| Porcine pancreatic lipase (PPL) is a secreted glycoprotein composed of a single chain of 449 amino acids, with a molecular weight of 50-52 kDa. It is an endo-lipase, and has high efficiency in catalyzing the hydrolysis of triglycerides and release free fatty acids.In animal the digestion of fat is to a large extent dependent on pancreatic enzymes. The digestibility of fat is of special concern because it has been demonstrated that piglets have a high demand for energy that is not met by the food consumed and consequently, body fat is mobilized to cover the energy requirement. During the postweaning period, the activation, secretion and function of pancreatic digestive enzymes has not yet completely developed, in particular the main lipolytic enzyme lipase and colipas. Insufficient production of PPL in early life, in particular at weaning, of pigs gives rise of a major stress that causes sudden pause or retardation of growth and leads to substantial economic loss. PPL has also been used for many years as aid for digestion of patients suffering from pancreas dysfunctions and malnutrition. But the commercial PPL mainly come from the crude extract preparation of animal pancreases contains a significant number of other enzymes as contaminants. The high cost of extraction and purification, limited availability of pig pancreatic tissues, and possible microbial contamination have precluded a large scale application of PPL in animal feed industry. Therefore, it is necessary to develop an efficient yeast expression system for economical and safety providition sufficient amounts of the enzyme.The objective of this study was to express and characterize a partial codon optimized porcine pancreatic lipase. A 534 bp cDNA fragment encoding N-terminal amino acid residue (no signal peptide) of porcine pancreatic lipase was synthesized according to the codon bias of Pichia pastoris and ligated to the full-length porcine pancreatic lipase cDNA fragment. The codon optimized PPL (rePPL) was cloned into the pPICZaA (Invitrogen, Beijing, China) vector. After the resultant pPICZaA-rePPL plasmid was transformed into P.pastoris, the over-expressed extracellular recombinant PPL protein (rePPL) containing a His-tag to the C terminus was purified using Ni Sepharose affinity column (GE Healthcare, Piscataway, NJ, USA), and was characterized against the native enzyme (PPL from porcine pancreas, Sigma). The rePPL exhibited a molecular mass of approximately 52 kDa, and showed optimal temperature (40℃), optimal pH (8.0), Km (15.4 mg/mL), and Vmax (432 U/mg) similar to those of the native enzyme. The recombinant enzyme was stable at 60℃, but lost 80%(P<005) after exposure to heating at=60℃ for 20 min. The codon optimization of PPL increased rePPL yield for ca four folds compared with those native pPICZaA-naPPL tranformant (146 mg/L vs 36 mg/L) and total enzyme activity increased about 5 fold (1900 IU/L vs 367 IU/L). The effect of metal ions on activity of the rePPL and commercial enzyme were compared and the activity of rePPL and commercial PPL enzyme manifested a dose-dependent decrease (P<0.05) by incubating with Ca2+, Zn2+, Cu2+, or Fe3+. In conclusion, we successfully optimized 5-terminal of porcine pancreatic lipase encoding gene and over-expressed the gene in P. pastoris as an extracellular, functional enzyme. |
PDF Full Text Request