spacer molecules have great importance in these modifications. Commonly used spacer arms are summarized in figure 8.3. 

Other plants such as potatoes, cauliflower, cherries, and soybeans and several fungi may also be used as sources of peroxidase enzymes. Soybeans, in particular, may represent a valuable source of peroxidase because the enzyme is found in the seed coat, which is a waste product from soybean-based industries [90]. In this case, it may be possible to use the solid waste from the soybean industry to treat the wastewaters of various chemical industries. In fact, the direct use of raw soybean hulls to accomplish the removal of phenol and 2-chlorophenol has been demonstrated [105]. However, it should be noted that this type of approach would result in an increase in the amount of solid residues that must be disposed following treatment. Peroxidases extracted from tomato and water hyacinth plants were also used to polymerize phenolic substrates [106], Actual plant roots were also used for in vivo experiments of pollutant removal. The peroxidases studied accomplished good removal of the test substrate guaiacol and the plant roots precipitated the phenolic pollutants at the roots surface. It was suggested that plant roots be used as natural immobilized enzyme systems to remove phenolic compounds from aquatic systems and soils. The direct use of plant material as an enzyme source represents a very interesting alternative to the use of purified enzymes due to its potentially lower cost. However, further studies are needed to confirm the feasibility of such a process. 

Weetall HH, Pitcher WHJ (1986) Scaling up an immobilized enzyme system. Science 232 1396-1403... 

Figure 10-23 Flow Diagram of an Immobilized Enzyme System (Column Operation of Lactase Immobilized on Phenol-Formaldehyde Resin with Glutaraldehyde). Source From W.L. Stanley and A.C. Olson, The Chemistry of Immobilizing Enzymes, J. Food Sci., Vol. 39, pp. 660-666, 1974.

The immobilization of invertase on aluminium hydroxide (2) was one of the earliest reports of adsorption technology. The use of aminoacylase adsorbed on DEAE-Sephadex for producing L-amino acids from a racemic mixture of their corresponding ethyl esters (4) was the first industrial application of an immobilized enzyme system. The basic disadvantage of this convenient technique is that binding is weak and the enzyme slowly leaches out. However, for many purposes, this slow leakage is not an important handicap. Immobilizing enzymes by adsorption has been extensively reviewed (5, 6, 27). Some special approaches are described (1, 28-30). 

Munnecke, D.M. (1979). Hydrolysis of organophosphate insecticides by an immobilized-enzyme system. Biotechnol. Bioeng. 21 2247-61. 

Advances in the understanding of structure-activity/selectivity relations for enzymes evolving from the use of x-ray, NMR, and other instrumental methods for characterization of enzyme structures should contribute to the development of improved immobilized enzyme systems for both analytical and industrial applications. Immobilized enzyme technology has enormous potential, but significant advances on several fronts are necessary prior to widespread industrial use of this technology. Katchalski-Katzir has discussed this problem in a review of past successes and failures in efforts to employ immobilized enzymes in the food, pharmaceutical, and chemicals industries. ... 

X 10 moles trypsin per liter fluid volume. To demonstrate the feasibility of using the Ford method to determine the active-site of our immobilized enzyme systems, trypsin CVB-PHEMA-PABS-carbamate was treated in a circulation reactor with NPGB and the titration is Illustrated in Figure 4. The amount of p-nitro-phenol produced by the burst is equal to the amount of the active immobilized trypsin which, for this particular system, turns out to be 31% of the total bound enzyme. Active-site titrations of soluble trypsin were performed according to Chase and Shaw (16), and the active molecules for free trypsin was found to be 70% of the total protein involved. Consequently, the retention of active molecules for the immobilized enzyme was calculated 45%. The specific activity is 17% (Table III) for the same system so the efficiency of the system, based on the actually available active sites, was 38%. Thus, 62% of the initially active trypsin bound has lost its activity upon binding. 

Enzyme electrodes are essentially immobilized enzyme systems. Crochet and Montalvo (C15) developed a technique for cholinesterase based on coupling a small pH electrode to a thin polymer membrane. At the electrode surface, cholinesterase interacted with acetylcholine to produce acetic acid, which was detected by the pH electrode. Excellent sensitivity was achieved by the use of a very thin film of enzyme solution, with extremely low strength of buffer containing the enzyme and almost complete suppression of spontaneous hydrolysis of the substrate. 

The deamination step has been carried out in broth, followed by extraction of the deaminated derivatives (51). Immobilized enzyme systems have been used for the 7ACA step (55). 

