3.5 CIRCUNSTANCIAS AGRAVANTES

With the biotransformation optimised to obtain a conversion >95 % on 0.4 mg of D- glucose 6-phosphate (18), it was now possible to investigate the scale-up of the process. The first target was to scale-up to using 10 mg of starting material, which would be a scale-up of 25 times magnitude.

There were no observed problems with this scale up. Using 300 µg of TbINO1, increasing the reaction volume to 600 µL and extending the reaction time to 3 days gave quantitative conversions.

2.4.2 100 mg

The initial attempt using 100 mg of D-glucose 6-phosphate (18) was not as successful as the 10 mg scale. In this experiment, the total reaction volume was increased to 4 mL, whereas the amount of TbINO1 was left at 300 µg. This amount of protein should be more than enough turnover 100 mg of substrate in a reasonable time period. The reaction was assayed every 24 hours, and the initial conversion calculated, 26 %, looked promising. However, the next day the reaction had only progressed to 32 % conversion and, after a further 2 days, the reaction did not progress any further. At this point, an extra 300 µg of TbINO1 was added to the reaction. Unfortunately, the additional protein only pushed conversion to 40 %. The D-glucose 6-phosphate (18) being used was in the form of a disodium salt, meaning that the high concentration of the starting material would not dramatically alter the buffer pH. Potential differences in pH were, therefore, not hypothesised as a reason for low conversion.

The second experiment on this scale was conducted with a higher concentration. Using only 1 mL for the total volume would make the D-glucose 6-phosphate (18) and protein 4 times as concentrated. To better accommodate this increase in enzyme concentration, the amount of NAD+ present was increased from 2 mM to 4 mM. The reaction was also placed within an incubator with shaking.

The results for this experiment are shown in Graph 2.4.1. There was a steady increase in conversion over the first 4 days, however after this point the conversion plateaued at 62 %. Extra NAD+ was added at this point, however this produced no further reaction. This addition of NAD+ was conducted because within the literature109 it is stated that NAD+ is subject to decomposition for prolonged periods in aqueous solution at basic pH. At pH 8.5, this reaction is slightly more basic than the recommended pH of 7 to 7.5.109 the reaction had gradually turned yellow over the course of the experiment. The major mechanism for NAD+ decomposition is from nucleophilic attack on C-4 of the nicotinamide moiety, leading to a 1,4-dihydropyridine.109

Graph 2.4.1 – Calculated conversion as time progressed. Reaction conditions: 100 mg

D-glucose 6-phosphate (18), 4 mM NAD+_{, 1 mM DTT, 50 mM ammonium bicarbonate}

(pH 8.5), 300 µg TbINO1, 37 °C with shaking, and 1 mL total reaction volume.

Conducting the reaction in the dark and under an inert atmosphere had a positive effect and experiment showed a steady increase of conversion for 6 days, allowing the reaction to reach 76 % conversion. At this point, the reaction gave no further increase in conversion and a white precipitate had formed within the reaction mixture. It is hypothesised that the white precipitate is denatured TbINO1 and that six days is the longest that this protein is stable to the conditions of the reaction. Indeed, with further experiments, the protein was never observed to survive longer than this. This problem meant that there was only a window of 6 days to achieve a quantitative conversion with this process.

The obvious way to achieve this would be to increase the protein to substrate ratio. Using 600 µg of TbINO1 in this system caused the protein to denature quicker, in only 2 days, and halted the reaction at 26 %. To accommodate the increase in protein, a separate experiment was conducted in which the total volume was increased to 5 mL. This allowed the reaction to progress to 50 % over the course of 5 days before the protein precipitated. 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 120 140 C on ve rs ion / % Time / hr

One factor that needed to be looked into was the mixing of the reaction mixture. The reaction definitely had a higher conversion when the reaction mixture was placed in an incubator with shaking, as evidenced in experiments after the initial one on this scale. However, with a total volume of 5 mL the reaction vessel being used was a 15 mL Falcon tube, which are very narrow. The Falcon tube was being stood vertically within the shaker, which did not allow for very thorough mixing. An experiment was conducted by placing the Falcon tube on its side within the shaking incubator, with the thought that this would lead to a more effective agitation of the reaction mixture. This was indeed the case, however the mixing was now too vigorous and all that this accomplished was the protein becoming denatured more quickly. Within 2 hours the protein had precipitated.

A potential solution to this problem was conducting the reaction mixture within a 50 mL Falcon tube. This vessel is much wider than its 15 mL counterpart and would allow the reaction mixture to mix more effectively, without being mixed too aggressively. Unfortunately, this had no positive effect and the reaction only gave a 32 % conversion.

A rotating wheel was also assessed as a potential solution to this problem. Placing the Falcon tube in this piece of apparatus allows it to be gently inverted, allowing for a non- vigorous mixing of the reaction mixture. Unfortunately, an incubated rotating wheel was not available, however the reaction was conducted at room temperature in parallel with a reaction being run in an incubator with shaking. After 2 days, the reaction in the incubator showed a conversion of 30 %, whereas the reaction in the rotating wheel gave a conversion of 28 %. Although this experiment contained two variables (method of mixing and temperature), it is known22 that TbINO1 has an optimum temperature of 37 °C, and a 12 °C decrease in temperature will slow down the reaction rate. The fact that the two conversion values are comparable suggests that the rotating wheel is a better method to mix the reaction.

As stated, an incubated rotating wheel was not available for use. As time progressed it became apparent that an “in-batch” process may not be viable for the scale-up of this process. Even if this piece of apparatus was available, there was no guarantee that this would allow for the achievement of quantitative conversion in this system. Protein lifetime under the reaction conditions would always remain the same, and scaling up

product inhibition, and as the concentration of product increases in the solution the protein becomes less active. The high concentration of D-glucose 6-phosphate (18) being used in these reactions could possibly explain why this was not observed on a smaller scale. Alternatives to an “in-batch” process were therefore sought, and the immobilisation of TbINO1 was identified as an attractive and accessible alternative.

Chapter Three 3 Biocatalysis in Flow