Difference between revisions of "Part:BBa K4652007"

Revision as of 09:37, 23 September 2023

T7-RBS-SpyTag-CALB-SpyCatcher-Tr

Lipases are a group of enzymes that break down triglycerides. They are present in numerous organisms and exhibit a broad range of substrate specificities. Pseudozyma (Candida) antarctica lipase B (CALB) stands out among the most renowned lipases worldwide due to its exceptional properties, such as high stereoselectivity and stability. Many researchers are intent on enhancing CALB's attributes through protein engineering techniques. Eukaryotic microbes, including Aspergillus oryzae, Pichia pastoris, Saccharomyces cerevisiae, and Yarrowia lipolytica, are frequently chosen for expressing recombinant proteins. These eukaryotic systems have played a significant role in lipase protein engineering.

This CALB lipase is commercially available in SIGMA-ALDRICH (Product Number: 62288)

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=== 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. doi: 10.1016/j.jbiosc.2015.07.001. Epub 2015 Aug 10. PMID: 26272415.

===Sequence and Features=== BBa_K4652007 SequenceAndFeatures