Havens, P.L., and H.F. Rase. 1991. Detoxification of organophosphate pesticide solutions Immobilized enzyme systems. Pp. 261-281 in Emerging Technologies in Hazardous Waste Management II, ACS Symposium Series No. 468. D.W. Tedder and F.G. Pohland, eds. New York, N.Y. Oxford University Press. 

Major breakthroughs that facilitated enzyme application on an industrial scale were improvements in the area of enzyme isolation, purification and immobilization. Thus, the development of genetically engineered microorganisms accounted for the high yield production of penicillin amidases. Also, the introduction of immobilized enzyme systems, both for whole cell systems and for the isolated and purified amidases[59, 62, 6i, resulted in prolonged enzyme stability enabling reuse and continuous process modes. As a result of this, the enzymatic routes currently display far better economics for both 6-APA and 7-ADCA production (Fig. 12.2-4) compared with their chemical counterparts. 

Thomas, D., 1975, Proc. of Int. Symp. on Analysis and Control of Immobilized Enzyme Systems, Compiegne, Edited by Thomas, D. and Kemevez, J., Elsevier. 

A variety of immobilization methods were tested for industrial purposes, from which aminoacylase immobilized by ionic binding to DEAE-Sephadex was chosen. Through chemical engineering studies on aminoacylase columns we designed an enzyme reactor for continuous production. Since 1969, we have been operating several series of enzyme reactors for the production of L-methionine, L-valine, L-phenylalanine and so forth. With this immobilized enzyme system, L-amino acids can be produced more economically con cured to the conventional batch system using native enzyme as shown in Fig. 1. 

A great deal more needs to be known about these important phosphoprotein phosphatases and the in vivo role they may play in the regulation of cellular activity and phosphate turnover. An immobilized enzyme system for the phosphorylation of proteins has been described (108a). 

In vitro studies on the ability of horseradish peroxidase or lacto-peroxidase to catalyze iodination of proteins at tyrosine residues are becoming quite frequent (244-248). The studies are designed either to provide better understanding of the incorporation of iodine into thyroid proteins or to develop better ways of producing radioactively labeled proteins for tracer studies. An immobilized enzyme system has been developed to achieve this second purpose (248). A large number of proteins incorporate iodine by this method which is superior to iodination with chloroamine-T or IC1. These iodinated proteins should prove invaluable in nutritional studies. 

For those reactions where the products are larger in molecular weight than the enzyme, one is forced to go to an immobilized enzyme system or to this system where the dextran-enzyme complex can be made larger than the products. 

In Japan, a leading country in the development of enzymatic engineering, more than seventy enzymes have already been immobilized, but only ten of them are used in most existing applications. This limitation, compared to the great potential of immobilized enzyme systems, requires that research efforts be directed towards the realization of more efficient immobilization techniques characterized by low carrier costs, and the development of practical equipment.4... 

Enzyme immobilization in the sponge of polymeric asymmetric and symmetric membranes has the advantage of a stable immobilized enzyme system along with the improved isolation of enzymatic proteins from immunodefensive system actions, high molecular weight inhibitors and proteolytic enzymes. A suitable choice of membranes based on their separation properties allows substrate molecules to permeate through the membrane while, at the same time, separating undesirable compounds from the enzymatic proteins. 

In previous papers and patents, we have introduced our novel system and provided proof of concept for the co-immobilized enzyme pellet system [29, 35, 36, 49, 50]. In this paper, we present results of experiments that illustrate the effectiveness of our co-immobilized enzyme system for isomerization under conditions optimal for fermentation by common S. cerevisiae. We have changed the media composition in an effort to both shift the equilibrium in favor of xylulose production and improve XI activity [51]. In addition to borate, we also investigated the effect of metal ion addition to the kinetics and equilibrium of the isomerization reactions as certain divalent metal ions have been shown to increase the long-term activity of XI [41,42]. This paper provides results of these experiments in our co-immobilized enzyme system and their potential impact on SIF enhancement. 

Fig. 2 Proof-of principle that two pH microenvironments are developed in the co-immobilized enzyme system via urea hydrolysis. Solid symbols are used for unaltered Sweetzyme open symbols are used for the Xl/urease co-immobilized pellets. The three experiments shown are A pH 7.5 B pH 4.5 with 0.01 M urea, and C pH 4.5 with no urea each used 0.13 g pellets. Unaltered Sweetzyme yielded no xylulose production at pH 4.5 (data not shown). Xylulose production shown for B indicates that XI has activity when urea is added...

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