Engineering

A critical objective of our project was to purify the phosphatase enzymes intended for testing. We initially designed a purification protocol incorporating sonication, ultracentrifugation and affinity chromatography using a nickel column to purify polyHis-tagged enzymes.

To implement our purification strategy, we constructed a nickel column using a PD10 column containing Chelating Sepharose, which was clamped to utilise gravity for pulling the sample through the matrix.

We applied this gravity-based system to purify our first sample of PafA, using the designed protocol (link). The system worked, allowing us to obtain purified enzyme samples for further experiments.

Through this initial test, we identified a key bottleneck; the gravity-driven process was not time-efficient, especially considering the multiple enzyme variants that needed to be purified. This realisation prompted us to reconsider our approach to increase throughput in future purification steps.

To address the time inefficiencies, we researched alternative methods to accelerate the nickel-column process. One promising approach was to modify the nickel column so it could be spun in a centrifuge, significantly speeding up the flow-through process without compromising sample quality.

We implemented this idea by drilling a hole in the cap of a Falcon tube to accommodate the P10 column, enabling us to spin the nickel column in a centrifuge.

Before applying this new method to enzyme samples, we conducted a test spin using a binding buffer to optimise centrifugation parameters. This experiment aimed to find the optimum speed and duration for the centrifugation to ensure efficient flow through the bead matrix without disturbing the column bed.

Centrifugation significantly improved efficiency over the gravity-driven method, allowing us to process samples faster. However, we also learned that excessive speed or prolonged centrifugation could disrupt the Sepharose bead matrix, underlining the importance of carefully balancing these variables in future protein purification steps.

As an early proposed implementation, we sought to develop a column device containing immobilised PafA enzymes capable of hydrolyzing organophosphates. We designed experiments to test this system using p-nitrophenyl phosphate (PNPP) as a substrate, which would pass through a nickel column with His-tagged PafA enzymes immobilised on the matrix.

We constructed a nickel affinity column containing purified PafA enzymes. This setup allowed us to immobilise the PafA enzymes on the column, creating a stable system for testing their ability to hydrolyse PNPP while immobilised.

To assess the functionality of the immobilised enzymes, we passed a 40 mM PNPP solution through the column. Our goal was to determine whether the immobilised PafA enzymes could hydrolyze PNPP into p-nitrophenol (pNP) and inorganic phosphate (P).

Our test confirmed that the immobilised PafA enzymes were still functional, successfully catalysing the hydrolysis of PNPP. However, we encountered a challenge: separating the inorganic phosphate from the rest of the flow-through was difficult. This realisation prompted us to consider additional strategies for phosphate capture.

To address the issue of phosphate capture, we researched various phosphate-binding proteins (PBPs) that could be immobilised in conjunction with PafA on the same nickel column. These PBPs would selectively bind inorganic phosphate, allowing for its separation from the flow-through.

We synthesised a gBLOCK containing the selected PBP gene and inserted it into our pET21a backbone. Following our established purification workflow, we purified the PBP and immobilised it on the same nickel column as the PafA enzyme, utilising its polyHis-tag for binding.

We repeated the experiment with the new setup, passing the 40 mM PNPP solution through the column to test whether the immobilised PBP could successfully capture inorganic phosphate. This was confirmed by the lower absorbance at 405 nm in the flow-through compared to the column containing only PafA, indicating that the PBP was effectively binding inorganic phosphate. (link to column section)

Our test demonstrated that the addition of PBP successfully enhanced phosphate capture, confirming the functionality of our dual-enzyme immobilisation system. This breakthrough marked a significant step toward optimising our phosphate recovery strategy, but further optimisation of stability and scalability will be required for practical applications.

As we progressed, concerns arose about the stability of PafA under the harsher conditions typical of wastewater treatment, which differed from the controlled lab environment with PNPP solution. To address this, we leveraged AI tools like LigandMPNN and AlphaFold 3 to redesign PafA, aiming to enhance its thermostability and pH tolerance for real-world applications.

We used these AI tools to generate six redesigned versions of PafA. We then ordered the redesigned genes and cloned them into the pET21a backbone. Following our standard recombinant protein production workflow, we purified these new PafA variants for testing.

To evaluate the improved stability of the redesigned enzymes, we performed circular dichroism analysis. This test confirmed that the redesigned enzymes were significantly more thermostable than the wild-type PafA. While the wild-type enzyme aggregated at 70°C, the redesigned variants remained stable and did not aggregate or unfold even at 95°C, demonstrating their enhanced robustness.

This iteration successfully improved the thermostability of PafA, generating more durable enzyme variants. However, due to time constraints, we were unable to test the immobilisation of these new variants onto the column. Future work will focus on integrating these variants into the column setup, which is crucial for practical implementation.

We sought external feedback on our column design and enzyme immobilisation strategy by consulting with Dr. Eleni Routoula from the University of Sheffield. She provided invaluable advice, recommending that we replace Sepharose beads with glass beads for enzyme immobilisation. Glass beads are more durable, especially in wastewater conditions, and reduce the risk of enzyme unfolding, which can occur with Sepharose.

To explore this new approach, we collaborated with Dylan Lewis, a PhD student, who allowed us to observe the process of enzyme immobilisation onto glass beads. This process involved the use of Piranha solution, which we could not handle ourselves due to safety restrictions. Unfortunately, we were unable to implement this method within our project timeframe, but it presents a promising avenue for future development.