WORKING CONC. (mM) BATCH FEED 4 10 BATCH FEED 2 5 BATCH FEED 0.4 1 NO FEED (CONTROL) 0 0 4.6.2 B iotransform ations
4.6.2.1 Benzoate Dihydrodiol Production
To ensure that production of benzoate dihydrodiol was not limited by the amount of recombinant cells, at least seven 400 mL cultures containing E. coli JM107 harbouring the plasmid pQR188 were prepared according to the method described in
Fiona Vanier_________________________________PhD Thesis Experimental Methods
section 4.3.3 using the complex medium. Induction took place 3 hours after inoculation using IPTG. After 15 -1 7 hours growth, the cells were harvested using the Beckman centrifuge at 2700g and 4“C for 20 minutes. The cells were resuspended in 100 - 200 mL phosphate buffer (100 mM at pH 7.5) and incubated at 37°C in a rotary incubating shaker.
A stock solution containing 1 M sodium benzoate and 1 M glucose was added to the resuspension such that the working concentration of benzoate in the shake flask was 1 mM. The cells were supplemented thereafter at 30 minute periods with 1 mM working concentrations of the stock solution. Samples were taken regularly (0.5 mL) and the supernatant was retained following microfugation at 135,500g for 5 min. The decrease in benzoate and increase in benzoate dihydrodiol was monitored using HPLC. Mobile phase 3) described in section 4.5.1.2 was used to detect sodium benzoate and benzoate dihydrodiol which had the retention times 11.5 and 9.7 minutes respectively. Dilution of the supernatant was carried out using 100 mM phosphate buffer pH 7.5. The substrate was added until a working concentration of 6 mM sodium benzoate was reached. At this point, substrate feeding was stopped and the biotransformation was followed to completion by monitoring as above at regular intervals for about 2 hours. After the biotransformation, the supernatant was retained using centrifugation as above and either used immediately or stored at -70°C.
4.6.2.2 2-Hydroxym uconic Semialdehyde Production
The preparation of a concentrated cell resuspension containing E. coli JM107 harbouring the plasmid pQR226 was carried out according the method described in section 4.6.2.1. A I M stock solution of catechol was added to the resuspension such that the working concentration of catechol in the shake flask was 0.25 mM. The resuspension was supplemented at regular periods with 0.25 mM aliquots of the stock solution and the increase in 2-HMSA was monitored by spectrophotometric analysis of supernatant from samples taken periodically. Dilution of the supernatant prior to analysis was carried out using 100 mM phosphate buffer pH 7.5. When the desired working concentration was reached, substrate feeding was stopped and the biotransformation was followed to completion by monitoring as above at regular intervals for about 2 hours. The supernatant was retained after centrifugation as before and used immediately.
4.6.3 Stability Studies 4.6.3.1 Benzoate Stability
Samples (1 mL) of a 2 M stock solution of sodium benzoate were subjected to the conditions shown in Table 4.8. All three samples were analysed in triplicate using HPLC. Mobile phase 3) described in section 4.5.1.2 was used to detect sodium benzoate and the average area under the peaks was calculated.
Table 4.8: Storage conditions of a 2 M sodium benzoate stock so lu tio n .
C O N D IT IO N T E M P E R A T U R E C O IN C U B A T IO N T IM E
(hr)
Freezing -70 66
Autoclaving 121 0.5
Control 28 (room temp) 15
4.6.3.2 Benzoate Dihydrodiol Stability
200 mL of a 6 mM stock solution of benzoate dihydrodiol produced using the method described in section 4.6.2.1 was diluted 2 fold and divided between four 1 L shake flasks. The pH was adjusted using concentrated phosphoric acid from pH 7.5 to pH 6.5 in two of the flasks. The flasks were then stored under the following conditions using incubating shakers:
1) pH 7.5 at 4 °C 2) pH 7.5 at 37 “C 3) pH 6.5 at 4 °C 4) pH 6.5 at 37 °C
Fiona Vanier_________________________________PhD Thesis Experimental Methods
Duplicate samples were taken immediately after incubation and every 20 minutes for 400 minutes. All samples were diluted 10 fold using 100 mM phosphate buffer at pH 7.5 and analysed using HPLC. Mobile phase 3) described in section 4.5.1.2 was used to detect the benzoate dihydrodiol.
