Revision as of 03:59, 30 September 2024

lipA

LipA is a lipase enzyme that hydrolyzes fats into fatty acids and glycerol. It is utilized in various industries, including food processing, detergents, and biofuel production, due to its ability to break down lipids efficiently under different conditions.

Description

We are concentrating on creating an enzyme that efficiently degrades fats for better jeans maintenance and cleaning. LipA, a lipase, hydrolyzes fats into fatty acids and glycerol, demonstrating effectiveness in various conditions. LipA's high efficiency is vital for optimizing production processes, improving product quality, reducing costs, and supporting sustainability initiatives.

Usage and Biology

The lipA gene(BBa_K5458004), derived from Pseudomonas sp. 7323, was synthesized and cloned into the pET23b plasmid, followed by overexpression in the BL21 strain.

Figure 1. The gene circuit of lipA.

Characterization

Analysis of Lipase Characteristics

The aim of this experiment is to construct a lipase-producing bacterial strain and evaluate the overexpression of lipase in the engineered strain.The lipA gene, derived from Pseudomonas sp. 7323, was synthesized and cloned into the pET23b plasmid, followed by overexpression in the BL21 strain. The soluble lipase in the supernatant was obtained through ultrasonic disruption of the engineered strain. Since lipids are insoluble in water and degrade at a very low rate, p-NPB (butyric acid p-nitrophenyl ester, Sigma) was used as the substrate due to its ester bond and similar properties to lipids. Lipase activity was measured using spectrophotometric methods. A 10 mM p-NPB solution (dissolved in acetonitrile) was prepared, and 0.1 mM p-NPB was added to 200 μL of the crude enzyme solution. The reaction was carried out at 37°C, and the hydrolysis rate was determined by measuring the absorbance of the produced p-nitrophenol at 405 nm using a microplate reader. Under the conditions of 37°C, pH=7, and 60 min of reaction time, the BL21/p23B-LipA group showed significantly higher absorbance than the control groups, indicating higher lipase activity and superior degradation efficiency.

Figure 2. Comparison of lipase activity among BL21, BL21/pET23B, and BL21/p23B-LipA. As shown in Figure 2, after 60 min of reaction at 37°C and pH=7, the absorbance at 405 nm in the BL21/p23B-LipA group was significantly higher than that in the BL21 and BL21/pET23B groups. This indicates that the BL21/p23B-LipA group has the highest degradation efficiency and possesses lipase activity.

The effect of temperature on lipase activity

The objective of this experiment is to study the effect of temperature on lipase activity and to determine the optimal reaction temperature. In the lipase crude enzyme solution and p-NPB reaction system, the environmental temperatures were adjusted to 16 ℃, 25℃, 30℃, 37℃, and 45℃. After incubating for 30 min, the effect of different temperatures on lipase activity was tested. The result shows that the optimal temperature for cellulase activity is 30℃.

Figure 3. The effect of temperature on lipase activity. Figure 3 shows the trend in lipase activity across different temperatures. Under the conditions of pH = 7 and a reaction time of 60 min, the lipase activity increased with temperature up to 30℃, where the highest activity was observed. Beyond 30℃, the activity declined, with significantly lower activity at both 16℃ and 45℃.

The effect of pH on lipase activity

To test the influence of pH on lipase, bacterial pellets were resuspended using citrate buffer (pH=3.0, 5.5), PBS (pH=7.4), or Tris-HCl buffer (pH=8.2, 10.8). After cell lysis, a crude enzyme solution was obtained, and 1 mM p-NPB was added. After incubating at 37℃ for 1 hour, the absorbance of the reaction system was measured at 405 nm. To achieve optimal reaction results, it is recommended to use the crude enzyme solution at pH=8.2.

Figure 4. The effect of pH on lipase activity. Under the conditions of incubation at 37 ℃ for 1 h with the addition of 1 mM p-NPB, the crude enzyme solution at pH=8.2 exhibited the highest absorbance, indicating that lipase has maximum activity under these conditions. Beyond this point, the activity decreased as pH increased further.

Potential application directions

The LipA expression vector has been shown to efficiently hydrolyze esters under various conditions. In consumer applications, such as denim cleaning, the enzyme improves the removal of oil and stains, enhancing fabric care and extending product life. In industrial settings, LipA offers significant advantages, such as increased efficiency in processes like bioremediation, waste treatment, and manufacturing, where ester breakdown is critical. This versatility allows LipA to contribute to advancements across multiple sectors, benefiting both industry and society. References Jonsson U, Snygg B G. Lipase production and activity as a function of incubation time, pH and temperature of four lipolytic micro‐organisms[J]. Journal of Applied Microbiology, 1974, 37(4): 571-581.

Sequence and Features