Enzyme stirrer

Fig. 40. Cross section of an enzyme stirrer containing immobilized GDH. (Redrawn from Kuan and Guilbault, 1977).

Kuan and Guilbault (1977) immobilized GDH on the surface of a stirrer and registered the liberated NADH fluorimetrically. The enzyme stirrer (Fig. 40) was stable for two months and permitted 500 analyses of glucose in plasma to be performed. 

A magnetic enzyme stirrer coupled with ADH immobilized on cellulose has been developed by Kuan et al. (1978). The immobilized enzyme was stable for 8 days. The reaction was indicated fluorimetri-cally. 

The coimmobilized enzymes have also been used for total cholesterol determination in serum by using an enzyme stirrer (Huang et al., 1977) and a reactor electrode (Karube et al., 1982b Yao and Wasa, 1988b). In... 

Immobilized enzyme stirrer for analytical determination of D-glucose in plasma... 

Immobilized-enzyme stirrer a specific enzyme electrode for urea Active immobilized enzyme Enzyme-reactor electrode used in a determination of urea Active immobilized enzyme determination of heat changes in the proximity of immobilized enzymes with an enzyme thermistor, and the use of an enzyme thermistor in assays for metabolites Comparisons of methods for the hydrophobic immobilization of enzymes with retention of activity... 

The effeet of temperature satisfies the Arrhenius relationship where the applieable range is relatively small beeause of low and high temperature effeets. The effeet of extreme pH values is related to the nature of enzymatie proteins as polyvalent aeids and bases, with aeid and basie groups (hydrophilie) eoneentrated on the outside of the protein. Einally, meehanieal forees sueh as surfaee tension and shear ean affeet enzyme aetivity by disturbing the shape of the enzyme moleeules. Sinee the shape of the aetive site of the enzyme is eonstrueted to eoirespond to the shape of the substrate, small alteration in the strueture ean severely affeet enzyme aetivity. Reaetor s stirrer speed, flowrate, and foaming must be eontrolled to maintain the produetivity of the enzyme. Consequently, during experimental investigations of the kineties enzyme eatalyzed reaetions, temperature, shear, and pH are earefully eontrolled the last by use of buffered solutions. 

The experimental results in Fig. 27 show the influence of the reactor system (see Fig. 28) on the disintegration of enzyme activity. It was found that the low-stress bladed impeller results in less activity loss than the propeller stirrer which causes much higher maximum energy dissipation ,. The gentle motion the blade impeller produces means that stress is so low that its disadvantage of worse micro mixing in NaOH (in comparison with the propeller) is more than compensated. 

Mechanical forces such as shear and surface tension affect enzyme activity by disturbing the shape of the enzyme molecule. Since the shape of the active site of the enzyme is specifically engineered to correspond to the shape of the substrate, even small changes in structure may drastically affect enzyme activity. Consequently, fluid flow rates, stirrer speeds, and foaming must be carefully controlled in order to ensure that an enzyme s productivity is maintained. 

HNL enzyme solution (Codexis Inc, 2.5 mL) trimethylsilylcyanide (1.25 mL) saturated ammonium sulfate solution (2.5 mL) ethyl acetate (25 mL) nitrogen gas fume hood cyanide detector 50 mL flask magnetic stirrer. 

The use of a monolithic stirred reactor for carrying out enzyme-catalyzed reactions is presented. Enzyme-loaded monoliths were employed as stirrer blades. The ceramic monoliths were functionalized with conventional carrier materials carbon, chitosan, and polyethylenimine (PEI). The different nature of the carriers with respect to porosity and surface chemistry allows tuning of the support for different enzymes and for use under specific conditions. The model reactions performed in this study demonstrate the benefits of tuning the carrier material to both enzyme and reaction conditions. This is a must to successfully intensify biocatalytic processes. The results show that the monolithic stirrer reactor can be effectively employed in both mass transfer limited and kinetically limited regimes. 

