Part:BBa_K1387006
The casette of degradation module is comprised of constitutive promoter (BBa_J23106), tet operator (BBa_K079036), RBS (BBa_B0034), outer membrane protein A (ompA) (BBa_K103006) fused with LC Cutinase (BBa_K936014) , and terminator (BBa_B1002). We modified the ompA by deleting the stop codon, so it can fused well with the LC cutinase. LC Cutinase part we got from UC_Davis 2012 part. Bba_K936014 from UC_Davis just contains pelB-LC Cutinase-with polyhistidine tag. But, the LC Cutinase from UC_Davis 2012 that we characterized is BBa_K936020, because it contains promoter-RBS-pelB-LC Cutinase-polyhistidine tag. The characterization data can be seen in experience page of BBa_K936020. By using constitutive promoter, we expect the recombinant protein, in this case ompA-LC Cutinase, will be synthesized indefinitely. ompA is outer membrane protein. It comprises of β–strand B3-B7. Rather than using full sequence of ompA, we use ompA (46-159) fused with lipoprotein signal sequence, thus exposing it on the cell surface of bacteria1. We fuse lpp-ompA with LC Cutinase, an enzyme that exhibit esterase activity. LC-Cutinase esterase activity will cleave the ester bond on PET and releasing terephthalic acid and ethylene glycol as a product. Fusion strategy of lpp-ompA on LC Cutinase gene is one of our way to create a whole cell biocatalyst which will be an effective molecular machinery to degrade PET on the environment. Due to constitutive expression of the recombinant protein in E.coli, there is a possibility of formation of insoluble protein inside the cell (inclusion body), because of that, we put tet operator upstream of RBS for further regulation.
For biodegrading experiments, we used 3x5 cm2 bottle plastics with average weight 0.05 g. Then, we washed it several times with water and ethanol.
Plastic sample was applying just before we inoculated the bacteria on the medium. Bacterial culture were grown at 37C in Luria-Broth medium. After 3 days of incubation, we washed the plastic with water and ethanol then dried for measurement of the weight loss.
Meanwhile, for UC Davis bacterial culture, we inoculated the bacteria in Luria-Broth medium supplemented with 170 ppm cloramphenicol. After an absorbance A600 nm of 0.6 was reached, we added 1% of arabinose and plastic sample. After 2 days of incubation, we washed the plastic with water and ethanol then dried for measurement of the weight loss. We used medium supplemented with antibiotics and plastic sample as control on this experiment.
The scaning electron microsccopy analysis of fractured surface of PET film was carried out using Scaning electron microscope. The surface of the treated PET samples were coated with conductive heavy metals such as gold/palladium.
SEM image shows that cracks were observed at the surface of a plastic sample (PET) after incubation on the bacterial culture. But, there is no significant weight decreased of the plastics samples. From this SEM study we conclude that Ompa-LcCut fussion ITB_Indonesia were able to degrade PET plastics samples. Figure 1. shown the control sampel that we could not observe any cracks. Figure 2 shown the PET degradation by OmpA-Lc cutinase from ITB_Indonesia.
Figure 1.Control sample (PET plastic incubated in medium)
Figure 2.PET plastic after treatment with ompA-LC-cutinase from ITB_Indonesia gene construct (BBa_K1387006)
Reference
Sepperumal, Umaheswari, Murali Markandan, and Anbusaravanan Natarajan. 2013. electron microscopic studies of Polyethylene terepthalate degradation potential of Pseudomonas species. J. Microbiol. Biotech. Res. (1): 104-110
Mittal,Alok, R.K. Soni, Khrisna Dutt, and Swati Singh. 2010. Scaning electron microscopy study of hazardous waste flakes of polyethylene terephthalate (PET) by aminolysis and ammonolysis. Journal of Hazardous Materials Volume 178, Issues 1-3
Poly(ethylene terephthalate) (PET) is a plastic that is a polymer of ethylene terephthalate (C10H8O4) units (Sarker et al., 2010). Polyethylene terephthalate (PET) can be degrade via limited enzymatic hydrolysis of the ester bond of the polymer backbone, one enzyme that have been assesed for this purpose is cutinase (Ribitsch et al., 2012). When polyethylene terephthalate degraded, it will produce terephthalic acid and ethylene glycol (Venkatachalam et al., 2012). Ethylene glycol is one of alcohol compound. Oxidation of alcohols by strong oxidants such as chromic acid (K2Cr2O7) in suphuric acid(H2SO4) is possible, but differs depending on the degree of alcohol. If a reaction has occurred using chromic acid in sulphuric acid, there is a color change from orange to green. In our project we use chromic acid to detect ethylene glycol as product of the PET degradation by LC cutinase enzyme.
