Enzyme loading dependence

In lab-scale hydrolysis and fermentation tests, between 10 and 35 FPU/g cellulose is commonly used as the optimum enzyme loading, depending on both substrate concentration and digestibility of the material used. However, considering both the overall cost of the... 

The procedure shows that it is feasible to combine racemization with the kinetic resolution process (hence the DKR) of R,S)- ethoxyethyl ibuprofen ester. The chemical synthesis of the ester can be applied to any esters, as it is a common procedure. The immobilized lipase preparation procedure can also be used with any enzymes or support of choice. However, the enzyme loading will need to be optimized first. The procedures for the enzymatic kinetic resolution and DKR will need to be adjusted accordingly with different esters. Through this method, the enantiopurity of (5)-ibuprofen was found to be 99.4 % and the conversion was 85 %. It was demonstrated through our work that the synthesis of (5)-ibuprofen via DKR is highly dependent on the suitability of the reaction medium between enzymatic kinetic resolution and the racemization process. This is because the compatibility between both processes is crucial for the success of the DKR. The choice of base catalyst will vary from one reaction to another, but the basic procedures used in this work can be applied. DKRs of other profens have been reported by Lin and Tsai and Chen et al. ... 

The amount of enzyme preparation added will depend on the relative activity of the enzyme preparation. A convenient starting point would be to add 40 pi enzyme preparation to enzyme load I and substrate blank 1, and 80 pi to enzyme load 2 and substrate blank 2. 

The left side summarizes the enzyme-dependent terms turnover number kcat and surface concentration renz- In case of enzyme-loaded films, renz should be replaced by the product of enzyme volume concentration in the film and the film thickness. The right side summarizes the experimental conditions diffusion coefficient D, concentration c of the mediator, and UME radius rT. The feedback mode always requires an as small as possible working distance d. The smaller the UME the more difficult it will be to detect the activity of the immobilized enzyme. 

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. 

Various authors have shown that non-ionic surfactants have a beneficial effect on the hydrolysis of cellulosic and lignocellulosic substrates, whereas anionic and cationic surfactants alone interfere negatively (Castanon and Wilke, 1981 Helle et al, 1993 Park et al, 1992 Ooshima et al., 1986 Traore and Buschle-Diller, 1999 Ueda el al., 1994 Eriksson el al., 2002). Increases in the amount of reducing soluble sugars and substrate conversion were reported. The effect depends on the substrate and is not observed for soluble substrates, such as carboxymethylcellulose or cellobiose. Nonionic surfactants increased the initial rate of hydrolysis of Sigmacell 100, and when they were added later in the process they were less effective (Helle et al, 1993). They same authors found also that the addition of cellulose increases the critical micelle concentration of the surfactant, which indicates that the surfactant adsorbs to the substrate. Surfactants are more effective at lower enzyme loads and reduce the amount of adsorbed protein (Castanon and Wilke, 1981 Ooshima et al, 1986 Helle et al, 1993 Eriksson et al., 2002) which can be used to increase desorption of cellulase from the cellulosic substrate (Otter et al., 1989). Anyhow, the use of surfactants to enhance desorption of cellulases from textile substrates in order to recover and recycle cellulases was not successful (Azevedo et al., 2002b). 

The specific activity and longevity of glucose oxidase intercalated in hectorite is dependent in part on the extent of surface coverage. As illustrated in Figure 3, the enzyme activity decays by two pathways a fast pathway which is loading dependent, and... 

The increase of the measuring value of the amperometric glucose electrode with increasing substrate concentration reflects the course of a Michaelis-Menten curve and reaches a concentration-independent saturation corresponding to the maximum rate, vmax. The sensitivity of the GOD electrode depends on the enzyme loading (Fig. 30) (Scheller et al., 1988). The substrate concentration giving rise to the half-maximum current in air saturated solution is between 1.4 and 1.8 mmol/1 glucose. 

Fig. 30. Dependence of the stationary current of a GOD electrode on glucose concentration at different enzyme loadings. Electrode surface 0.22 mm2 electrode potential +600 mV vs. Ag/AgCl conditions as in Fig. 28 curves 1-3 oxygen-saturated solution curves 4—6 air-saturated solution.

Fig. 116. Inhibition of urease as it depends on the concentration of fluoride and the enzyme loading of the sensor. (Redrawn from Tran-Minh and Beaux, 1979).

Figure 17.3 The pH dependence of a glucose oxidase membrane electrode at different enzyme loadings and different glucose concentrations electrode surface, 0.22 mm electrode potential, +600 mV against Ag/AgCl phosphate buffer, 0.66 molT (reproduced with the permission of Elsevier Science Publishers BV). 

Provided that external diffusion is not limiting, P depends linearly on S and the ratio of the substrate and product diffusion coefficients (/> and Dp), and nonlinearly on a square-root expression, the so-called Thiele modulus (the square of which is the enzyme loading factor, 4). 

Arrhenius plot of the tmperature dependence of the response of a GOD electrode with different enzyme loadings to different giucose concentrations and to hydrogen peroxide. 

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