4.6.3.3 Catechol Stability
A I M stock solution of catechol was made and left to stand at room temperature. Samples were taken every 40 minutes for 4 hours and analysed using CE. All samples were diluted 1000 fold using RO water and injected through 0.2 pm PTFE filters into 200 pi CE vials. Electrophoresis was run at 15 kV (24 pA) for 10 minutes using a 47 cm long silicon fused capillary with a 50 pm internal diameter and a 40 cm length to the detector window. The running buffer, wash and prerinse conditions were as previously described in section 4.5.1.1. Acetone treatment was unnecessary so a
1% solution of acetone used as an electroosmotic marker was run in tandem with each sample. The electroosmotic marker and sample were subjected to air pressure injection for 3 and 5 seconds respectively.
4.6.3.4 2-Hydroxym uconic Semialdehyde Stability
Three cultures containing E. coli JM107 harbouring the plasmid pQR226 were prepared according to the method described in section 4.3.3 using the defined medium. Induction in all three cases occurred 4 hours after inoculation using IPTG and two of the cultures were left to grow for a further 20 hours after induction. The third culture was grown for a further 14 hours and then fed with a 2 M stock solution of sodium benzoate at a rate of 1.5 mM (working concentration) per hour for 4 hours. The cells in all three cultures were harvested after centrifugation at 2700g and 4°C for 20 minutes and the supernatant obtained from the two cultures not subjected to benzoate feeding was also retained. A 2 mM stock solution of 2-HMSA was produced using the method described in section 4.6.2.2. This stock solution (500 mL) was divided between five 1 L shake flasks into which were also added the components shown in Table 4.9. All five shake flasks were incubated at 37°C and samples were taken every 30 minutes for 6 hours. The concentration of 2-HMSA present in each sample was determined spectrophotometrically. In a similar experiment, a 1 mM stock solution of 2-HMSA (300 mL) was divided between three 1 L shake flasks and the pH of the stock solution in two of the shake flasks was adjusted to pH 6 and pH 6.5 using concentrated
phosphoric acid. All three flasks were agitated at 37°C and samples were taken every 30 minutes for 4 hours to test for the presence of 2-HMSA using spectrophotometry.
Table 4.9: Contents of shake flasks used to test product degradation.
T E S T 2 -H M S A stock (mL) 100 mM P h osph ate buffer pH 7.5 (mL) Growth supernatant (m L) W et weight induced cells (g) Wet weight c e lls subjected to benzoate (g) C o n tro l 10 0 100 - - - In flu en ce o f c e lls 100 100 - 1 .2 - In flu en c e o f broth su pern atant 100 - 100 - - In flu en ce o f c e l l s & broth su pern atant 100 - 1 0 0 1 .2 - In flu en ce o f c e l l s su b jected to b e n z o a te 100 100 - - 1 .2
4.6.4 Product Removal Experiments 4.6.4.1 Resin Description
An anion exchange resin was used to investigate the design of a product removal strategy. This resin is made of translucent beads covalentely bound to quaternary ammonium ions which are bound to hydroxyl groups. Treatment of the resin with a sodium metabisulphite stock solution was required to convert the resin to the bisulphite form used during anionic exchange as shown in Figure 4.4.
Fiona Vanier PhD Thesis Experimental Methods i) RESIN PREPARATION NH4+ OH NH4+ OH NH4+ OH ■ RESIN BEADS IN PURCHASED FORM
Incubation in sodium bisulphite solution fo r 2 hours NH4+ BS- NH4+ BS- NH4+ BS* RESIN BEADS IN USED FORM ii) RESIN USE NH4+ BS* NH4+ BS- NH4+ BS- RESIN BEADS IN USED FORM
Incubation in semialdehyde solution
N a+ B S* Na+ BS - Na+ BS • BISULPHITE IONS (SODIUM SALT) Na+OH* Na+OH* Na+OH SODIUM HYDROXIDE SA SA SA 2-HYDROXYMUCONIC SEMIALDEHYDE MOLECULES NH4+ BS - SA NH4+ BS *SA NH4+ BS - SA
RESIN BEADS BOUND TO A FULLY