Figure 1. Ethylene glycol assay using chromic acid)
Each sample used for measure the absorbance contain a fixed amount of chromic acid. The absorbance of the chromic acid will represent the total amount of ethylene glycol inversely. It means that if the absorbance is high, the amount of ethylene glycol in solution is low, and if the absorbance is low, the amount of ethylene glycol in solution is high. If the absorbance is high, the amount of chromic acid is high, which means that the amount of ethylene glycol being oxidized by chromic acid is low and vice versa. Both of LC Cutinase UC Davis and OmpA-LC Cutinase ITB_Indonesia shown the degrading activity of PET become ethylene glycol as once of the product.The optimum time to produce ethylene glycol is on the fourth hour after inoculation.
Reference
Ribitsch D, Enrique HA, Katrin G, Anita D, Sabine Z, Annemarie M, Rosario DR, Georg S, Karl G, Helmut S and Georg MG. 2012. A New Esterase from Thermobifida halotolerans Hydrolyses Polyethylene Terephthalate (PET) and Polylactic Acid (PLA). Polymers. 4f: 617-629
Sarker M, Aminul K, Mohammad MR, Mohammed M and ASMD Mohammad. 2011. Waste Polyethylene Terephthalate (PETE-1) Conversion into Liquid Fuel. Journal of Fundamentals of Renewable Energy and Applications. 1
Venkatachalam S, Shilpa GN, Jayprakash VL, Prashant RG, Krishna R and Anil KK. 2012. Degradation and Recyclability of Poly (Ethylene Terephthalate). Intech
Blastp program shows that LC cutinase shows high amino acid sequence similarity (54 to 60%) to lipase (Sulaiman, 2012). So, we tried to measure LC-cutinase activity using p-nitrophenylpalmitate (pNPP) as a substrate. pNPP typically used for measuring lipase activity.
In this assay, we used bacterial culture because in real application we would like to use bacterial culture to degrade PETdirectly. As negative control, we used E.coli BL21 without plasmid. We also included LC-cutinase UC Davis 2012 gene construct (Bba_K936020) in this experiment. LC-cutinase UC Davis 2012 bacterial culture is induced after 2 hours of growth (after OD 600 nm ±0.6 was reached) using 1% of arabinose. All of the bacterial culture were harvested after 4 hours of growth.From ethylene glycol assay using chromic acid, we found that ompA-LC-cutinase from ITB_Indonesia construct (BBa_K1387006) has the highest activity after 4 hours of growth.
At first, we measured the activity at 60 degrees Celsius. We found that both ompA-LC-cutinase from ITB_Indonesia and LC-cutinase UC Davis 2012 still performed small activity. Then, we tried measure the activity at 25 degrees Celsius. The ompA-LC-cutinase from ITB_Indonesia’s activity was 0.001 U/mL. While, LC-cutinase UC Davis 2012 has ten fold higher (0.01 U/mL). The LC cutinaseactivity was determined based on the standard curve of p-nitrophenol. One unit of cutinase activity was defined as the amount of enzyme releasing 1 μmolpNP per minute under the assay conditions.
From this experiment, we are able to confirm that ompA-LC-cutinase from ITB_Indonesiasuccessfully perform activity. But, its suggest that we still have to check the possible activity in different time of bacterial growth, the expression rate, the LC-cutinase presentation by ompA and activity assay using different substrate.
Figure 1.LC Cutinase activity. The enzymatic activity was determined at 60C in assay condition using pNP-palmitate (C16) as a substrate. The experiment was carried out twice.
Figure 2.LC Cutinase activity. The enzymatic activity was determined at 25 C in assay condition using pNP-palmitate (C16) as a substrate. The experiment was carried out twice.
Reference
Sulaiman, Sintawee, SayaYamat, EikoKanaya, Joong-Jae Kim, Yuichi Koga, Kazufumi Takano and Shigenori Kanaya. 2012. Isolation of a Novel Cutinase Homolog with Polyethylene
Terephthalate-Degrading Activity from Leaf-Branch Compost by Using a Metagenomic Approach. Applied and Environmental Microbiology p 1556-1562.
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