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Cutinase-like Enzyme

Encodes a lipase that hydrolyses compound polylactic acid from Cryptococcus sp. strain S-2

UManitoba Additional information

Description:
CLE is a lipase that is capable of degrading PLA-based plastics, and other high molecular weight polymers. Below, we characterized CLE activity against PLA including kinetic data and degradation of solid PLA films.

CLE is a lipase that was isolated from Cryptococcus sp. strain S-2 and was documented to degrade various biodegradable plastics, including PLA2. We modelled the important interaction sites of CLE with the ester substrates, 4-nitrophenyl butyrate (pNPB), 4-nitrophenyl octanoate (pNPO) and 4-nitrophenyl dodecanoate (pNPD) (Figure 1) and determined their corresponding binding affinities (Figure 2).

Figure 1. 2D representation of the protein-ligand binding of CLE [wt] with pNPB, pNPO and pNPD.

Figure 2. Binding affinity (from AutoDock Vina) of wild-type CLE docked with pNPB, pNPO, and pNPD as ligands.

By modelling CLE affinity for the ester substrates, there was a slight preference for the longer, twelve-carbon substrate pNPD, suggesting a potential preference for longer chains of PLA. This is one of the reasons we decided to pursue this enzyme. Other iGEM teams may use this modelling data to determine sites in CLE that can be mutated to improve enzyme activity or binding affinity under specific conditions of interest or to better understand the interactions between substrate and enzyme if they choose to use pNPB, pNPO, or pNPD for testing other esterases.

Results

We constructed our CLE plasmid in pET22 and confirmed our genetic sequence via double digestion (Figure 3).

Figure 3. Double digest confirming the presence of H6-CLE. A band can be observed at ~700 bp, corresponding to the expected fragment of CLE. The agarose gel was run at 100 V for 45 minutes.

We then transformed our CLE construct into Escherichia coli BL21(DE3) and overexpressed the enzyme using an IPTG-induced expression system. CLE was then purified by Ni-NTA affinity chromatography (Figure 4), confirmed by the presence of a band at 24 kDa.

Figure 4. Ni-NTA affinity chromatography of purified CLE fractions. 12.5% SDS-PAGE gel was run for 50 minutes at 20 milliamp.

Once we had CLE expressed and purified, we first tested it against pNPB for esterase activity. pNPB contains ester bonds that when cleaved, release 4-nitrophenol, which absorbs light at 410 nm. We were able to obtain Michaelis-Menten Kinetic parameters that show CLE can bind pNPB with 165.5 µM affinity (Km) and a substrate turnover rate of 7.75 s-1(kcat) (Figure 5 and Table 1).

Figure 5. Esterase activity of CLE against pNPB. A final concentration of 75 nM CLE 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 – 800 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.

Next, we characterized the catalytic activity of ABO2449 with pNPD, a twelve-carbon length long substrate (Figure 2), and compared the activity between each substrate (Table 1). CLE had a binding affinity (Km) of 175.6 µM and a substrate turnover rate of 4.95 s-1(kcat). Overall CLE appeared to cleave shorter carbon-chain substrates more efficiently based on the Michaelis-Menten Kinetic parameters (Table 1). Interestingly, however, CLE later demonstrated the ability to better cleave higher molecular weight PLA as shown by the HPLC experiments and results (Figure 7).

Figure 6. Esterase activity of CLE against pNPD. A final concentration of 60 nM CLE 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 – 800 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.

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

After confirming esterase activity, we were interested in the ability of CLE to degrade PLA powder that consisted of a mixture of amorphous and crystalline PLA. From our HPLC results, which detected the concentration of lactic acid produced upon incubating our enzyme with PLA powder, 0.21 mg/mL lactic acid was produced in the presence of CLE. This outperformed our positive control, proteinase K, which has been documented to degrade PLA in literature (Figure 7). The ability of CLE to degrade PLA powder suggests activity against crystalline PLA.

Figure 7. High-Performance Liquid Chromatography results in PLA degradation. PLA degradation was measured based on the amount of lactic acid produced.

Sequence and Features