Process constraints and implications

The relationship o f specific CHMO activity to pH shows a maximum activity at pH 9, with good retention of activity found from pH 8-10. These data are valuable since they highlight the pH range within which both enzyme and reaction component stability studies need to be performed. In this way the structured approach is helpful in leading the design process to more focused experimentation.

The solubility of the asymmetric reaction components was an order o f magnitude greater than the kinetic resolution reaction components. This immediately identifies probable divergent process designs for the two systems even from this limited data, highlighting the importance of the characteristics of the components. The 2-HCP system has veiy low solubility (<lgL'^) and so realistically could not be operated as a batch reaction unlike the 4-MCH system which theoretically could produce product titres in excess of 30gL'\ There was no constraint found due to volatilisation o f the substrates and products or by the stability o f the ketones. Both lactones were found to be alkali labile, probably as a consequence of base catalysed hydrolysis (Marsden, 1963). Significant losses over 24 hours were shown for both ketones at pH 7.5 and above. This constraint conflicts with the region of high enzyme activity, high reaction rates are seen at pH 8 and above. Degradation was particularly marked with

6-HTPO. This would suggest that product removal techniques need particular attention with this reaction.

The choice o f reaction pH will have to be a compromise. The compromise must maximise product yield, the compromise being between product formation rate and product degradation rate. The point o f highest difference between the two rates is the ideal condition, although this is true only if productivity over time is not a consideration. Upon inspection pH 7.5-8 seems to be the most satisfactory to allow

this. The stability o f the catalyst is also of importance, and follows a similar pattern to the region o f high activity, suggesting the process should not be operated below pH 8 (if the enzyme is considered to be a significant cost).

The choice of reaction pH can be illustrated diagramatically by constructing a window of operation (Woodley and Titchener-Hooker, 1996) which plots key variables to show the feasible limits o f operation in the system under investigation. By assuming a basis (arbitrarily) of no greater than 10% 5-MOP lost due to degradation and either a CHMO activity o f 30% or 50% o f it’s maximum (which occurs at a product concentration o f zero and pH9) it is possible to plot out a series o f windows, as shown in Figures 2.12. and 2.13. It can be seen that the window is larger if the constraint o f CHMO activity is relaxed. It is possible to plot other important variables such as substrate concentration or CHMO degradation rate, however pH and 5-MOP concentration are likely to be the most important variables and as such illustrate this technique.

Enzyme stability was shown to be a strong function o f cofactor concentration. After 24 hours the residual activity o f CHMO in a crude extract and whole cells was roughly equal at 25-30%. Deactivation in whole cells is more rapid than in homogenate, possibly due to active proteolysis. The addition o f cofactor to the isolated enzyme increased stability markedly, with the addition of 2mM NADPH nearly all inactivation was prevented. It has been suggested that there is a catalytically important cysteine residue at the active site (Donoghue and Trudgill, 1976) and these residues are known to be susceptible to oxidation (Wells and Estell, 1988). It is possible that the cofactor is able to bind to the active site and prevent access by oxygen to the cysteine. The protective effect appears to be a function of concentration, but not a function of the cofactor form as NADP^ offers equivalent protection. This could offer another explanation as to instability o f the enzyme within whole cells, as the intracellular concentration of cofactor may be very low and therefore not prevent oxidation. The role of oxygen in enzyme deactivation is reinforced by the lower rate o f inactivation found when free enzyme is exposed to nitrogen compared to air. The effect of increased interfacial contact by bubbling gasses through a solution of the enzyme is to accelerate inactivation, however a

significantly greater amount of activity remains in the oxygen free state (using nitrogen) than the use o f air. CHMO deactivation would thus seem to be a combination of oxidation and physical deactivation due to interfacial effects. This has important implications for the choice of catalyst form, whole cells would protect the enzyme firom interfacial damage but will be inherently unstable Free enzyme would be protected by cofactors but aeration damage will dominate and require artificial regeneration of NADPH. One solution may be to use immobilised CHMO that should be generally more stable to these effects.

With the 4-MCH system significant substrate and product inhibition was observed, as was enzyme degradation due to 4-MCH and 5-MOP. The type of inhibition was not elucidated. However due to the irreversible kinetics of the reaction it is not an equilibrium effect. The nature of the compounds involved may affect the monooxygenase by distorting the tertiary protein structure and disrupting intramolecular hydrogen bonds. No such effects were noted for the 2-HCP system. The probable reason for the difference is simply the maximum achievable aqueous concentration of the compounds. 2-HCP and 6-HTPO cannot exist in a high enough concentration within the enzymes environment to have a detrimental effect.

Performing whole cell reactions with both systems showed that the observed reaction rate was unexpectedly low, compared to the potential reaction rate. The potential reaction rate was easily determined by quantifying the activity of CHMO within the cells after cell disruption and clarification using the spectrophotometric rate assay. With both the 2-HCP and 4-MCH systems the reaction rate observed was less than 2% o f the potential velocity. This may be due to several factors; the internal pH o f E.coli may not suit CHMO activity; the cell’s metabolism may not be able the recycle cofactor quickly enough; 4-MCH/2-HCP may be extremely toxic to cells or there is a diffusional limitation, bottlenecking the transmission of ketone across the cell membrane. Repeating the reactions in the presence of toluene or ethanol in an attempt to permeablise the cell membrane showed no improvement in reaction rate, suggesting that diffusion was not limiting. This is supported by the fact that the two ketones used differ greatly in hydrophobicity but showed no significant difference in reaction rate. The reaction rate with 2-HCP would be higher if diffusion limited

because the more hydrophobic the molecule the easier the passage across the membrane should in theory be.

Intracellular CHMO activity seemed to be much more unstable than in resting cells, complete loss o f active CHMO occurred in under five hours. This is possibly due to the cells degrading CHMO in an attempt to generate energy for NADPH turnover to drive the reaction, although it would be expected that supplying glucose would have limited this effect. As such no satisfactory answer to this phenomenon could be produced.

It would appear that a whole cell catalyst is not appropriate and so even with the disadvantages associated with an isolated enzyme (principally the recycle o f NADPH) the increase in catalyst stability and reaction rates achievable with an isolated enzyme make this the catalyst form o f choice.

150

Product inhibition

Base catalysed 5-MOP degradation [5-MOP], mM WINDOW 25 g p H too low for CHMO 10.0

Figure 2.12 Operating window for 90% residual 5-MOP over 24 hours and a CHMO efficiency of >50%.

Figure 2.13 Operating window for 90% residual 5-MOP over 24 hours and a CHMO efficiency of >30%.

150

Product inhibition

Base catalysed 5-MOP degradation WINDOW [5-MOP], mM pH too low for CHMO 10.0 97