We amplified the lipase gene from the lactobacillus Plantarum lipase gene W1-ligase and pseudomonas lipase gene SP-ligase, and inserted them in the XhoI and HindIII sites of pET28a, respectively (Figure 1).

In order to build our plasmids, we let the synthetic company synthesize two target gene fragments (W1-lipase and SP-lipase). We digested the target fragments and the pET28a vector with XhoI and HindIII (Figure 2), 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.

We verified the colonies through colony-PCR (Figure 3), and then we inoculated single colonies and we send the constructed recombinant plasmid to a sequencing company for 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.

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

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.

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.

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.

As shown in Figure7, when changed the pH value of the buffer, the activity of lipase is changed compared with the negative control, and the W1-lipase and SP-lipase showed no obviously different. And when the pH value is 9, the lipase exhibited the highest activity.

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 ℃.

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, while 40℃ may be the optimum temperature for W1-lipase.