Results

Overview

Our wet lab team chose five potentially plastic-degrading enzymes (Est 119, GEN0105, MGS0156, RPA1511, and TTL) to focus their efforts for our project PLAnet Zero. This project has 2 objectives:

1. To engineer an organism that is capable of surface-displaying improved polylactic acid (PLA) degrading enzymes for application in Manitoban composting facilities
2. To study how incorporating non-canonical amino acids (ncAAs) can potentially improve enzyme activities and explore the possibility of expanding the amino acid repertoire for biocatalysis application

By employing optimized PLA-degrading enzymes that allow for an improved rate of PLA degradation, PLAnet Zero offers a unique biological solution to tackle the current issues of PLA composting in Manitoba. Thermoanaerobacter thermohydrosulfuricus lipase (TTL) is known to degrade polyethylene terephthalate (PET) plastic and was used as our model enzyme for the engineering of our improved PLA-degrading enzymes. Based on the work by Haernball et al., it is known that the incorporation of the non-canonical amino acid, norleucine, to replace all methionine residues, including those close to the active site, improves the activity of TTL in degrading PET plastic (Akutsu-Shigeno et al., 2003). Using this knowledge, we aim to improve our PLA-degrading enzymes with the incorporation of ncAAs that can modify enzymatic properties and interactions with the plastic polymer. Below, you will find the detailed workflow for each respective enzyme including the characterization of esterase activity using Michaelis Menten kinetic assays, the surface-display mechanism using the Lpp-OmpA anchor in Escherichia coli, and the results of the crude-plastic PLA assays. Due to the limited time, we divide our enzyme collections into 2 parts:

1. Est119 and RPA1511 are used as models for surface displacement approach. We use a part (BBa_K2302003), found in the iGEM registry, which encodes Lpp-OmpA for surface displacement.
2. MGS0156 and GEN0105 are used as models for genetic code engineering approaches. The literature reports that the global replacement of methionine with norleucine is shown to improve TTL’s ability to degrade PET plastic (Haernvall et al. 2022). Hence, we used TTL as our model study and then applied the same principle to engineer MGS0156 and GEN0105.

Experimental Methods

Genetic Constructs

The genetic constructs containing the PLA-degrading enzymes were obtained from various sources. The construct containing the TTL gene of interest was gifted to our team in the expression vector pQE80L from Dr. Nedjiliko Budisa’s lab group. The constructs containing codon-optimized Est119 and RPA1511with Lpp-OmpA secretion signal were both purchased from the gene synthesis company, Integrated DNA Technologies (IDT), in the pUCIDT cloning vector (Hu et al. 2009; Hajighasemi et al. 2016). The construct containing the MGS0156 gene of interest without Lpp-OmpA secretion signal, and the GEN0105 gene of interest without Lpp-OmpA secretion signal was gifted to our team in the expression vector p15TV-L from Dr. David Levin’s lab group.

Cloning

To confirm the genetic sequence of interest, the vectors outlined were chemically transformed into competent Escherichia coli (E. coli) DH5α cells. The transformation mixtures containing KCM, the plasmid DNA and the competent E. coli DH5α cells were incubated on ice for 30 minutes, then heat shocked for 45 seconds at 42℃. The mixtures were placed on ice for two minutes, and LB media was added. The mixtures were incubated at 37℃ for 1 hour shaking at 250 rpm. The cell suspensions containing TTL, MGS0156, and GEN0105 were plated on LB and 50 ug/mL ampicillin plates, while Est119 and RPA1511 were plated on LB and 50 ug/mL kanamycin plates. All plates were incubated overnight at 37℃. Overnight cultures were then prepared for each construct with LB and 50 ug/mL of the appropriate antibiotic and left at 37℃, 250 rpm. Plasmid DNA was then isolated using a homemade miniprep kit equivalent to the Qiagen Miniprep Kit using the same protocol as outlined in the manufacturer’s instructions.

The components are as follows: resuspension solution (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 µg/mL RNase A), lysis solution (200 mM NaOH, 1% SDS), neutralization buffer (4.2 M guanidinium-HCl, 0.9 M potassium acetate, pH 4.8), wash buffer (10 mM Tris-HCl pH 7.5, 80% ethanol), elution buffer (10 mM Tris-HCl, pH 8.5) and EZ-10 Spin Column & Collection Tube (DNA). Cat.:SD5005 (BioBasic)

Double digestion reaction mixtures were then prepared with the appropriate restriction enzymes as follows. For TTL, Xho1 (NEB, Cat.: R0146) and PstI-HF (NEB, Cat.:R3140) were combined with the plasmid DNA and rCutSmart buffer (NEB, Cat.: B6004). For Est119 and RPA1511, EcoRI-HF (NEB, Cat.: R3101) and PstI-HF were combined with plasmid DNA and rCutSmartTM buffer (NEB, Cat.:B6004). For MGS0156 and GEN0105, NcoI-HF (NEB, Cat.:R3193) and EcoRI-HF were combined with plasmid DNA and rCutSmartTM buffer. For each reaction, a single-digestion control was also performed with only one of the respective enzymes. Reaction mixtures were incubated at 37℃ for 1 hour, with the exception of TTL which was left overnight. All samples were prepared with NEB Gel Loading Dye, Purple (6X) (NEB, Cat.: B7024) and run on 1% agarose gel prepared in TAE buffer and visualized with SYBERSafe DNA (ThermoFisher, Cat.: S33102) stain for 45 minutes at 100 V. Quick-Load Purple 1 Kb Plus DNA Ladder(NEB, Cat.L N3200) was used as a reference.

