Part:BBa_K4652010
T7-RBS-SpyTag-PCLase1-SpyCatcher-Tr
Polycaprolactone (PCL) is a biodegradable plastic material that has received FDA approval. It's extensively used in various applications such as tissue engineering, drug delivery, food packaging, and agricultural coverings1. Typically, products made from PCL take 2-3 years to decompose. As a result, there's a growing interest in identifying novel enzymes that can degrade PCL or in optimizing those that are currently available commercially2. However, most of them can’t tolerate at extremely high temperatures, including the boiling temperatures encountered during the thermoforming process of shaping PCL plastic.
In Prof. Fan Li's laboratory, novel PCL-degrading enzymes, PCLase I and PCLase II, were identified and purified from Pseudomonas hydrolytica3. This discovery was made possible by cultivating the bacteria in a PCL-emulsified medium. The team conducted an in-depth study of the PCLase enzymes, examining aspects such as enzyme activity, the influence of pH and temperature, substrate specificity, degradation products, as well as the associated gene sequences and protein structures. Learned with this comprehensive data and the enzymes' impressive PCL-degrading efficiency, our aim is to enhance their thermostability.
SpyRing cyclization technique to enhance enzyme thermal resilience was clarified by Dr. Mark Howarth’s team4. SpyRing harbors genetically modified SpyTag (13 amino acids) on the N-terminus and SpyCatcher (12kDa) on the C-terminus on the protein of interest. This context spontaneously reacts together through an irreversible isopeptide bond. SpyRing cyclization was demonstrated successfully to increase stress resilience of β-lactamase and some industrially important enzymes. With this synthetic biology tool, we plan to employ SpyRing cyclization techniques and expect to make PCLase thermal resistent, as demonstrated in our work with the SpyTag-GFP-SpyCatcher construct (BioBrick Part: BBa_K4652002)5.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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