PROGRAMACIÓN NO LINEAL (PNL)

The product concentration-time profiles depicted in figure 3.6 show results for reactions carried out at a phase ratio of 0.4 and an [Ea] of 2 g experiments were repeated at agitation rates of 1000 and 1800 rpm respectively. In order to compare the effect on the stability of the enzyme at these two rates o f agitation the reaction was monitored, under pH controlled conditions, over a period of time, (250 mins), where eventually very little enzyme activity remained, evidenced by no further increase in the product concentration in the reactor. Product concentrations, as given here, represent the overall product concentration based on medium volume since the phase ratio was the

■k 900

750

^ 600

O 450

m 300

150

250

200

150

50

Figure 3.6 Product concentration in the STR with time for the esterase hydrolysis. Reactor conditions: 0 = 0.4. [Ea] = 2 g n = 1000 (■), n = 1800 (O).

same and thus the true amount of enzyme in the reactor identical. The slope o f the line represents the rate at which product formation is occurring. Previous measurements based on the initial rate of the reaction, i.e. the first 3 minutes of reaction, indicated slightly higher rates at the stirrer speed of 1800 rpm. As reaction proceeds beyond the initial 3 minute period a reduction in the rate of product formation at 1800 rpm compared to 100 0 rpm is observed, the comparative reduction becoming more marked as reaction proceeds. At 46 minutes, for the reaction at 1800 rpm, enzyme has ceased to be active in contrast to the activity for the enzyme at 1 0 0 0 rpm which at a decreasing rate for a further 50 minutes up until 100 minutes at which very little further activity is observed. An effect on enzyme stability can thus be attributed to the rate o f agitation, higher rates resulting in reduced stability.

From the previous experiments it was established, by examining the specific reaction rate and relating this to substrate mass transfer in the reactor, an increase interfacial area due to the creation o f smaller organic droplets at higher agitation rates. Exposure o f the enzyme to such increased organic/aqueous interface has a comparative detrimental effect upon the enzyme stability. We also observed that at the higher stirrer speed air was drawn into the reactor as a separate gaseous phase and thus having its own distinct interface thus this may help to magnify the instability of the enzyme.

Due to the difference in stability a further 200 mM o f product was generated for the same amount o f enzyme in the reactor operated at 1 0 0 0 rpm compared with that operated at 1800 rpm. This required a further 50 minutes o f operation time. The final yield o f product from reactors operated under these conditions based on the percentage o f total substrate available in the reactor, which at a phase ratio o f 0.4 is 2.6 M, was at

1000 rpm 30 % and at 1800 rpm 23 %.

3.1.4. Phase Ratio

The initial product concentration time profiles depicted in figure 3.7 show results o f reactions carried out an agitation rate o f 1000 rpm and [Ea] o f 2 g 1*^ experiments being repeated at different phase ratios of 0.2, 0.4, 0.5, 0.6, and 0.75. With

E

Figure 3.7 Initial two liquid phase reaction kinetics for esterase hydrolysis in the STR. Reactor conditions; n =1000 rpm, [Ea] = 2 g 1*^; 0 = 0.2 (À), 0.4 (■), 0.5 ( • ) , 0.6 (□), 0.75 (O).

the exception of the phase ratio of 0.75 the reactions were carried out under a regime of pH maintenance, at high phase ratios the degree of inversion makes pH measurement impractical. However as previously discussed in section 2.3.3 measurements o f initial activities under pH and non pH maintained regimes correlate well and thus the comparison is valid. The [Ea] was selected so that the reactor was operated in a mass transfer limited regime as for the experiments at different agitation rates. The results reflect steady state measurements. The product concentrations are expressed on an aqueous phase volume basis, particularly important under changing conditions o f phase ratio as previously discussed.

Figure 3.7 was used to plot figure 3.8 which shows the specific activity expressed as a function o f phase ratio at an agitation rate o f 1000 rpm. The maximum specfic activity observed is lower than that previously determined in solely aqueous medium confirming the reactor was operated in a mass transfer limited regime. Therefore any changes in the specific activity are a result of increased availabilty of substrate. Highest activity is at a phase ratio of 0.4. Substrate mass transfer in the reactor is best facilitated at a phase ratio o f 0.4 when agitated at 1000 rpm.

Initially at low phase ratios only a small amount o f organic volume is present. The interfacial area is small resulting in low substrate KlA resulting in low enzyme activity. As the phase ratio is increased then the volume of organic phase relative to the volume o f aqueous phase is increased. The organic/aqueous interfacial area increases providing greater area and a higher substrate KlA resulting in higher enzyme activity. As we increase the phase ratio beyond 0.4 a resultant decline in activity is observed due to a lowering o f KlA.