Enzyme loading test

The variation of the enzyme loading is a means of determining the minimum amount of enzyme required for maximum sensitivity. Furthermore, this test reveals the magnitude of the enzyme reserve of diffusion controlled sensors. 

As is evident from Fig. 29, the transient from the linear region to saturation occurs at /e values between 7 and 20. This agrees with the 

Apparent Enzyme Activity and Km Value of Adsorbed Layers and Enzyme Membranes 

Enzyme Immobilization Apparent enzyme activity (mU/cm2) Apparent Am values soluble immob. (mmol/1) References 

Owing to differences in the Kyi values and the layer thickness, the transient from kinetic to diffusion control of different enzyme electrodes takes place at rather different enzyme activities. Gelatin-entrapped enzymes exhibit transient values of 0.17 U/cm2 (uricase,iifM= 17 pmol/1), 16 U/cm2 (urease, Kyi = 2 mmol/1) and 1.0 U/cm2 (lactate monooxygenase, Km = 7.2 mmol/1). 

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Fig. 29. Enzyme loading test of gelatin entrapped GOD as performed at pH 7 and 25°C. (Redrawn from Scheller et al., 1988).

Weigelt et al. (1987a) optimized an LMO-gelatin membrane for application in the Glukometer analyzer (ZWG, GDR). The enzyme loading test (Fig. 56) shows that above 1 U/cm2 the sensor is diffusion controlled,... 

Figure 17.2 An enzyme loading test of a lactate oxidase membrane electrode e 2.5 mmoir lactate + 5.0 mmoir lactate (reproduced with the permission of Elsevier Science Publishers BV).

A commercial pectinase, immobilised on appropriately functionalised y-alumina spheres, was loaded in a packed bed reactor and employed to depolymerise the pectin contained in a model solution and in the apple juice. The activity of the immobilized enzyme was tested in several batch reactions and compared with the one of the free enzyme. A successful apple juice depectinisation was obtained using the pectinase immobilised system. In addition, an endopolygalacturonase from Kluyveromyces marxianus, previously purified in a single-step process with coreshell microspheres specifically prepared, was immobilised on the same active support and the efficiency of the resulting catalyst was tested. 

If checking the PGase activity associated with a single enzyme preparation, there will be five test solutions. The enzyme load and substrate blanks are done at two different enzyme concentrations. With duplicate or triplicate analyses, there will thus be ten or fifteen tubes in total. 

The performance of cellulase and amylase immobilized on siliceous supports was investigated. Enzyme uptake onto the support depended on the enzyme source and immobilization conditions. For amylase, the uptake ranged between 20 and 60%, and for cellulase, 7-10%. Immobilized amylase performance was assessed by batch kinetics in 100-300 g/L of com flour at 65°C. Depending on the substrate and enzyme loading, between 40 and 60% starch conversion was obtained. Immobilized amylase was more stable than soluble amylase. Enzyme samples were preincubated in a water bath at various temperatures, then tested for activity. At 105°C, soluble amylase lost -55% of its activity, compared with -30% loss for immobilized amylase. The performance of immobilized cellulase was evaluated from batch kinetics in 10 g/L of substrate (shredded wastepaper) at 55°C. Significant hydrolysis of the wastepaper was also observed, indicating that immobilization does not preclude access to and hydrolysis of insoluble cellulose. 

Metabolic loading tests and the determination of enzyme saturation with cofactor measure the ability of an individual to meet his or her idiosyncratic requirements from a given intake, and, therefore, give a nearly absolute indication of nutritional status, without the need to refer to population reference ranges. A number of factors other than vitamin intake or adequacy can affect responses to metabolic loading tests. This is a particular problem with the tryptophan load test for vitamin Be nutritional status (Section 9.5.4) a number of drugs can have metabolic effects that resemble those seen in vitamin deficiency or depletion, whether or not they cause functional deficiency. 

At One time it was thought that women taking oral contraceptives were at risk for B deficiency. This notion seem-S to have been in error. The error was due to a misinterpretation of the tryptophan load lest. As mentioned earlier, a deficiency in vitamin B(,can induce the accumulation of specific intermediates of the tryptophan catabolic pathway and enhanced excretion in the urine. Oral contraceptives can also induce ar increase in the formation and excretion of specific intermediates by stimulating the activity of specific enzymes of the tryptophan catabolic pathway, This stimulation was responsible for the false indications of deficiency. Independently of the tryptophan load test, there continues to be some evidence for risk associated with the use of oral contraceptives. Oral contraceptive use may result in lowered levels of plasma vitamin Bf, Tlicsc lowered levels may result in a vitamin deficiency when coupled with pregnancy and lactation. 

To test the model predictions, the single-pass clearance of heparin was determined as a function of time in three different sheep. For each experiment, the volume of beads, the initial enzyme loading, the animal s antithrombin level, the hematocrit, the inlet heparin concentration, and the half-life of the enzyme were used to generate model predictions for the clearance of heparin as a function of time. 

Reusability of immobilized CALB was tested in subsequent cycles of methyl butyrate hydrolysis. It can be observed in Fig. 7 that CALB-7A retained less than 50% of its initial hydrolytic activity after the third cycle of reaction whereas Novozyme 435 retained almost 70% after the tenth cycle (Fig. 7). Other authors [36] observed that CALB immobilized on activated carbon retained more than 55% of its initial activity after the sixth cycle of methyl butyrate hydrolysis. The worse operational stability of CALB immobilized on coconut fiber, when compared to CALB immobilized on activated carbon and to Novozyme 435, may be due to enzyme desorption during reaction, induced by the hydrophobic substrate, and by the low enzyme load adsorbed. As discussed before, the driven forces of CALB adsorption on coconut fiber are electrostatic interactions that are weaker than hydrophobic interactions, which predominate on Novozyme 435 and CALB adsorbed on activated carbon. Furthermore, both aetivated carbon and the resin used in the preparation of Novozyme 435 are porous support with high superficial area available for enzyme immobilization, allowing obtaining of high enzyme load. Coconut fiber, on the other hand, does not have a porous structure, and it has a low surface area [27], making it difficult to achieve high enzyme loads. 

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