Project Description














Plastics Concerns in SDGs


The mass consumption of plastics, especially single-use products, is a significant concern highlighted in Sustainable Development Goal 12: Responsible Consumption and Production. Fewer than 10% of these plastics are recycled. The diversity of plastic materials complicates the gathering and categorization processes during recycling1. Consequently, most plastic waste ends up in landfill and incineration, leading to land, sea and climate pollution. Biodegradable plastics have gained increasing interest in recent years. However, various materials such as PLA, PBAT, PHA, PBS, and PCL decompose under distinct conditions, including specific temperatures, durations, and the presence of certain microbes and their plastic-degrading enzymes (Table 1)2. It's a substantial challenge and impractical to sort each type of plastic individually into recycling bins.

Current Studies and Bottlenecks



Scientists have studied plastic-degrading microbes and discovered novel potential enzymes, including lipases, cutinases, esterase-like enzymes, and more3. In a groundbreaking study published in Nature in 2021, Dr. Ting Xu from UC Berkeley successfully embedded and dispersed enzymes into PCL plastic material using nanoparticles, achieving near-complete depolymerization of polyesters4. However, in the real world, plastic product manufacturing may involve a high-temperature process called thermoforming (Table 2). This process could inactivate the bioactive enzymes, rendering them non-functional. Although PCL is the only plastic material that becomes pliable under 100°C, this near-boiling temperature still challenges the thermal stability of most natural enzymes.



Synthetic Biology and Inspiration

The fibronectin-binding (FbaB) protein in Streptococcus pyogenes is characterized by its thermostable and pH-resistant features, attributed to the CnaB domains. These domains spontaneously form an isopeptide bond, cyclizing the protein through post-translational modification5. In 2015, Dr. Samuel C. Reddington and Dr. Mark Howarth of the University of Oxford in the UK isolated the CnaB domains, dividing them into SpyTag and SpyCatcher components6. They experimented on beta-lactamase by attaching SpyTag to the N-terminus and SpyCatcher to the C-terminus. This technique, termed SpyRing, was illustrated by the isopeptide formation mechanism that circularizes the protein through Asp7 on SpyTag and Lys31 / Glu77 on SpyCatcher. The cyclized protein was then confirmed through protein structure analysis and a bioactivity test under boiling conditions7.



Why PCL?

Polycaprolactone (PCL) is a promising biomaterial highly regarded for its biocompatibility and applications in tissue engineering and medical implants8. Additionally, due to its biodegradable nature, PCL is gaining traction in sustainable packaging solutions and agricultural applications like controlled-release fertilizers and compostable mulch films. However, PCL's degradation is influenced by environmental conditions, typically requiring specific microbes and prolonged durations up to 2-3 years9. We selected PCL as our target for a biodegradable plastic because it is not only FDA-approved10 but also because PCL-based products can be processed under 100°C. This temperature range provides the potential to engineer a thermostable protein for incorporation.



Perspective

In our search for PCL-degrading enzymes, we cloned four potential lipase genes. Two of these (PCLase I and PCLase II)11 are novel discoveries by Dr. Fan Li, while the other two (BCLA12 and CALB13) are commonly cited in numerous studies and are commercially available. After using the pNPB assay, a standard test for lipase activity, PCLase I showed the highest activity in our lab. We then characterized SpyTag and SpyCatcher-incorporated PCLase I (referred shortly as to PCLase from this point) in detail. This characterization included its thermostability using the pNPB assay, its protein structure as revealed by SDS-PAGE and Coomassie Blue staining, and its features related to PCL degradation.



References

  1. Tiwari R, Azad N, Dutta D, Yadav BR, Kumar S. A critical review and future perspective of plastic waste recycling. Sci Total Environ. 2023 Jul 10;881:163433. PMID: 37061055.
  2. Al Hosni AS, Pittman JK, Robson GD. Microbial degradation of four biodegradable polymers in soil and compost demonstrating polycaprolactone as an ideal compostable plastic. Waste Manag. 2019 Sep;97:105-114. PMID: 31447017.
  3. Tournier V, Duquesne S, Guillamot F, Cramail H, Taton D, Marty A, André I. Enzymes' Power for Plastics Degradation. Chem Rev. 2023 May 10;123(9):5612-5701. PMID: 36916764.
  4. DelRe C, Jiang Y, Kang P, Kwon J, Hall A, Jayapurna I, Ruan Z, Ma L, Zolkin K, Li T, Scown CD, Ritchie RO, Russell TP, Xu T. Near-complete depolymerization of polyesters with nano-dispersed enzymes. Nature. 2021 Apr;592(7855):558-563. PMID: 33883730.
  5. Amelung S, Nerlich A, Rohde M, Spellerberg B, Cole JN, Nizet V, Chhatwal GS, Talay SR. The FbaB-type fibronectin-binding protein of Streptococcus pyogenes promotes specific invasion into endothelial cells. Cell Microbiol. 2011 Aug;13(8):1200-11. PMID: 21615663; PMCID: PMC4754676.
  6. Reddington SC, Howarth M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Curr Opin Chem Biol. 2015 Dec;29:94-9. PMID: 26517567.
  7. Schoene C, Fierer JO, Bennett SP, Howarth M. SpyTag/SpyCatcher cyclization confers resilience to boiling on a mesophilic enzyme. Angew Chem Int Ed Engl. 2014 Jun 10;53(24):6101-4. PMID: 24817566; PMCID: PMC4286826.
  8. Ilyas RA, Zuhri MYM, Norrrahim MNF, Misenan MSM, Jenol MA, Samsudin SA, Nurazzi NM, Asyraf MRM, Supian ABM, Bangar SP, Nadlene R, Sharma S, Omran AAB. Natural Fiber-Reinforced Polycaprolactone Green and Hybrid Biocomposites for Various Advanced Applications. Polymers (Basel). 2022 Jan 3;14(1):182. PMID: 35012203; PMCID: PMC8747341.
  9. Bartnikowski M, Dargavilleb TR, Ivanovskia S, Hutmacher DW. Degradation mechanisms of polycaprolactone in the context of chemistry, geometry, and environment. Progress in Polymer Science. 2019 May 20;96:1-20.
  10. U.S. Food and Drug Administration. December 2, 2021. "Polycaprolactone (PCL) Safety Profile"
  11. Li L, Lin X, Bao J, Xia H, Li F. Two Extracellular Poly(ε-caprolactone)-Degrading Enzymes From Pseudomonas hydrolytica sp. DSWY01T: Purification, Characterization, and Gene Analysis. Front Bioeng Biotechnol. 2022 Mar 18;10:835847. PMID: 35372294; PMCID: PMC8971842.
  12. Yang J, Guo D, Yan Y. Cloning, expression and characterization of a novel thermal stable and short-chain alcohol tolerant lipase from Burkholderia cepacia strain G63. Journal of Molecular Catalysis B: Enzymatic. 2007 Jan 9;45(3-4):91-96.
  13. Ujiie A, Nakano H, Iwasaki Y. Extracellular production of Pseudozyma (Candida) antarctica lipase B with genuine primary sequence in recombinant Escherichia coli. J Biosci Bioeng. 2016 Mar;121(3):303-9. PMID: 2627241