To transfer Lpp-OmpA-Est119 and Lpp-OmpA-RPA1511 from the pUCIDT vector to the pET28b(+) backbone, Gibson assembly was performed 10 µM of primers were used and the sequences are outlined in Table 1. PCR is done using Q5® High-Fidelity 2X Master Mix (NEB, Cat.:M0492) and reaction mixtures were made following the manufacturer’s instruction PCR conditions were set up for the protein inserts with an initial denaturation of 95℃for five minutes and the following parameters for 30 cycles: fifteen seconds at 98℃ for denaturation, fifteen seconds at 59℃ for annealing, one minute at 72℃ for extension. A final extension was performed for three minutes at 72℃. For the pET28b(+) backbone, PCR conditions were set up with the following parameters for 30 cycles: five minutes at 98℃ for denaturation, fifteen seconds at 67.2℃ for annealing, three minutes at 72℃ for extension. A final extension was performed for 9 minutes at 72℃. The PCR products were then run on 1% agarose gel as described above and gel extraction was performed using the NEB Monarch Gel Extraction Kit (NEB, Cat.: T1020) according to the manufacturer’s instructions. After extraction, DNA samples were precipitated by adding 1/10 volume of 3 M sodium acetate, 2x volume of 95% ethanol and left at -20℃ overnight. Then, samples are centrifuged, washed with ice-cold 70% ethanol, left to air dry for 5-10 minutes, and then resuspended using the elution buffer (10 mM Tris-HCl, pH 8.5). After resuspension, Gibson assembly was performed using the NEBuilder HiFi DNA Assembly Kit (NEB, Cat.: E5520) according to manufacturer's instructions with reaction mixtures incubated at 50℃ for 1 hour. Transformation into E.coli DH5α cells, miniprep and confirmation by double digestion and agarose gel electrophoresis were then performed to confirm successful assembly of Lpp-OmpA-Est119 and Lpp-OmpA-RPA1511 as previously described.

Table 1. Lpp-OmpA-Est119 and Lpp-OmpA-RPA1511 PCR Primer Sequences for Gibson Assembly Cloning

After Gibson Assembly, the vectors were chemically transformed into competent E. coli BL21(DE3) cells, following the same protocol for transformation into E. coli DH5α cells, but the mixtures were instead heat shocked for 30 seconds at 42℃. The cell suspensions containing TTL, MGS0156, and GEN0105 were plated on LB and 50 ug/mL ampicillin plates, while Lpp-OmpA-Est119 and Lpp-OmpA-RPA1511 were plated on LB and 50 ug/mL kanamycin plates. All plates were incubated overnight at 37℃. Note that for Lpp-OmpA-Est119 and Lpp-OmpA-RPA1511, transformation was also performed into E. coli BL21(DE3) (ΔLPP) for the whole-cell activity assay. E. coli BL21(DE3) (ΔLPP) strain was kindly gifted by Dr. Karbalaei-Heidrari from the Budisa Group. This strain is genetically modified by removing native Lpp secretion signal in the genome with aims to reduce competition for secretion machinery and increase secretion of our Lpp-OmpA-enzymes.

Mutagenesis

Mutagenesis is done to remove Lpp-OmpA from Lpp-OmpA-Est119 and Lpp-OmpA-RPA1511 to generate stand alone enzymes (Est119 and RPA1511). 10 µM of primers were used and the sequences are outlined in Table 2. PCR was done using Q5® High-Fidelity 2X Master Mix (NEB, Cat.:M0492) and reaction mixtures were made following manufacturer’s instructions. PCR conditions were set up for the protein inserts with an initial denaturation of 95℃ for five minutes and the following parameters for 30 cycles: fifteen seconds at 95℃ for denaturation, fifteen seconds at 53℃ (Est119) 55℃ (RPA1511) for annealing, three minutes and thirty seconds at 72℃ for extension. A final extension was performed for ten minutes at 72℃. Agarose gel electrophoresis, gel extraction, and DNA clean up were performed as described above. Then, a one-pot digestion and ligation was performed by mixing DNA, BsaI-HF®v2 (NEB, Cat.: R3733), T4 DNA ligase (NEB, Cat.: M0202), rCutSmartTM buffer, ligase buffer, and sterile water. The reaction was performed on a thermocycler by incubating at 37℃ for 45 minutes and then, 16℃ for 30 minutes. The mixture was transformed into E.coli DH5α cells, miniprepped, double digested, and run on agarose gel for identity confirmation. The plasmids were then transformed into E. coli BL21(DE3) as previously described.

Table 2. Est119 and RPA1511 Primers for Lpp-OmpA Anchor Deletion

Growth and Purification

Overnight cultures were prepared for each of the constructs in E. coli BL21(DE3) and E. coli BL21(DE3) (ΔLpp) with LB and 50 ug/mL of the appropriate antibiotic at 37℃, 250 rpm. The cells from the overnight culture were used to inoculate fresh LB media in a 1 in 100 dilution and incubated at 37℃, 250 rpm for three hours until an OD₆₀₀ of 0.6-0.8 was reached. 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was used to induce protein expression. All expression was done overnight with 250 rpm shaking. The expression conditions are as follows: TTL, MGS0156, and RPA1511: 30℃, Lpp-OmpA-Est119, Lpp-OmpA-RPA1511, Est119: 16℃

Non-canonical amino acid incorporation utilized methionine-auxotrophic cells (E. coli BU14) that were kindly gifted by the Budisa group and used for selective pressure incorporation (SPI) to incorporate norleucine and methoxinine into TTL and MGS0156. Cells were transformed with appropriate plasmids and used to make overnight cultures and glycerol stock. Overnight cultures were then washed three times with sterile water and then incubated in New Minimal Media (NMM). The media composition as followed: salts (8.6 mM NaCl, 22 mM KH₂PO₄, 50 mM K₂HPO₄, 7.5 mM (NH₄)₂ SO₄, 1 mM MgSO₄), cofactors (10 µg/mL thiamine, 10 µg/mL biotin), 20 mM D-Glucose,trace metals (Teknova, Cat.: T1001), 50 mg/L 19 amino acids (excluding methionine), 6.7 g/L yeast extract without amino acids, 35 µM methionine (limiting concentration), and appropriate antibiotics. Cells were grown at 37oC for 8-10 hours and then induced with 1 mM IPTG and 1 mM of non-canonical amino acids (norleucine or methoxinine) are added. Then, cells are incubated overnight at the appropriate temperature as mentioned above.

