Difference between revisions of "Part:BBa K4652005"

Latest revision as of 09:48, 23 September 2023

T7-RBS-SpyTag-BCLA-SpyCatcher-Tr

The Burkholderia cepacia lipase (BCLA), previously identified as Pseudomonas cepacia lipase, is a commercially available enzyme, known for its heat resistance and compatibility with various solvents and short-chain alcohols. This enzyme, available in both soluble and immobilized forms, is predominantly utilized in transesterification processes and drug synthesis, owing to its distinct properties.

This BCLA lipase is commercially available in SIGMA-ALDRICH (Product Number: 534641)

PCL-DEGRADING LIPASE COMPARISON

To compare the lipase activities of PCLase I, PCLase II, and other commercially available PCR-degrading enzymes such as BCLA⁵ and CALB⁶, we conducted a pNPB assay. In this assay, a potential lipase breaks down the ester bond of p-nitrophenylbutyrate (pNPB), producing p-Nitrophenol. The concentration of p-Nitrophenol can be measured at 405nm, and these measurements are corresponding to the lipase activity.

Lysates from E. coli BL21, which carried the T7 promoter-driven expression plasmid with the indicated genes in the same context, were collected after being induced with 0.3 mM IPTG at 25°C for 20 hours. The lipase activities within these lysates were assessed using the pNPB assay⁵. As shown in Figure 2, PCLase I exhibited the significantly highest readings at 405 nm. This suggests that under our experimental conditions, PCLase I is the most effective lipase, demonstrating potential activity in decomposing PCL through the hydrolysis of the ester bonds between polymers. Consequently, we chose to investigate the characteristics of PCLase I (hereafter referred to as PCLase for short) in terms of its thermostability, protein structure, PCL degradation capability, and its potential use in real-world products.

Figure 2. Comparison between lipase activities of BCLA, CALB, PCLase I, and PCLase II using pNPB assay. E. coli BL21 was transformed using the indicated T7 promoter-driven gene expression plasmid. The bacteria were 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). 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. The obtained readings were normalized with the OD600 values at the time of bacterial lysate collection.

Reference

Sánchez DA, Tonetto GM, Ferreira ML. Burkholderia cepacia lipase: A versatile catalyst in synthesis reactions. Biotechnol Bioeng. 2018 Jan;115(1):6-24. doi: 10.1002/bit.26458. Epub 2017 Oct 30. PMID: 28941272.

Sequence and Features