T7-RBS-SpyTag (D7A)-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 (Part:BBa_K4652002).

PROTEIN STRUCTURE & ACTIVITY

The mechanism in SpyRing for the spontaneous cyclization reaction involves Asp7 on the SpyTag at the N-terminus forming double hydrogen bonds with Glu77 on the SpyCatcher at the C-terminus. This interaction strengthens the creation of an irreversible isopeptide bond between Asp7 on SpyTag and Lys31 on SpyCatcher. As a result, the protein is seamlessly cyclized, enhancing its resistance to chemical, thermal, or enzymatic degradation⁷.

To verify the protein structure and its correlation to thermal tolerance, in a similar approach on T7-RBS-SpyTag (D7A)-GFP-SpyCatcher-Tr (Part:BBa_K4652003), we engineered a SpyTag mutation of PCLase and designated as T7-RBS-SpyTag (D7A)-PCLase1-SpyCatcher-Tr (Part:BBa_K4652011). The lysates, as collected previously, were subject to SDS-PAGE and Coomassie Blue staining. In Figure 4, the result showed a distinct band corresponding to the linear form of SpyTag (D7A)-PCLase1-SpyCatcher, with an expected size of 46.53 kDa. However, the lysates from SpyTag-PCLase1-SpyCatcher displayed somewhat fuzzy bands representing incomplete cyclized or fully cyclized forms. This ambiguity might arise from aggregation or polymerization due to isopeptide bond formation⁸. It's worth noting that the presence of incompletely cyclized PCLase could partially account for the reduced lipase activity after heating observed in the experiment in Figure 3.

Figure 4. Protein structure analysis of cyclized form and linear form of SpyTag-PCLase1-SpyCatcher. 15 µg of IPTG-induced lysates were run on a 4-12% gradient SDS-PAGE (BIO-RAD) and stained with standard Coomassie Blue. The AceColor Prestained Protein Marker (10 - 180 kDa) was used for size reference. Lane 1 refers to the linear proteins derived from lysates of SpyTag (D7A)-PCLase1-SpyCatcher (BBa_K4652011). Lane 2 refers to the cyclized proteins derived from lysates of SpyTag-PCLase1-SpyCatcher (BBa_K4652010). The predicted molecular weight for the SpyTag-PCLase1-SpyCatcher protein is 46.53 kDa.

In addition, we compared the thermostabilities between lipase activities in lysates with linear PCLase (SpyTag (D7A)-PCLase1-SpyCatcher) and cyclized PCLase (SpyTag-PCLase1-SpyCatcher) using the pNPB assay. After being treated at temperatures ranging from 70°C to 100°C for 10 min, the cyclized lipase retained higher activity compared to the linear form of PCLase (Figure 5). It maintained over 50% activity up to 90°C and retained up to 20% at 100°C. It's worth exploring whether purifying and concentrating the fully cyclized PCLase could enhance the lipase activity in the lysates for thermal testing. Taken together, the experimental data demonstrated successfully making a thermostable PCLase by cyclization through SpyRing technique.

Figure 5. Thermostability of PCLase lipase activities between cyclized PCLase (SpyTag-PCLase1-SpyCatcher, Part:BBa_K4652010) and linear PCLase (SpyTag (D7A)-PCLase1-SpyCatcher, Part:BBa_K4652011). E. coli BL21 was transformed with the indicated plasmid and then was induced by 0.3 mM of IPTG at 25°C for 20 hours. Subsequently, the lysates were harvested using 0.1 mm Disruptor Beads (Scientific Industries, Inc). After treated at 100°C for the indicated time, a 20 µL aliquot of these lysates was combined with 175 µL of Tris-HCl buffer (20mM, pH=8) and 5 µL of pNPB (40 mM dissolved in 2-methyl-2-butanol). Lipase activity was read at 405 nm based on p-Nitrophenol production. All values were divided by the average of the untreated control, with the resulting ratio representing the lipase activity fold change.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]