Non-IPTG-induced and IPTG-induced protein samples were taken, normalized to OD ₆₀₀=1, and prepped for expression testing by re-suspending in SDS-PAGE sample buffer and heating at 95℃ for 10 minutes. The samples were run on 12.5% SDS-PAGE gels at 20 milliamps/gel for 50 minutes and stained using Coomassie Brilliant Blue R-250 in 30% methanol, and 10% acetic acid. The gel was then de-stained for six hours in a de-staining buffer (30% methanol, and 10% acetic acid).

Following expression, the cell cultures were harvested by centrifugation at 6000xg for 15 minutes at 4℃. The cell pellet for Est119 and RPA1511 were used immediately in the whole-cell activity assay outlined in (v), while that of TTL and MGS0156 proceeded to cellular lysis. The cell pellets were resuspended in 1/10 of the culture volume in lysis buffer (20 mM Tris, 200 mM NaCl, 0.05% azide, pH 8.0) and sonicated at 50% amplitude for 15 seconds on, 30 seconds off. Samples were then centrifuged at 18,000xg for 45 minutes at 4℃. The supernatant was then purified by immobilized metal affinity chromatography (IMAC) using commercially available nickel resin via the Äkta Fast Protein Liquid Chromatography (FPLC) machine (Cytiva, Cat.: 29022094). A 1 or 5 mL column (HisTrap HP, Cytiva, Cat.: 17524801 and 29051021) was used as appropriate and the flow rate was set according to column manual, and absorbance was monitored at 280 nm. The column was equilibrated with 10 CV binding buffer (20 mM Tris, 200 mM NaCl, 0.05% azide, pH 8.0), washed with 5 CV washing buffer (20 mM Tris, 200 mM NaCl, 0.05% azide, 15-30 mM imidazole) pH 8.0, and 5 CV elution buffer (20 mM Tris, 200 mM NaCl, 0.05% azide, 300 mM imidazole). Aliquots were taken of the cell lysate, flow-through, wash and elution and prepared with SDS-PAGE sample buffer. The samples were run on 12.5% SDS-PAGE gel and stained as outlined above.

Size-exclusion chromatography (SEC) was used to further purify the TTL and MGS0156 elution samples. HiLoad 16//600 Superdex 75 pg column was equilibrated with 1.2 CV (144 mL) of vacuum-degassed PBS overnight one day prior to purification. The Ni-NTA purified samples were ultracentrifuged at 17,000xg for 10 minutes at 4℃ and injected on the second day into the column using a 2 mL injection loop at the flow rate of 0.5 mL/min at 4℃ for 1.2 CV. Aliquots were taken of the cell lysate, flow-through, wash and elution and prepared with SDS-PAGE sample buffer. The samples were run on 12.5% SDS-PAGE gel and stained as outlined above to confirm successful purification.

Michaelis Menten Kinetic Assay

Michaelis-Menten parameters were extracted by incubating the purified lysate (of either TTL or MGS0156) with a chromogenic compound either, 4-nitrophenyl octanoate (NPO) or 4-nitrophenyl butyrate (pNOB). TTL was diluted to 1.5 µM in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) containing either NPO or pNOB at varying concentrations ranging from 0.003 µM - 2 µM. The absorbance rate per minute was monitored for 1 minute at 410 nm using the Jasco V-770 spectrophotometer. The reactions were measured at both 60℃ and at 25℃ . For the 60 reactions, the TTL or MGS0156, PBS and the 1 mL glass cuvette were pre-heated prior to measurement. The initial rates were extracted using the first 3 seconds of the data using the kinetic function in Spectra Manager. MGS0156 was diluted to concentrations ranging from 0.0365-0.11 µM in PBS containing either NPO or pNOB at concentrations ranging from 0.0003µM-2µM. Absorbance per minute was monitored for 1 minute at 410 nm and 0.2 seconds per data stamp. Note that for all reactions a PBS buffer blank was used.

To analyze the change in the Michaelis-Menten parameters of TTL and MGS0156 with norleucine (Nle) incorporation, Nle was incorporated into the enzymes by selective pressure incorporation (SPI) as outlined above. All purification procedures as outlined above were performed and Michaelis-Menten parameters were extracted following the same procedure outlined above.

Whole-Cell Activity Assay

The cell pellets containing either Lpp-OmpA-Est119 or Lpp-OmpA-RPA1511 were washed and resuspended with PBS and normalized to OD ₆₀₀ of 1.0. The reaction mixture was prepared with PBS, E.coli BL21(DE3) containing the plasmid with Lpp-OmpA-Est119 or Lpp-OmpA-RPA1511, and 100 µM NPO or pNOB. After the addition of the substrate, the samples were incubated for five minutes at room temperature before 100 uL were transferred to a microfuge tube and centrifuged at either 17,000xg or 6,500xg for five minutes. The absorbance at 410 nm was then recorded and the process was repeated in five-minute intervals. Two types of controls were prepared: a negative bacterial control and a negative substrate control. The bacterial control contained PBS, E.coli BL21(DE3) with the plasmid containing Est119 or RPA1511, and 100 µM NPO or pNOB. The negative substrate control contained PBS, and 100 µM NPO or pNOB. A reaction mixture was also prepared for Lpp-OmpA-Est119 and Lpp-OmpA-RPA1511 using E.coli BL21(DE3) (ΔLpp) as outlined above.

PLA Coupon Assay

Our team was gifted PLA coupons from Dr. David Levin’s lab group. These coupons were incubated with the various enzymes at room temperature (20-23℃) for 72 hours with gentle shaking as outlined in Table 3. All trials were performed in triplicate.

