Enzyme Activity Assay Protocols

2.20.1 Quantification of N-acetyl-D-neuraminic acid aldolase (NanA) Activity

N-acetyl-D-neuraminic acid aldolase (NanA) activity was quantified by measuring the amount of N-acetyl-D-neuraminic acid (Neu5Ac) produced by the enzyme from the condensation of Sodium pyruvate and N-acetyl-D-mannosamine (ManNAc). The protocol used to quantitate NanA activity was adapted from conditions described by Xu et al. (X. Xu et al., 2011).

Pyruvate + ManNAc Neu5Ac

Figure 5. Diagram detailing the production of Neu5Ac from the condensation of Pyruvate and ManNAc catalysed by NanA. Adapted from Xu et al (P. Xu et al., 2007)

52 2.20.1.1 General Quantification of Enzyme Activity Protocol (NanA)

Prior to testing, the beads/ particles were stored in 20% Ethanol in 50mM Potassium phosphate buffer which needed to be removed by a wash step prior to testing enzyme activity. Aliquots of PHA bead and GFP particle slurries containing 25 μg of fusion

protein were mixed with 50 mM Potassium phosphate buffer, pH 7.50, to a total volume of ~1 ml. The samples were centrifuged at 16000 x g for 5 min in a Heraeus Pico 17 Centrifuge. The supernatant was then removed and the PHA bead/ GFP particle pellets resuspended in their original volume of 50 mM Potassium phosphate buffer, pH 7.50. A substrate solution consisting of 1.0 M Sodium pyruvate and 0.2 M ManNAc in 50 mM Potassium phosphate buffer, pH 7.50, was made, and aliquots of this solution were added to the PHA bead/ GFP particle samples to a final reaction volume of 500 μl. This

made the final concentration of fusion protein in the samples to be 0.05 mg/ml per reaction. The samples were then mixed by vortex, and incubated at 50 °C on a Gyrotory Shaker (New Brunswick Scientific Company, USA) set at 200 rpm, under aerobic conditions. Following incubation, the samples were centrifuged at 16000 x g for 10 min in a Heraeus Pico 17 Centrifuge and the supernatants removed. Aliquots of the

supernatants were then diluted and assessed by HPLC (2.19). All PHA bead/ GFP particle samples were assayed in triplicate.

2.20.2 Quantification of α-Amylase (Bla(-ss)) Activity

α-Amylase (Bla(-ss)) activity was quantified using a colorimetric assay that measured the amount of maltose liberated by the enzyme through the hydrolysis of starch. This method was adapted from previously described protocols (Sigma-Aldrich; Yamabhai et al., 2008).

Starch + H2O Reducing Groups (Maltose)

Figure 6. Diagram detailing the production of maltose from the hydrolysis of starch by Bla(-ss). (Sigma-Aldrich)

53 2.20.2.2 General Quantification of Enzyme Activity Protocol (Bla(-ss))

Aliquots of PHA bead and GFP particle slurries containing 50 μg of fusion protein were

mixed with 20 mM Sodium phosphate buffer plus 6.7 mM of Sodium chloride, pH 6.90,

to a total volume of ~1000 μl. The samples were centrifuged at 16000 x g for 5 min in a

Heraeus Pico 17 Centrifuge, and the supernatants were then removed in order to extract the 20% Ethanol in 50mM Potassium phosphate buffer used for bead/ particle storage. The PHA bead/ GFP particle pellets were resuspended in their original volume of 20 mM Sodium phosphate buffer plus 6.7 mM Sodium chloride, pH 6.90. A 1.0% Soluble Starch solution was prepared as described above (2.4.8). Once the 1.0% Soluble Starch solution was prepared aliquots were dispensed into the PHA bead/ GFP particle tubes to a final volume of 1000 μl. This made the final concentration of fusion protein in the samples to be 0.05 mg/ml per reaction. Aliquots were also dispensed into tubes that did not contain PHA beads or GFP particles, and were to serve as blank controls. All samples were incubated at 25 °C, on a Lab-Line Orbit Shaker (Lab-Line