The monolithic stirrer reactor (MSR, Figure 2), in which monoliths are used as stirrer blades, is a new reactor type for heterogeneously catalyzed liquid and gas-liquid reactions (6). This reactor is thought to be especially useful in the production of fine chemicals and in biochemistry and biotechnology. In this work, we use cordierite monoliths as stirrer blades for enzyme-catalyzed reactions. Conventional enzyme carriers, including chitosan, polyethylenimine and different are used to functionalize the monoliths. Lipase was... 

In summary, it can be concluded that the monolithic stirrer reactor is a convenient reactor type both for the laboratory and the production plant. It is user-friendly and can be used to compare different catalysts in the kinetically limited regime or hydrodynamic behavior in the mass transfer controlled regime. Stirrers or monolith samples can be easily exchanged and reloaded to suit the desired enzyme and/or reaction conditions. 

Most liquid phase chemical and biochemical reactions, with or without catalysts or enzymes, can be carried out either batchwise or continuously. For example, if the production scale is not large, then a reaction to produce C from A and B, all of which are soluble in water, can be carried out batchwise in a stirred tank reactor that is, a lank equipped with a mechanical stirrer. The reactants A and B are charged into the reactor at the start of the operation. The product C is subsequently produced from A and B as time goes on, and can be separated from the aqueous solution when its concentration has reached a predetermined value. 

When the production scale is large, the same reaction can be carried out continuously in the same type of reactor, or even with another type of reactor (Chapter 7). In this case, the supplies of the reactants A and B and the withdrawal of the solution containing product C are performed continuously, all at constant rates. The washout of the catalyst or enzyme particles can be prevented by installing a filter mesh at the exit of the product solution. Except for the transient start-up and finish-up periods, all the operating conditions such as temperature, stirrer speed, flow rates, and the concentrations of incoming and outgoing solutions remain constant - that is, in the steady state. 

On occasion, solid particles - such as catalyst particles, immobilized enzymes, or even solid reactant particles - must be suspended in liquid in stirred-tank reactors. In such cases, it becomes necessary to estimate the dimension and speed of the stirrer required for suspending solid particles. The following empirical equation [15] gives the minimum critical stirrer speed (s ) to suspend the particles. 

The enzyme-agarose conjugate, a gel, is stored as a suspension in the immobilization buffer. This gel is rather mechanically fragile. Magnetic stirrers should be avoided, and the contents of reaction vessels gently stirred on a rotary shaker. Attention is drawn to the poisonous nature of cyanogen bromide. 

Pour 100 mL of cellobiose solution with a certain concentration (20, 10, 5, 2, or ImM) into the reactor, turn on the stirrer, and wait until the solution reaches 50°C. Initiate the enzyme reaction by adding 1 mL of cellobiase solution to the reaction mixture and start to time. 

Pour 300 mL 0.1M Sodium acetate buffer solution (pH 4.7) into the vessel and start the stirrer and add 2.5 g of cellulase. (Typical FPU of commercially prepared enzyme is about 100 FPU/g.)... 

Fig. 1. Schematic diagram ot high-pressure apparatus tor enzyme activity tests. A, C02 cylinder B, syringe pump C, equilibrium cell D, sapphire windows E, magnetic stirrer F, white light source G, pressure transducer H, ball valve I, micrometering valve J, relief valve.

Reactions were carried out in the 140 ml reaction vessel. ( )-menthol and 200 mg of the enzyme peparation were placed in the reactor and the reactor connected to the system. The whole system was flushed with CO2 after which pressure and temperature were adjusted to 100 bar and 50°C. Water activity was set to the desired value by adding portions of water via the HPLC valve. The reaction was started by the addition of 1 ml of isopropenyl acetate once again via the HPLC valve. Final substrate concentrations were 20 mM menthol and 54 mM isopropenyl acetate. Stirring of the enzyme reactor was accomplished by a magnetic stirrer. In addition the reaction medium was pumped in a circle using a gear pump. The enzyme was retained in the enzyme reactor by a nylon membrane. Samples were taken via the HPLC-valve with a 500 pi sample loop, the content of which was expanded into hexane, and analyzed on a HP 5890 Series II gas chromatograph. 

---