Table 3. Treatment Conditions for PLA Coupon Incubation

Following incubation, the samples were sent to Dr. Kathleen Gough’s lab for FTIR microscopic analysis, which is still in progress…

TTL

Description

The potential utility of TTL lipase for the breakdown of synthetic polymers is already suggested in the literature. This lipase has been observed to catalyze the hydrolysis of ester linkages in many synthetic condensation polymers, including poly-L-lactic acid (PLLA) (Haernvall et al. 2022). Our group utilized TTL lipase as the point of reference in determining the viability of the other enzymes under investigation, specifically when investigating the incorporation of non-canonical amino acids to improve esterase activity.

Wild Type (WT) TTL

The expected bands for the TTL fragment and vector backbone are 925 bp and 4.6 kb, respectively. As seen in Figure 1, the presence of a band at ~900 bp and ~ 5 kb, corresponds to the expected fragment and vector size, allowing confirmation regarding the identity of our plasmid of interest.

Figure 1. Double digest confirming the presence of H6-TTL. A faint band can be observed at 900 bp, corresponding to the expected fragment of TTL. The agarose gel was run at 100 V for 1 hour.

Following confirmation of our TTL genetic sequence, the construct was transformed into E. coli BL21(DE3) and underwent protein expression. Figure 2 depicts the SDS-PAGE results following the expression test. The presence of an overexpressed band between 25 kDa and 35 kDa corresponds to the expected value of 30 kDa, suggesting that the expression was successful and purification procedures can proceed.

Figure 2. 12.5% SDS-PAGE confirming expression of H6-TTL. E.coli BL21(DE3) was induced with 1 mM IPTG and incubated at 30oC at 250 rpm overnight. The gel was run for 50 minutes at 20 milliamp.

Following expression, TTL was ready to be purified in order to prepare the sample for Michaelis Menten kinetics characterization. Figure 3 shows the fractions collected during Ni-NTA affinity chromatography. The presence of the TTL band in the elution fraction between 25 kDa and 35 kDa corresponds to the protein of interest, and the lack of contaminating bands suggests pure protein.

Figure 3. Ni-NTA affinity chromatography purified TTL fractions. 12.5% SDS-PAGE gel was run for 50 minutes at 20 milliamp.

TTL (Nle)

Following the incorporation of Nle into the TTL enzyme, size-exclusion chromatography purification was performed. The presence of the TTL (Nle) band in the elution fractions between the 25 kDa and 35 kDa lanes in Figure 4 suggests successful purification and Michaelis-Menten characterization of esterase activity may proceed.

Figure 4. Size-Exclusion Chromatography fractions confirming purification of TTL (Nle). 12.5% SDS-PAGE gel was run for 50 minutes at 20 milliamps.

Characterization of Michaelis Menten kinetics

Table 4. Michaelis-Menten parameters for TTL using NPO chromogen.

Table 5. Michaelis-Menten parameters for TTL using pNOB chromogen.

Figure 6. Michaelis-Menten Esterase Activity Assay of Thermoanaerobacter thermohydrosulfuricus lipase with 4-nitrophenyl butyrate (pNOB). Assays were conducted with 1.5 µM of lipase in PBS with pNOB concentration ranging from 0.0365-0.11 µM. The rate of change of absorbance was monitored at 410 nm for 1 min. A) Wild-type TTL lipase chromogenic assay with initial rate as a function of substrate concentration at 25 B) Wild-type TTL lipase chromogenic assay with initial rate as a function of substrate concentration at 60 ℃ C) TTL lipase chromogenic assay with initial rate as a function of substrate concentration, with norleucine introduced by residue specifically by SPI at 25 ℃ D) TTL lipase chromogenic assay with initial rate as a function of substrate concentration, with norleucine introduced by residue.

Figure 7. Michaelis-Menten Esterase Activity Assay of Thermoanaerobacter thermohydrosulfuricus lipase with 4-nitrophenyl octanoate (NPO). Assays were conducted with 1.5 µM of lipase in PBS with pNOB concentration ranging from 0.0365-0.11 µM. The rate of change of absorbance was monitored at 410 nm for 1 min. A) Wild-type TTL lipase chromogenic assay with initial rate as a function of substrate concentration at 25 ℃ B) Wild-type TTL lipase chromogenic assay with initial rate as a function of substrate concentration at 60 ℃. C) TTL lipase chromogenic assay with initial rate as a function of substrate concentration, with norleucine introduced by residue specifically by SPI at 25℃. D) TTL lipase chromogenic assay with initial rate as a function of substrate concentration, with norleucine introduced by residue specifically by SPI at 60 ℃.

Conclusions for TTL

As observed by the graph slopes in Figure 6.A and 6.B, the enzyme affinity (Km) of wild-type TLL on the NPO substrate did not change at 25℃ in comparison to the assay done at 60℃. However, the catalytic rate (k<sub<cat) of TTL on NPO increased from 2.0s^-1 to 6.3s^-1 at a higher temperature. This suggests that at 60°C the wild-type TTL is converting a larger ammount of NPO into products per second. The wild-type TTL also showed an increased catalytic efficiency at 60℃ in comparison to 25℃ (Table 4).

The enzyme affinity of (Km) wild-type TTL to the pNOB substrate was observed to be lower at 60℃, as reported in Table 5. The Km value at 25℃ of 1.7x10^-3µM. decreased to 2.4x10^-2µM at 60℃. This indicates that at 25℃ more of the substrate must be present to saturate the enzyme thus having a lower affinity for the substrate. However, the catalytic activity of wild-type TTL did not change in response to temperature (Table 5). The wild-type catalytic increased at 60℃ in comparison to 25℃. Both observations of TTL catalytic efficiency towards NPO and pNOB are consistent with TLL having maximal activity at thermophilic temperatures (Haernvall et al. 2022). The trends observed in the wild-type TTL were also observed in the TTL with norleucine introduced (Nle TTL), apart from the catalytic activity with the pNOB substrate. The catalytic activity of Nle TTL to pNOB increased from 1.3s^-1 at 25℃ to 6.9s^-1 at 60℃.