Instruments Inc., USA), at 200 rpm, under aerobic conditions. Following incubation the samples were centrifuged at 16000 x g for 5 min in a Heraeus Pico 17 Centrifuge, and

500 μl of each supernatant was removed to separate glass Kimax tubes (16 x 125 mm, Kimble Glass Inc., Mexico). 500 μl of α-Amylase Colour Reagent Solution (2.4.7) was added, and the tubes mixed by swirling. The tubes were then heated for 15 min at

100 ˚C in a memmert OB 10 oil bath (memmert, Germany). After heating the tubes

were placed on ice to cool to room temperature- approximately 3 min. Finally, the tubes were brought to a final volume of 6 ml with Milli-Q water and mixed by inversion. 1 ml of the mixture in each tube was pipetted into separate 2-ml cuvettes, and the absorbance measured at 540 nm using an Ultrospec 2000 spectrophotometer (Pharmacia Biotech, UK). All PHA bead/ GFP particle samples were assayed in triplicate.

2.20.3 Quantification of Organophosphohydrolase (OpdA) Activity

Organophosphohydrolase (OpdA) activity was quantified using a colorimetric assay that measured the amount of para-nitrophenol liberated by the enzyme through the

hydrolysis of the substrate Methyl parathion. This method was adapted from previously described protocols (Blatchford et al., 2012; Dumas, Caldwell, Wild, & Raushel, 1989).

54 Methyl parathion para-nitrophenol + Dimethyl phosphorothioic acid

Figure 7. Diagram detailing the hydrolysis of methyl parathion by OpdA to para-nitrophenol and dimethyl

phosphorothioic acid. Adapted from Gresham et al. (Gresham, Rosenbaum, Gaspari, Jackson, & Bird, 2010)

2.20.3.1 General Quantification of Enzyme Activity Protocol (OpdA)

Aliquots of PHA bead and GFP particle slurries containing 50 μg of fusion protein were

mixed with 20% (v/v) Methanol in 50 mM HEPES Buffer, pH 8.00, to a total volume of

~1000 μl. The samples were then centrifuged at 16000 x g for 5 min in a Heraeus Pico

17 Centrifuge. As described above, the beads/ particles were suspended in 20% Ethanol in 50 mM Potassium phosphate buffer for storage which needed to be removed. The supernatant was removed and the PHA bead/ GFP particle pellets resuspended in their original volume of 20% (v/v) Methanol in 50 mM HEPES Buffer, pH 8.00. A stock solution consisting of 10 mM Methyl parathion was made by dissolving the appropriate amount of Methyl parathion crystals in 20% (v/v) Methanol in 50 mM HEPES Buffer, pH 8.00, that had been heated to 37 °C-45 °C to aid the dissolving process (the melting

point of Methyl parathion is 35 ˚C-38 °C). A working solution of 200 μM Methyl

parathion was made my diluting from the 10 mM Methyl parathion stock. Aliquots of

the 200 μM Methyl parathion solution were added to the PHA bead/ GFP particle

samples to a final reaction volume of 1000 μl. This made the final concentration of

fusion protein in the samples to be 0.05 mg/ml. Aliquots were also dispensed into tubes that did not contain PHA beads or GFP particles, and were to serve as blank controls. All samples were incubated at 25 °C, on a Lab-Line Orbit Shaker set at 200 rpm, under aerobic conditions. Following incubation the samples were centrifuged at 16000 x g for 5 min in a Heraeus Pico 17 Centrifuge, and the supernatants removed. Absorbance measurements of the supernatants were then conducted using an Ultrospec 2000

spectrophotometer (Pharmacia Biotech, UK) set at 405nm. All PHA bead/ GFP particle samples were assayed in triplicate.

In order to prevent the loss of Methanol during the pre-incubation steps of the stability studies, samples were washed in 50 mM HEPES Buffer, pH 8.00 that lacked the 20% (v/v) Methanol, and then resuspended in their original volume of the same buffer. The

55 pre-incubation steps of the temperature and pH stability assays were then performed, and the activity of the OpdA enzyme displayed on the PHA beads/ GFP particles assessed as described above.