Biotransformation process design and operating conditions

Previous work on a wide range o f transformations led to the identification o f a systematic design procedure for biocatalytic processes (Woodley and Lilly, 1992). This was based upon analysing the properties o f each component in the system and their interactions in a series of defined experiments. Table 4.2 indicates the key features of the design procedure and summarises the results obtained with the current biotransformation. Clearly the major process constraints are the low aqueous solubility of the substrate molecule, which led to the selection of a two-phase process, and the need to minimise the activity o f the amidase in order to maximise production of the desired 3-cyanobenzamide product.

Once the process has been defined, in this case a whole-cell catalysed conversion in a two- liquid-phase reactor, it is necessary to rapidly establish suitable operating conditions or a ‘window of operation’ (Woodley and Titchener-Hooker, 1996). A window is an area of operational space determined by the constraints of the bioprocess whether they are biological, physical, chemical, engineering or economic. Although the vsdndow does not define the optimum conditions for a process they place the process in an area that is close to this without the need for extensive factorial experiments which can be expensive and time consuming. Given the data presented in Figures 4.6-4.8 it is logical to define the operating window for the current process based on substrate concentration, catalyst concentration and phase volume ratio as all these parameters have a significant effect on the productivity of the transformation. These parameters were also the important variables in an operating window developed for the production of cis- glycols in a similar two-liquid phase process (Collins et aL, 1995).

Figure 4.9 shows a three-dimensional operating window for the two-phase hydration of 1,3-DCB by the whole cell NHase biocatalyst. The scales on the axes represent the ranges over which

§ 1 s ob §■

I

M T S L

i

L P M T \

Figure 4.9 Operating window for the Rhodococcus R312 catalysed transformation of 1,3-DCB in an aqueous-toluene two-phase system at pH 7 and 30°C. Axes indicate range over which variables were investigated: (MT) substrate mass transfer limitation; (LP) low amide

productivity (< 30 pmol min'^ mg"^); (T) toluene toxicity limitation; (SL) substrate solubility limit. The window was compiled from data shown in Figures 4.6-4.S.

5 - E

1 1

_{ro JO}

I §

I S

oo 0 20 40 60 80 100 120 140 Time (min)

Figure 4.10 Two-phase Rhodococcus R312 biotransformation kinetics for final optimised conditions; (■) 1,3-DCB,

(O)

3-CB,

(0)

3-CA. Cells produced as described in Section 2.3.1 and harvested at 30 hours. The biotransformation was as described in Section 2.6.1.2.6 with a phase volume ratio of 0.2 and the initial [1,3-DCB] concentration in the toluene phases of 25 g.L’VXhe biocatalyst concentration was 12.5 gww-L ^

conditions under which; (1) the system is not substrate mass transfer limited during amide synthesis, (2) amide synthesis is kinetically favoured over its further conversion to the corresponding acid and (3) the minimum specific initial rate of amide synthesis is 30 pmol min ' mg '. Substrate mass transfer limitations are avoided by operation at relatively low biocatalyst concentrations (Figure 4.6) and high concentrations of substrate in the organic phase (Figure 4.8). Damage to the catalyst due to phase toxicity effects is avoided by not operating at large values o f

Vr

(Figure

4.7).

Currently the boundaries o f the window are defined by straight lines which assumes the three parameters can be considered independently. Clearly this is an initial approximation but the operating window allows us to rapidly define the region of experimental space in which the process can be most feasibly operated.

To confirm the utility o f the operating window approach. Figure

4.10

shows the time course of a biotransformation carried out at a position within the defined window of operation for this process. The particular conditions chosen were those where the rate of amide production was expected to be highest i.e. a biocatalyst concentration of

12.5 gww-L'', Vr = 0.2,

and an initial organic phase substrate concentration o f

25 gww-L"'.

After

60

minutes o f operation a maximum

concentration of 6 mM 3- cyanobenzamide was obtained. This represents a 12 fold improvement in what was achieved in the single-phase biotransformation process {c.f data in Figure 4.2). The low rate of acid by-product formation during this time, due to inhibition o f the amidase activity by the amide product (Foumaud et a l, 1998), meant that less than 8 % w/w o f the amide produced was lost due to further metabolism by the amidase.

4.4 CONCLUSIONS

This chapter covered the application of a stmctured approach (Woodley and Lilly, 1992) to the design of a biotransformation process for the specific case o f aromatic nitrile hydration. It has shovm that the use of a two-phase process leads to a considerable enhancement in both the rate o f product synthesis and the final product yield compared to what could be achieved in a single­ phase process.

Isolation o f the NHase at process scale however was not feasible due to the rigid cell wall of the bacteria and the poor stability of the isolated enzyme. The whole cell form of the biocatalyst was thus used even though the activity of the associated amidase could overmetabolise the amide product into the corresponding acid. To overcome productivity limitations imposed by the characteristically low aqueous solubility of this class of substrate (-0.34 g 1"^ in the case of 1,3- DCB) the use o f an aqueous-organic two-phase bioreactor was investigated. After screening a wide range of solvents to act as a substrate reservoir toluene was selected as the organic phase due to the most favourable combination of Log P value (2.9) and 1,3-DCB saturation concentration (-30 g 1'^). The effects of phase volume ratio (0.05-0.3), wet weight biomass concentration (1.25-200 gww T^) and substrate concentration in the organic phase (5-25 g 1'^) were then combined to define a suitable operating window where the maximum space-time yield o f amide formation could be obtained. Compared to a single-phase transformation, the two- phase process yielded 12 times as much o f the amide product of which less than 8% w/w was lost due to overmetabolism.

Having established and optimised the two-phase biotransformation o f 1,3-DCB the hydrodynamics o f the system can now be investigated as a basis for the successful scale-up of the process. This will be discussed in Chapter 6. Before that, however, the issue of replacing

toluene (relatively low Log P value) as the substrate reservoir will be addressed in Chapter 5 where room temperature ionic liquids will be examined as possible solvent replacements.

5. USE OF ROOM TEMPERATURE IONIC

In document Process selection and design for the microbial biotransformation of poorly water-soluble nitrile compounds (Page 95-100)