The results from the NPO assays suggest that the incorporation of norleucine greatly improved the catalytic of TTL. When comparing the results of the 60°C assays the enzyme affinity decreased from 1.7x10^-3µM for the wild type to 2.0x10^-4µM for the Nle TTL. However, the catalytic activity of Nle TTL decreased from 6.3s^-1 for wild-type to 5.3s^-1 for Nle TTL. In comparison, to the assays at 25°C the catalytic efficiency decreased (Table 6). Although 25°C is much lower than the optimal temperature of TTL, resulting in smaller values for catalytic activity and enzyme specificity for both the wild type and modified Lpp.

The results from the pNOB assays at 60°C suggest a small decrease in catalytic efficiency after the incorporation of norleucine. However, at 25°C the catalytic efficiency was shown to improve in the norleucine-modified TTL, from 19.0 to 31.0 s^-1µM^-1. Note the substrate binding affinity decreased from 1.7x10^-3 µM to 4.2x10^-2 µM, although the catalytic activity was still quite low in both the wildtype and modified TTL which is consistent with assays being done below TTL’s optimal temperature.

The results from the pNOB assays showed lower activity across all conditions, likely due to its shorter and less hydrophobic chain. Overall, the norleucine incorporation assays at 60°C showed an overall improvement of enzymatic activity. This is consistent with the increased hydrophobicity of Nle TTL likely improved the interactions with the substrate. The results from the assays at 25°C decreased catalytic efficiency due to the increased substrate binding affinity (Km) value. Outside the optimal temperature range, the norleucine modification did not appear to improve.

MGS0156

Description

MGS0156 is a serine-dependent ⍺/β hydrolase originally identified from an environmental metagenomic analysis study in which various microbial genomes were sequenced for the identification of proteins with strong degradative activity against synthetic polyesters (Hajighasemi et al. 2016). MGS0156 has been shown to hydrolyze PLA into a mixture of lactic acid monomers, dimers, and higher oligomers as products, with a maximal activity between 35 and 40 °C (Hajighasemi et al. 2016). Our team was interested in MGS0156 due to its functionality in a lower temperature range, making it potentially useful in local composting facilities that often cannot maintain the sustained, high temperatures currently required for PLA degradation. Based on the results seen with TTL, MGS0156 was used as the PLA-degrading enzyme model to test if the similar incorporation of norleucine would result in increased esterase activity.

Wild type (WT) MGS0156

The expected bands for the MGS0156 fragment and vector backbone are 1.45 kb and 5.3 kb, respectively. As seen in Figure 8, the presence of a band at ~1.5 kb and one just under ~6 kb, corresponds to the expected fragment and vector size, allowing confirmation regarding the identity of our plasmid of interest.

Figure 8. Double digestion confirms the presence of MGS0156. A faint band can be observed at 1.5 kb, corresponding to the expected fragment of MGS0156. The agarose gel was run at 100 V for 1 hour.

Following confirmation of our MGS0156 genetic sequence, the construct was transformed into E.coli BL21(DE3) and underwent protein expression. Figure 9 depicts the SDS-PAGE results following the expression test. The presence of an overexpressed band between 35 kDa and 40 kDa corresponds to the expected value of 37.1 kDa, suggesting that expression was successful and purification procedures can proceed.

Figure 9. A 12.5% SDS-PAGE confirming expression of MGS0156. E.coli BL21(DE3) was induced with 1 mM IPTG and incubated at 30oC at 250 rpm overnight. The gel was run for 50 minutes at 20 milliamps.

Following protein expression, MGS0156 was ready to be purified. Figure 10 shows the fractions collected during Ni-NTA chromatography purification. The presence of the MGS0156 band in the elution fraction between 35 kDa and 40 kDa corresponds to the protein of interest. However, the presence of other bands in this lane suggests that there is still contamination and further purification is needed. The presence of a band between 35 kDa and 40 kDa corresponds to MGS0156 in the fraction elution lanes following size-exclusion chromatography (Figure 11). Note that the presence of bands in some of the lanes corresponding to 70 kDa suggests dimer formation. With the MGS0156 purified, we can now proceed to the Michaelis-Menten characterization of esterase activity.

Figure 10. Ni-NTA affinity chromatography purified MGS0156 fractions. 12.5% SDS-PAGE gel was run for 50 minutes at 20 milliamps.

Figure 11. Size-Exclusion Chromatography fractions confirming purification of MGS0156. 12.5% SDS-PAGE gel was run for 50 minutes at 20 milliamps.

MGS0156 (Nle)

Following the incorporation of Nle into the MGS0156 enzyme, size-exclusion chromatography purification was performed. The presence of the MGS0156 (Nle) band in the elution fractions between the 35 kDa and 40 kDa lanes in Figure 12 suggests successful purification and Michaelis-Menten characterization of esterase activity may proceed.

Figure 12. Size-Exclusion Chromatography fractions confirming purification of MGS0156 (Nle). 12.5% SDS-PAGE gel was run for 50 minutes at 20 milliamps.

Characterization of Michaelis Menten Kinetic

Table 6. Michaelis-Menten parameters for MSG0156 using NPO chromogen.

Table 7. Michaelis-Menten parameters for MSG0156 using pNOB chromogen.

Figure 13. Michaelis-Menten Esterase Activity Assay of MGS0156 with 4-nitrophenyl octanoate (NPO). Assays were conducted with 1.5 µM of lipase in PBS with NPO concentration ranging from 0.0365 - 0.11 µM. Rate of change in absorbance was monitored at 410 nm for 1 min. A) Wild type MGS0156 chromogenic assay with initial rate as a function of substrate concentration at 25 ℃ B) Wild type MGS0156 chromogenic assay with initial rate as a function of substrate concentration at 60 ℃ C) MGS0156 chromogenic assay with initial rate as a function of substrate concentration, with norleucine introduced by residue specifically by SPI at 25 ℃.

