Part:BBa_K4279003

pET28a-W1 lipase

Profile

Name: pet28a-W1-lipase

Origin: Lactiplantibacillus Plantarum, genome

Properties: a lipase for triacylglyceride digestion.

Usage and Biology

Figure 1. The structural modeling and fuction of bacterial lipase.

BBa_K4279003 is the coding sequence of W1-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 is a lipase amplified from Lactiplantibacillus Plantarum (LP1406), which is a gram-positive lactic acid bacteria species and exhibits ecological and metabolic adaptability and is capable of inhabiting a range of ecological niches including fermented foods, meats, plants, and the mammalian gastrointestinal tract. The W1-lipase is made up of 265 aa [6].

Construct design

1. Construction of the lipase expression plasmids

We amplified the lipase gene from the lactobacillus Plantarum lipase gene W1-ligase, and inserted them in the XhoI and HindIII sites of pET28a (Figure 2).

Figure 2. W1-lipase expression plasmids in this project.

We digested the target fragments and the pET28a vector 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 results of target gene fragments.

A. double-enzyme digested with the W1-ligase, B. double-enzyme digested with the pET28a vector.

We verified the colonies through sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region, and the plasmid was successfully constructed (Figure 4). So far, we have successfully obtained two recombinant plasmids, which were respectively on the pET28a vector, which can be used to express lipase proteins.

Figure 4. Mapped the sequencing of pET28a-W1-lipase.

As a result, the amplified target gene W1-lipase and the double digested vector pET28a were ligated with T4 ligase to obtain recombinant plasmids pET28A-W1-lipase, so that the recombinant protein had 6-His tags at the carboxyl terminus which could be used to purify the corresponding proteins.

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.

3. Lipase activity detection at different pH and temperature

Standard curve measurement

a) 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 Figure 7, when changed the pH value of the buffer, the activity of lipase is changed compared with the negative control, and the W1-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 8. 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 Figure 8, when changed the reaction temperature, the activity of lipase is different compared with the negative control. 40℃ may be the optimum temperature for W1-lipase.

Improvement of an existing part

Compared to the old part projects BBa_K1671001，which is a biological part submitted by iGEM15_Hangzhou-H14Z in 2015, they only provided a lipase expression plasmid. However, the activity of the lipase didn’t be verified. So it is really important to detect the activity of the lipase and optimize the in vitro reaction system. In this part, we used numerical models to simulate the influence of temperature and pH on e W1-lipase activity. Then, the optimal temperature and pH corresponding to the peak activity of the enzymes were predicted according to the numerical results. Table 1 presents the experimental data of the effect of temperature on the activities of W1-lipase. Table 2 shows the experimental results of the influence of pH on the activities of W1-lipase.

Table 1. Activities of W1-lipase under different temperature.

Table 2. Activities of W1-lipase under different pH.

Here, we used different models to characterize the activities of W1-lipase.

Model (1) to simulate the effect of temperature and pH on the activities of W1-lipase and SP-lipase.

Where p1，p2，p3，q1 and q2 are the parameters need to be determined.

Figure 9. Comparison of numerical and experimental results for the influence of temperature on W1-lipase activity.

Model (2) was applied to simulate the relationship between pH and W1-lipase activities.

Where p'1，p'2，p'3，q'1，q'2 and q'3 are the parameters need to be determined.

Figure 10. Comparison of numerical and experimental results for the effect of pH on W1-lipase 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] S.P.S. Chundawat, V. Balan, L. Da costa Sousa, B.E. Dale, Thermochemical pretreatment of lignocellulosic biomass. Bioalcohol Production. 2010, 24-72.