Difference between revisions of "Part:BBa K3040501"

Figure 6 Lipase activity assay analysis was performed to check the activity of lipase at different temperature varies with time at pH9.0. The protein was first incubated at the experiment temperature (10, 20, 25, 30 and 40°c) for 30 minutes. Then 4-nitrophenyl decanoate (pND) mixture was added into the protein lysate. The mixture was then detected at 405nm in continuous duration (0, 20, 40, 60 mins) at the temperature required. The fluorescence level in the graph was subtracted with the background fluorescence of protein. Histograms represent normalized means±s.e.m. (n=3).

Figure 7 Lipase activity assay analysis was performed to check the activity of lipase at different temperature for 40 minutes at pH9.0. 40 minutes data was chosen because this time duration is best fitted to the activity curve reported previously in the paper. Thus, our lipase is proved to be functional.

Characterization by 2022 iGEM Team SubCat_China

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SP-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].

Figure 1. The structural modeling of an extremophilic bacterial lipase isolated from saline habitats.

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).

Figure 2. W1-lipase and SP-ligase expression plasmids in this project.

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.

Figure 3. Gel electrophoresis of double-enzyme digested pET28a-SP-lipase.

The returned sequencing comparison results showed that there were no mutations in the ORF region, and the plasmid was successfully constructed (Figure 4).

Figure 4. Mapped the sequencing data of the pET28a-SP-lipase.

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.

Figure 5. SDS-PAGE detection of lipase protein.

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).

Figure 6. The standard curve of p-nitrophenol.

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.

b) Measure the activity of lipase at different pH

Esterase activity was assayed in the pH range from 3.0 to 12.0, and at temperatures of 25 to 70°C. Enzyme thermostability was measured by incubation of the enzyme in 50 mM sodium phosphate buffer (pH 9.0) at 25-70°C for 5 min, 15 min, 30 min, and 1, 2, 4, 6, and 20 h. After incubation, the residual activity of lipase was measured as described above. To test the effects of metals, ions, and additives on the activity of the esterase, lipase was incubated in their presence at a final concentration of 1 mM for 5 min at room temperature. Then, the substrate (p-nitrophenyl acetate) was added, and the reaction mixture was incubated at 37°C. The experiments were performed in triplicate.

Figure 7. The enzyme activity of lipase at different pH..

As shown in Figure7, when changed the pH value of the buffer, the activity of lipase is changed compared with the negative control, and SP-lipase showed no obviously different. And when the pH value is 9, the lipase exhibited the highest activity.

c) Measure the activity of lipase at different temperature

When pH=9, the recombinant enzyme activity reached the highest, 36.-40U/mL, and decreased when pH=9, so the optimal pH of the recombinant enzyme was 9. According to the standard curve, the enzyme activity at the optimum pH and different temperatures are shown in Figures 3-10 (right). At 40℃, the recombinant enzyme activity reached the highest, 36-40U/m L, and decreased when the temperature was higher than 40℃. Therefore, the optimal temperature for the recombinant enzyme was 40℃, but it had higher activity at 30-40 ℃.

Figure 8. the enzyme activity of lipase at different temperatures.

As shown in Figure8, when changed the reaction temperature, the activity of lipase is different compared with the negative control. And when the temperature is around 35℃, the SP-lipase showed the highest activity.

Reference

[1] Paul Joyce, Catherine P. Whitby, Clive A. Prestidge, Nanostructuring Biomaterials with Specific Activities towards Digestive Enzymes for Controlled Gastrointestinal Absorption of Lipophilic Bioactive Molecules, Advances in Colloid and Interface Science,2016, 237; 52-75.

[2] Khan I, Nagarjuna R, Dutta JR, Ganesan R Enzyme-Embedded Degradation of Poly(ε-caprolactone) using Lipase-Derived from Probiotic Lactobacillus plantarum. ACS Omega. 2019, 4(2):2844-2852

[3] H. L. Brockman, lipase.Encyclopedia of Biological Chemistry (Second Edition), 2013.

[4] Birari RB, Bhutani KK. Pancreatic lipase inhibitors from natural sources: Unexplored potential. Drug Discov Today. 2007;12:879–889

[5] Kim S, Lim SD. Separation and Purification of Lipase Inhibitory Peptide from Fermented Milk by Lactobacillus plantarum Q180. Food Sci Anim Resour. 2020, 40(1):87-95.

[6] Kim, K.K., Song, H.K., Shin, D.H., Hwang, K.Y. and Suh, S.W. The crystal structure of a triacylglycerol lipase from Pseudomonas cepacia reveals a highly open conformation in the absence of a bound inhibitor. Structure 5 (1997) 173–185.

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