Difference between revisions of "Part:BBa K5089012"

Latest revision as of 23:18, 1 October 2024

ABO2449 is a α/β hydrolase which enzymatically cleaves covalent bonds using water molecules. Here, we show that ABO2449 is capable of degrading poly-lactic acid (PLA). iGEM teams can use ABO2449 to target PLA or other ester substrates under mesophilic to cold temperatures.

PLA consists of repeating chains of naturally produced lactic acid covalently bonded via an ester bond, resulting in a plastic that is generally regarded as biodegradable. The latter is not entirely true as PLA has been found to require sustained exposure to high temperatures and moisture before showing signs of degradation. Enzymatic degradation offers a possible solution to aiding in PLA breakdown. We believe that by elucidating the kinetic parameters associated with the enzymatic activity of ABO2449 against PLA, these enzymes can be further modified to enhance their activity in a compost setting.

ABO2449 is naturally synthesized in the alkaline-degrading marine bacterium Alcanivorax borkumensis and belongs to the α/β hydrolase class which enzymatically cleaves covalent bonds using water molecules (1). It is a cold-adapted esterase, native to bacteria grown at 4C. As such the literature shows activity across a broader range of temperatures, including stability at 5C (1).

When we decided to work with ABO2449 we wanted to model the important interaction sites required for esterase activity. Because there is currently no experimentally determined structure for ABO2449 we decided to model the enzyme structure (Figure 1).

Figure 1. 3D representation of the ABO2449 Alphafold 3.0 generated structure. Catalytic trade residues are shown in green (S120, D247, H275).

Next, we modelled the important interaction sites of ABO2449 with the ester substrates, 4-nitrophenyl butyrate (pNPB), 4-nitrophenyl octanoate (pNPO) and 4-nitrophenyl dodecanoate (pNPD) (Figure 2) and determined their respective binding affinities (Figure 3).

Figure 2. 2D representation of the protein-ligand binding of ABO2449 [wt] with pNPB, pNPO and pNPD.

Figure 3. Binding affinity (from AutoDock Vina) of wild-type ABO2449 docked with pNPB, pNPO, and pNPD as ligands.

The modelling of ABO2449 for various ester substrates showed stronger binding to longer carbon chain substrates compared to shorter chains. This suggested that ABO2449 may be better suited for cleaving longer chains of poly-lactic acid. Our wet lab experiments confirmed that ABO2449 has a higher binding affinity for longer carbon chains, however, the overall rate of degradation was slower possibly due to the slow release resulting from more hydrophobic interactions. These modelling results may help guide future iGEM teams as to which amino acids may be substituted to improve the overall performance of ABO2449. Further, these results can help future teams to better understand the interactions between the pNPB, pNPO and pNPD substrates with ABO2449.

Results Understanding the potential of this enzyme, our team cloned ABO2449 into the pET22 vector and confirmed the sequence of interest using restriction digestion (Figure 4)

Figure 4. Double digest confirming the presence of ABO2449. A faint band can be observed at 900 bp, corresponding to the expected fragment. The agarose gel was run at 100 V for 45 minutes.

We then overexpressed ABO2449 from Escherichia coli BL21(DE3) using an IPTG inducible expression vector. A C-terminal histidine tag was incorporated into the coding sequence to then allow us to purify the enzyme via nickel affinity chromatography. The enzyme was then further purified with size-exclusion chromatography. To check for the presence of the enzyme, we examined it using SDS-PAGE. We consistently observed throughout the purification procedure that a 30 kDa band, corresponding to the theoretical molecular weight of ABO2449 (32.5 kDa; Uniprot ID: Q0VLQ1) (Figure 5) was present.

Figure 5. Purification of ABO2449. The following lanes are as follows from left to right: Protein ladder, E. coli BL21(DE3) cell pellet overexpressing ABO2449, cell lysate, nickel affinity chromatography elution fraction containing ABO2449, size exclusion chromatography fractions (A11 – D7).

After successfully purifying ABO2449, the enzyme was first characterized against para-nitrophenyl butyrate (pNPB) for esterase activity. We were able to obtain Michaelis-Menten kinetic parameters that show that ABO2449 could bind to pNPB with high affinity (KM=15.2 µM) with a high substrate turnover rate (kcat=4.65 s-1) (Figure 6 and Table 1). The results led us to examine whether ABO2449 had a preference for shorter carbon-chain length substrates. To do so, we tested para-nitrophenyl dodecanoate (pNPD), a substrate that is twelve carbon lengths long compared to pNPB which is four carbon lengths (Figure 7). Comparison between both substrates indicated that ABO2449 cleaves pNPB faster than pNPD (Table 1), but has looser binding. Longer chain substrates may reach deeper into the binding pocket of ABO2449, resulting in more hydrophobic interactions that lead to slower release from the active site. This suggests that ABO2449 preferentially cleaves shorter chained substrates.

Figure 6.Esterase activity of ABO2449 against pNPB. A final concentration of 13 nM ABO2449 was prepared in a 1 cm pathlength cuvette with phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4 (pH 7.5)). pNPB dissolved in acetonitrile was added to a final concentration tested above (2.5 – 100 µM). Triplicates of the reaction were measured for 100 seconds each to obtain linearity. The initial rate (Vmax) obtained from 0 – 30 s was plotted against pNPB concentration and was fitted with a hyperbolic function.

Figure 7. Esterase activity of ABO2449 against pNPD. A final concentration of 26 nM ABO2449 was prepared in a 1 cm pathlength cuvette with phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4 (pH 7.5)). pNPD dissolved in acetonitrile was added to a final concentration tested above (2.5 – 100 µM). Triplicates of the reaction were measured for 100 seconds each to obtain linearity. The initial rates (Vmax) obtained from 0 – 30 s were plotted against pNPD concentration and were fitted with a hyperbolic function.

Table 1. Michaelis-Menten kinetic parameters of ABO2449 esterase activity against ester bond substrates.

ABO2449