Figure 14. Michaelis-Menten Esterase Activity Assay of MGS0156 with 4-nitrophenyl butyrate (pNOB). Assays were conducted with 1.5 µM of lipase in PBS with pNOB concentration ranging from 0.0365 - 0.11 µM. Rate of change in absorbance was monitored at 410 nm for 1 min. A) Wild-type MGS0156 chromogenic assay with initial rate as a function of substrate concentration at 25 degrees C. B) Wild-type MGS0156 chromogenic assay with initial rate as a function of substrate concentration at 60 ℃. C) MGS0156 chromogenic assay with initial rate as a function of substrate concentration, with norleucine introduced by residue specifically by SPI at 25℃.

Wild-type MGS0156 enzyme demonstrates better catalytic efficiency (k_cat/k_m) when binding to NPO in comparison to pNOB, indicating a preference for NPO binding at 25℃. Enzyme-substrate binding exhibits a higher catalytic rate when the enzyme binds to 4-nitrophenyl octanoate (NPO) as opposed to 4-nitrophenyl butyrate (pNOB). It is hypothesized that this phenomenon can be attributed to the increased hydrophobicity and longer alkane chains present in NPO compared to pNOB. The hydrophobic binding of the substrate in the enzymatic active site contributes to enhanced enzyme-substrate binding affinity. The specificity of wild-type MGS0156 for NPO is 4.8x104 s^-1µM^-1 at room temperature (Table 6), while its specificity for pNOB is 2.2x104 s^-1µM^-1 (Table 7). The heightened specificity of wild-type MGS0156 for NPO substrates relative to pNOB can be attributed to the larger size and increased hydrophobicity of the NPO substrate.

Wild-type MGS0156 exhibits a higher Km value than mutated MGS0156 Nle at 25℃, indicating that MGS0156 Nle demonstrates better substrate binding affinity than MGS0156 Nle. The Km value for MGS0156 Nle to NPO binding is approximately 4.82-fold smaller than wild-type MGS0156. The Km value for MGS0156 Nle to pNOB binding is approximately 4.68-fold smaller than wild-type MGS0156. Nevertheless, the catalytic efficiency of wild-type MGS0156 is still better than MGS0156 due to higher Kcat in wild types. Our initial expectations were for MGS0156 Nle to exhibit improved catalytic efficiency and Km due to the substitution of Nle, which is less electronegative than the corresponding sulphur atom in Met, leading to increased hydrophobicity in the methylene group and favourable hydrophobic effects for enhanced binding and catalysis. Our experimental results indicate that although Nle MGS0156 exhibited better substrate binding affinity, WT MGS0156 maintained superior catalytic efficiency (k_cat/k_m) compared to MGS0156 Nle.

Finally, wild-type enzyme activity is tested with varying temperatures. Results reveal that the wild-type MGS0156 enzyme, when acting on NPO, exhibits significantly higher catalytic efficiency at 60 ℃ compared to wild-type MGS0156 at 25 ℃ and both mutated forms of MGS0156 Nle at both room temperature and 60℃.

Overall Summary on MGS0156 Enzyme

Catalytic properties of the wild-type MGS0156 enzyme and its mutated variant, MGS0156 Nle are investigated by focusing on their interactions with substrates NPO (4-nitrophenyl octanoate) and pNOB (4-nitrophenyl butyrate) at 25℃ and 60℃. In summary, wild-type MGS0156 enzyme demonstrates better catalytic efficiency (k_cat/k_m) and specificity when binding to NPO in comparison to pNOB at 25℃, signifying a clear preference for NPO. This preference is attributed to the increased hydrophobicity and longer alkane chains in NPO. While wild-type MGS0156 exhibits a lower k_m value than MGS0156 Nle at 25℃, indicating better substrate binding affinity for MGS0156 Nle, the catalytic efficiency of wild-type MGS0156 remains superior due to higher k_cat/ values.

Despite initial expectations favouring MGS0156 Nle for improved catalytic efficiency and k_m due to the substitution of Nle, which is less electronegative than the corresponding sulphur atom in Met and results in increased hydrophobicity in the methylene group, and the anticipated favourable hydrophobic effects enhancing binding and catalysis, our experimental results indicate that wild-type MGS0156 exhibits better catalytic efficiency (k_cat/k_m) but weaker substrate binding affinity when compared to MGS0156 Nle. Furthermore, wild-type MGS0156 enzyme activity across a range of temperatures is investigated. It is observed that the wild-type MGS0156 acting on NPO at 60 degrees shows substantially high catalytic activity (1.4x105 s^-1µM^-1), while MGS0156 acting on pNOB at 60 degrees shows slightly higher catalytic activity.

Overall Summary on both TTL enzyme MGS0156 enzyme and Nle modification

In comparison with the TTL Nle and the MGS0156 Nle , the k_m of TTL Nle has a substantially lower k_m than MGS0156 Nle ranging from 2.0x10_-4µM (Table 4) to 1.1x10_-3(Table 6 row 3). The results illustrated that TTL Nle has a more effective binding affinity toward the substrate and enzyme than the MGS0156 Nle. With regards to the k_cat , the MGS0156 is approximately four-fold higher than the TTL Nle.

In addition to the specificity constant, wild-type MGS0156 in 60 ℃ has superior performance than all the TTL variants, with an extremely high specificity constant of 1.4x10⁵ s^-1µM^-1. It surpasses at least the TTL variants over five-fold. This significant result can lead to the further experiments on the PLA degradation by MGS0156 in 60 ℃ which suits the environment on the PLA degradation.

Est119

Description

Est119 is an esterase originally identified in the Thermobifida alba strain AHK119 (AB298783). Est119 has been shown to degrade aliphatic-aromatic copolyesters and decrease the size of polymer particles of other biodegradable plastics, with an optimal temperature range of 45-55°C (Hu and al. 2009). Similarly to MGS0156, Est119 is interesting due to its potential temperature compatibility with Manitoban composting methods. Our team modified the Est119 genetic sequence to include the Lpp-OmpA anchor to allow for the characterization of the surface-display mechanism of PLAnet Zero.

