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5.7.1 Free enzyme studies

From the studies conducted using free enzyme it is clear there are a number of benefits to this form of catalyst over whole cell and immobilised enzyme. The reaction rates seen were much closer to the maximum possible c.f. whole cell studies (where reaction rates were found to be very low). There is however the extra expense of additional cofactor and of the second enzyme. On lOOmL scale using 4-MCH as substrate the reaction rate was observed to be initially rapid, becoming more linear. This may be due to a limitation in the removal o f acetone from solution, particularly in shake flasks where the mass transfer rate may be low. Similar experiments using 2-HCP, which forms a second phase did not indicate the slowing o f product formation. This may be because acetone partitions in some degree into the organic phase and removes some of the inhibitory effect on TBADH. The use o f a second phase was successful in raising product yields above the aqueous solubility limit. The formation of an emulsion was observed, which was not unexpected as there was a significant amount of dissolved protein. This did not appear to affect the formation of product at lOOmL scale although the purification of 6-HTPO is made more challenging than from a whole cell or immobilised enzyme reaction. It is also unclear as to the mass transfer characteristics of the system, the rate of mass transfer from the organic to aqueous phase may or may not be limiting and so measurement of the mass transfer coefficient should be a target for future studies. At 1.5L scale the increased agitation and aeration coupled with a second liquid phase seemed to cause the rapid deactivation of CHMO, the yield produced from the 2-HCP reaction was very low and the reaction mixture became visibly more opaque. The single phase of the equivalent 4-MCH reaction did not seem to cause the same effect on the protein, and the yield produced was more satisfactory. There was however the limitation on agitation rate in both reactions due to excessive foaming above 250rpm, a serious

bottleneck in supplying oxygen to the reaction. Using homogenate also presents the problem of maintaining sterility, homogenate is extremely vulnerable to microbial attack, the implication is that the reaction may be halted prematurely limiting the yield, the process may also be difficult to validate on a large scale.

5.7.2 Immobilised enzyme studies

Initial attempts to immobilise CHMO were not successful, retained activities upon immobilisation were disappointingly low. The inclusion of trace amounts o f reaction components acted to preserve the integrity of the active site during the immobilisation procedure, although the retained activities only improved to a maximum of 12%. The optimisation of all the variables that affect the retained activity (temperature, pH, time, buffer molarity, protein loading, protein purity and trace compound concentration) is very much still a ‘hit and miss’ procedure with little in the way o f a systematic approach. The retained activity of 12% therefore still represents an unoptimised system but it does permit the use of the immobilised enzyme to perform a number of studies. Judicious selection of the immobilisation conditions can increase activity of the enzyme upon immobilisation, such as Transketolase from E.coli (Brocklebank et al, 1996) which initially produced residual activities of 2 0% and after a period of development work this had increased to 60%. The specific activity of immobilised CHMO produced was only in the region l-1.2Ug ’ which is also low. Possibly this is due to the dilute nature o f CHMO within the clarified lysate, where it represents cal% of the total protein. Immobilisation o f both enzymes simultaneously from one solution produced a catalyst with a specific activity of 0.6U g'\ this is lower due to the compromise in conditions that have to be used for both enzymes resulting in lower binding for both. However this does illustrate the potential for immobilising a catalyst in a single procedure from a lysate of a recombinant E.coli strain containing both enzyme activities.

The stability o f the immobilised form of CHMO illustrated the increase in stability that is often generated by this technique. Enzyme activity decreased only gradually in the presence o f elevated levels of both 4-MCH and 5-MOP. This compares well with soluble enzyme, which under similar conditions would be completely deactivated in

1-2 days. However this increase in stability must be offset against the drop in activity seen during the immobilisation procedure. The overall effectiveness of either form o f the enzyme is thus the amount o f product it will form per unit of activity initially produced within the cell. This can be calculated by integrating under a curve of activity as a function of time. This is shown in Figure 5.12. The basis used is 1 unit o f CHMO. This becomes 0.12 units after immobilisation. Although the immobilised form is likely to be active beyond the 14 days o f the study, this was considered the limit of the use o f the immobilised form, as it is unwise to extrapolate the activity beyond the timescale actually measured.

Free enzyme theoretically will produce 1 44mmol product per unit of activity whereas immobilised enzyme produces 1.88mmol, an increase of 30%. The increase is only modest due to the loss of activity upon immobilisation. If the residual activity of the immobilised enzyme were to be increased to e.g. 45% the increase in productivity rises to 360%. The enzyme was only assayed over 14 days and from inspection of the stability plot it is more likely that the figure of 30% would rise if a longer stability trial were conducted. This is not the entire picture as free enzyme can realistically only be used once whereas the immobilised form allows more flexible processing by introducing the possibility of repeated batches.

Losses of immobilised TBADH activity over time are reported to be extremely low under processing conditions (Keinan et al, 1986) such that periodically a percentage of the catalyst would have to be re-charged to make up for declining levels o f immobilised CHMO. TBADH was immobilised with much higher residual activity (42%) and to a higher specific activity (13.6Ug'^). The higher retained activity is to be expected as the protein is highly resistant to dénaturation and conformational change. The specific activity is higher as the source o f the protein was initially much purer than the preparation o f CHMO.

Reactions performed with the immobilised enzymes illustrate some of the potential benefits and disadvantages of using CHMO in this form. Separation of catalyst is much less problematic than using soluble enzyme. The beads o f Eupergit C easily settle out from solution and product rich liquor can simply be decanted. The product stream thus only contains a small quantity o f salts (fi’om pH control) and cofactor. Enzyme kinetics often change when immobilised due to mass transfer limitations of the substrates gaining access to the enzyme within the bead, for example the value of apparent Km often rises. In the case of cofactor the Km value was shown to be critical in running a recycle system efficiently (chapter 4), any increase in this value due to immobilisation (for either CHMO or TBADH) may necessitate running the reaction with additional cofactor to compensate, lowering the TTN achievable. The low retained activity could also be due to a change in the kinetics, a 4-MCH concentration of 2mM is used in both the soluble and immobilised assay. This is far in excess of the Km value for soluble enzyme (0.016mM) but the value for immobilised enzyme may be higher and the assay may be kinetically limited. Upon closer inspection this is unlikely because the 2L reactions contained higher 4-MCH concentrations and the reaction rate did not appear to be significantly higher. The distribution of immobilised protein has previously been shown to occur evenly throughout an Eupergit C bead (Brocklebank, 1998) this offers CHMO protection from gas/liquid and liquid/liquid interfaces. Higher agitation rates can be used without fear of foaming or protein damage, the result being a ‘de-bottlenecking’ of the supply of oxygen to the reaction. The rapid deactivation o f CHMO in the 2-phase agitated reaction would also not occur.

c 3 X O 1.0 0.8 Free enzym e 0.6 0.4 Immobilised enzym e 0.2 0.0 400 300 100 200 0 Time, h