Part:BBa_K4279001
W1-lipase
W1-lipase
Profile
Name: SP-lipase
Base Pairs: 1854 bp
Origin: Lactiplantibacillus Plantarum, genome
Properties: a lipase for triacylglyceride digestion.
Usage and Biology
BBa_K4279000 is the coding sequence of SP-lipase. Lipase is a primary lipase critical for triacylglyceride digestion in humans and is considered a promising target for the treatment of obesity [1]. Triacylglycerol lipase is the primary lipase secreted by the pancreas, and is responsible for breaking down dietary lipids into unesterified fatty acids (FAs) and monoglycerides (MGs). Medically, lipases are targets for therapeutic intervention in the treatment of obesity. The focus of applied research with lipases has been to exploit the unusual properties of lipolytic systems for the production of chiral pharmaceuticals, improved detergents, and designer fats [2]. Obesity is a medical condition in which excess body fat accumulates to the extent that it may have a negative effect on health, leading to reduced life expectancy and/or increased health problems. Diverse approaches to the prevention and treatment of obesity have been reported [3-5]. W1-lipase (EC 3.1.1.3) is a lipase amplified from Pseudomonas sp. 7323. The SP-lipase is made up of 265 aa [6].
Construct design
1. Construction of the lipase expression plasmids The SP-lipase gene was amplified from the pseudomonas and then inserted in the XhoI and HindIII sites of pET28a (Figure 2).
In order to build our plasmids, plasmid pET28a was digested with XhoI and HindIII (Figure 3), and we used T4 DNA ligase to ligate the fragments and the vector. Then we transformed the recombinant plasmids into E. coli DH5α competent cells and coated on the LB (Kanamycin) solid plates.
The returned sequencing comparison results showed that there were no mutations in the ORF region, and the plasmid was successfully constructed (Figure 4).
2. Protein lipase expression
The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and cultured overnight in the medium containing resistance. When the OD600 was around 0.4-0.5, the IPTG was added to induce the expression of recombinant protein W1-lipase/SP-lipase, and then the strains were cultured at 16℃ for 20h. After that, the collected bacterial solution was cracked by Ultrasonic crushing. SDS-PAGE was used to analyze the recombinant proteins. Figure 5 showed the electrophoretic results of the protein gel.
6.Lipase activity detection at different pH and temperature
a)Standard curve measurement
In order to measure the standard curve of the activity of lipases, we chose p-nitrophenol as the substrate and detected its absorbance value of it when adding lipases. 0.02789g of p-nitrophenol (p-np) was weighed and dissolved in 100mL of solution B, and stored in a brown reagent bottle after configuration and stored at 4°C. 0.02, 0.04, 0.06, 0.08, 0.12, 0.16mL of p-nitrophenol solution (2mmol/L) was diluted to 4mL, and the absorbance value at 410nm was measured successively. The standard curve was drawn with p-nitrophenol (0.01, 0.02, 0.03, 0.04, 0.06, 0.08, mmol/L) as the abscissa and absorbance value Y as the ordinate (Figure 6).
According to the standard curve determination method, the standard curve is drawn as shown in Figure 6. Regression coefficient R2=0.9979, the results are credible. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 777
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 46
- 1000COMPATIBLE WITH RFC[1000]
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