Results

Cloning and Expression

The expected bands for the Est119 fragment and vector backbone are 1.1 kb and 5.5 kb, respectively. As seen in Figure 15, the presence of a band at ~1.2 kb and ~ 5 kb, corresponds to the expected fragment and vector size, allowing confirmation regarding the identity of our plasmid of interest.

Figure 15. Double digestion confirming the presence of Est119. A band can be observed at 1.2 kb, corresponding to the expected fragment of Est119. The agarose gel was run at 100 V for 1 hour.

Following confirmation of the Est119 fragment, Gibson Assembly was performed to transfer Est119 from the pUCIDT cloning vector to the pET28b(+) expression vector. Double digestion was performed following cloning and the presence of a band at 1.5 and 5.0 kb representing the Est119 fragment and pET28b(+) vector, respectively, suggests successful assembly (Figure 16).

Figure16. Double digestion confirming the presence of Est119. A band can be observed at ~1.5 kb, corresponding to the expected fragment of Est119, following Gibson assembly into the pET28b (+) vector. The agarose gel was run at 100 V for 1 hour.

Following confirmation of our Est119 genetic sequence, the construct was transformed into E. coli BL21(DE3) and underwent protein expression. Figure 17 depicts the SDS-PAGE results following the expression test. The presence of an overexpressed band between 25 kDa and 35 kDa in lane 3 containing the induced sample, compared to the non-induced sample in lane 4, corresponds to the expected literature value of 33.3 kDa, suggesting that expression was successful and we may proceed to the whole-cell activity assay.

Figure17. 12.5% SDS-PAGE confirming expression of Est119. E.coli BL21(DE3) was induced with 1 mM IPTG and incubated at 16℃ at 250 rpm overnight. The gel was run for 50 minutes at 20 milliamps.

Characterization of Whole-Cell Activity

Regarding the whole-cell activity assay of Lpp-Est119, Figures 18 and 19 show increased absorbance at 410 nm after incubation of Lpp-Est199 with the chromogenic substrate. This increase in absorbance corresponding to enzyme activity suggests a functional surface-display mechanism of Lpp-Est119. It should be noted that similar results were seen in the E. coli BL21(DE3)ΔLpp strain, meaning that the Lpp-Est119 is not competing with the native Lpp in the cell.

Further, Figures 20 and 21 support a functional Lpp-Est119 surface display mechanism. A similar increased absorbance at 410 nm when incubated with Lpp-Est119 was observed, and no absorbance is seen when the Est119 protein alone, with no Lpp anchor, was transformed. As a result, it can be assumed that the absorbance corresponding to the enzymatic activity from the whole-cell assay is not due to free enzyme.

Figure18. Absorbance as a function of time for the whole cell esterase activity assay of Est119. After induced expression, E.coli BL21 (DE3) + Est 119 or E.coli BL21 (DE3)ΔLpp + Est119, a native Lpp deficient stain, were washed and resuspended in PBS with 100µM NPO with a cell density normalized to OD600 = 1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.

Figure 19. Absorbance as a function of time for the whole cell esterase activity assay of Est119. After induced expression E.coli BL21 (DE3) + Est119 or E.coli BL21 (DE3)ΔLpp +Est119, a native Lpp deficient stain, were washed, and resuspended in PBS with 100µM pNOB with a cell density normalized to OD600 = 1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.

Figure 20. Absorbance as a function of time for the whole cell esterase activity assay of Est119. After induced expression E.coli BL21 (DE3) + Est119 or E.coli BL21 (DE3) + Est119(ΔLpp), bacteria with with a Lpp deficient plasmid, were washed, and resuspended in PBS with 100µM pNOB with a cell density normalized to OD600 = 1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.

Figure21. Absorbance as a function of time for the whole cell esterase activity assay of Est119. After induced expression, E.coli BL21 (DE3) + Est119 or E.coli BL21 (DE3) + Est119(ΔLpp), bacteria with an Lpp deficient plasmid, were washed and resuspended in PBS with 100µM NPO with a cell density normalized to OD600 = 1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.

RPA1511

Description

RPA1511 is a carboxyl esterase originally identified in Rhodopseudomonas palustris. RPA1511 has been shown to degrade a variety of polymers, including PLA, with an optimal temperature range of 50-60℃ (Hajighasemi et al. 2016). Our team modified the RPA1511 genetic sequence to include the lpp-OmpA anchor in order to characterize the surface-display mechanism of PLAnet Zero.

Results

Cloning and Expression

The expected bands for the RPA1511 fragment and vector backbone are 1.7 kb and 2.8 kb, respectively. As seen in Figure 22, the presence of a band at ~1.5 kb and ~3 kb, corresponds to the expected fragment and vector size, allowing confirmation regarding the identity of our plasmid of interest.

Figure22. Double digestion confirming the presence of RPA1511. A band can be observed at 1.5 kb, corresponding to the expected fragment of RPA1511. The agarose gel was run at 100 V for 1 hour.

Following confirmation of the RPA1511 fragment, Gibson Assembly was performed to transfer RPA1511 from the pUCIDT cloning vector to the pET28b(+) expression vector. Double digestion was performed following cloning and the presence of a band at 1.5 kb and 5 kb representing the RPA1511 fragment and pET28b(+) vector, respectively, suggests successful assembly (Figure 16).

Following confirmation of our RPA1511 genetic sequence, the construct was transformed into E. coli BL21(DE3) and underwent protein expression. Figure 23 depicts the SDS-PAGE results following the expression test. The presence of an overexpressed band between 25 kDa and 35 kDa in lane 3 containing the induced sample, compared to the non-induced sample in lane 4, corresponds to the expected literature value of 32.7 kDa, suggesting that expression was successful and we may proceed to the whole-cell activity assay.

Figure23. 12.5% SDS-PAGE confirming expression of RPA1511. E.coli BL21(DE3) was induced with 1 mM IPTG and incubated at 16℃ at 250 rpm overnight. The gel was run for 50 minutes at 20 milliamps.

