Fats are an intake in everyday diet. Regardless of their nutritional value, they must be properly broken down for use or removal from the human body. Dietary fats can be difficult to break down correctly since they are lipids that can’t dissolve in liquid and the production of fatty acids in the digestion system may be insufficient. Therefore, we are here to produce an oral solution that contains lipase to help lipid digestion to plausibly eliminate common stomach and intestine symptoms and other health issues including but not limited to abdominal distention, diarrhea, gastropasm, and vitamin deficiency.

Lipase is an enzyme that catalyzes the hydrolyses of triglycerides (oils and lipids), which release fatty acids, diglycerol, glycerol monoesters, and glycerol at different stages, among which fatty acids and glycerol are used to produce energy or used for energy storage. Compared with animal-produced lipase, plant or microbial lipases (Figure 2) have advantages in terms of better substrate specificity and a broader range of tolerance for temperature and pH due to characteristics of a greater diversity of secretion pathways and external excretion.

In our experiment, the method of genetic engineering was chosen to construct an E. coli system capable of overexpressing lipase.
The Lactobacillus W1 and Pseudomonas SP genomes lipases were synthesized by the company. Next, the lipase gene was inserted into the plasmid pET28a to construct an E. coli expression vector in E. coli DH5α and sent to the company for Sanger sequencing. Then, the recombinant expression vector was transformed into E. coli BL21 (DE3), so that the lipase gene could be expressed heterologously in E. coli. We used IPTG to induce protein expression and obtained the lysate to measure the activity of the lipases. Finally, the optimum temperature and pH of the two lipases were preliminarily tested before implementation on humans.

In the experiment, we cultured competent monoclonal E. coli DH5α cells, extracted plasmids from the two gene template cells, double-enzyme digested the targeted gene segment and plasmid pET28a, and used the gel electrophoresis technique to verify the fragments, then ligated the fragments and vector through T4 DNA ligase, transformed the recombinant plasmid into E. coli DH5α competent cells, extracted the recombinant plasmids from the transformed monoclonal bacteria, and transformed into E. coli BL21 (DE3). After inducing the expression of lipase by adding IPTG, we purified the lysate, measured the activity of the lipases, and chemically modified the lipase solution to test enzymatic activity under different pH and temperatures.

1. Successfully construct pET28a-W1-lipase and pET28a-SP-lipase.
2. Expressed and purified lipase lysate.
3. The expressed lipase can catalyze the hydrolyses of triglycerides.

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