Characterization of Whole-Cell Activity

In comparison to Est119, the results regarding the Lpp-RPA1511 surface display mechanism remain inconclusive. Although a spike in absorbance can be observed in the Lpp-RPA1511 that is not present in the control with the RPA1511 protein alone, there is a drop in absorbance as it reaches twenty minutes (Figures 24, 25). However, in Figures 26 and 27 it can be seen that Lpp-RPA1511 is resulting in increased absorbance at 410 nm and that similar results were seen in the BL21(DE3)ΔLpp strain also suggesting no competition with native Lpp. Paired with the results of Lpp-Est119, it is of interest to repeat the experiment to further characterize the surface-display mechanism in Lpp-RPA1511.

Figure 24. Absorbance as a function of time for the whole cell esterase activity assay of RPA1511. After induced expression E.coli BL21 (DE3) + RPA1511 or E.coli BL21 (DE3) + RPA1511(ΔLpp), bacteria with with a Lpp deficient plasmid, were washed, and resuspended in PBS with 100µM NPO with a cell density normalized to OD600 = 1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.

Figure 25. Absorbance as a function of time for the whole cell esterase activity assay of RPA1511. After induced expression E.coli BL21 (DE3) + RPA1511 or E.coli BL21 (DE3) + RPA1511(ΔLpp), bacteria with with a Lpp deficient plasmid, were washed and resuspended in PBS with 100µM pNOB with a cell density normalized to OD600 = 1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.

Figure 26. Absorbance as a function of time for the whole cell esterase activity assay of RPA1511. After induced expression, E.coli BL21 (DE3) + RPA1511 or E.coli BL21 (DE3)ΔLpp +RPA1511, a native Lpp deficient stain, were washed and resuspended in PBS with 100µM NPO with a cell density normalized to OD600 = 1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.

Figure 27. Absorbance as a function of time for the whole cell esterase activity assay of RPA1511. After induced expression, E.coli BL21 (DE3) + RPA1511 or E.coli BL21 (DE3)ΔLpp +RPA1511, a native Lpp deficient stain, were washed and resuspended in PBS with 100µM pNOB with a cell density normalized to OD600 = 1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.

GEN0105

Description

GEN0105 is a PLA depolymerase originally identified in Paenibacillus amylolyticus, strain TB-13. This strain has been observed to degrade a variety of aliphatic polyesters, including PLA plastics with maximal activity occurring in the range of 45-55℃ (Hajighasemi et al. 2016). Our team modified the GEN0105 genetic sequence to include the Lpp-OmpA anchor in order to characterize the surface-display mechanism of PLAnet Zero.

Results

Cloning

The expected bands for the GEN0105 fragment and vector backbone are 1.38 kb and 5.3 kb, respectively. As seen in Figure 13, the presence of a band at ~1.4 kb and ~6 kb, corresponds to the expected fragment and vector size, allowing confirmation regarding the identity of our plasmid of interest.

Figure 28. Double digestion confirming the presence of GEN0105. A band can be observed at 1.5 kb, corresponding to the expected fragment of GEN0105. The agarose gel was run at 100 V for 1 hour.

After multiple attempts to express GEN0105 and due to time constraints, we were unsuccessful in achieving protein expression.

Protocols

Molecular Biology (DNA)

Transformation

Restriction Digestion of DNA

Agarose Gel Electrophoresis

DNA Precipitation

Molecular Biology (Proteins)

SDS Page

Protein Expression

DNA SDS Page Expression Test

Protein Purification Using IMAC

Activity Assays

Michaelis-Menten Enzyme Activity Assay

Whole Cell Assay

References

Akutsu-Shigeno, Y., Teeraphatpornchai, T., Teamtisong, K., Nomura, N., Uchiyama, H., Nakahara, T., & Nakajima-Kambe, T. (2003). Cloning and sequencing of a poly(DL-lactic acid) depolymerase gene from Paenibacillus amylolyticus strain TB-13 and its functional expression in Escherichia coli. Applied and environmental microbiology, 69(5), 2498–2504. https://doi.org/10.1128/AEM.69.5.2498-2504.2003

Haernvall, K., Fladischer, P., Schoeffmann, H., Zitzenbacher, S., Pavkov-Keller, T., Gruber, K., Schick, M., Yamamoto, M., Kuenkel, A., Ribitsch, D., Guebitz, G. M., & Wiltschi, B. (2022). Residue-specific incorporation of the non-canonical amino acid norleucine improves lipase activity on synthetic polyesters. Frontiers in Bioengineering and Biotechnology, 10. https://doi.org/10.3389/fbioe.2022.769830

Hajighasemi, M., Nocek, B. P., Tchigvintsev, A., Brown, G., Flick, R., Xu, X., Cui, H., Hai, T., Joachimiak, A., Golyshin, P. N., Savchenko, A., Edwards, E. A., & Yakunin, A. F. (2016). Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases. Biomacromolecules, 17(6), 2027–2039. https://doi.org/10.1021/acs.biomac.6b00223

Hajighasemi, M., Tchigvintsev, A., Nocek, B., Flick, R., Popovic, A., Hai, T., Khusnutdinova, A. N., Brown, G., Xu, X., Cui, H., Anstett, J., Chernikova, T. N., Brüls, T., Le Paslier, D., Yakimov, M. M., Joachimiak, A., Golyshina, O. V., Savchenko, A., Golyshin, P. N., Edwards, E. A., … Yakunin, A. F. (2018). Screening and Characterization of Novel Polyesterases from Environmental Metagenomes with High Hydrolytic Activity against Synthetic Polyesters. Environmental science & technology, 52(21), 12388–12401. https://doi.org/10.1021/acs.est.8b04252

Hu, X., Thumarat, U., Zhang, X., Tang, M., & Kawai, F. (2010). Diversity of polyester-degrading bacteria in compost and molecular analysis of a thermoactive esterase from Thermobifida alba AHK119. Applied microbiology and biotechnology, 87(2), 771–779. https://doi.org/10.1007/s00253-010-2555-x