Abstract
Pancreatic lipase plays a key role in intestinal digestion of feed fat, and is often deficient in young animals such as weaning piglets. The objective of this study was to express and characterize a partial codon optimized porcine pancreatic lipase (opPPL). A 537 bp cDNA fragment encoding N-terminus amino acid residue of the mature 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 was cloned into the pPICZαA (Invitrogen, Beijing, China) vector. After the resultant opPPL/pPICZαΑ plasmid was transformed into P.pastoris, the over-expressed extracellular opPPL 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 (commercial PPL from porcine pancreas, Sigma). The opPPL exhibited a molecular mass of approximately 52 kDa, and showed optimal temperature (40°C), optimal pH (8.0), Km (0.041 mM), and Vmax (2.008 µmol.mg protein −1.min−1) similar to those of the commercial enzyme with p-NPP as the substrate. The recombinant enzyme was stable at 60°C, but lost 80% (P<0.05) of its activity after exposure to heat ≥60°C for 20 min. The codon optimization increased opPPL yield for ca 4 folds (146 mg.L−1 vs 36 mg.L−1) and total enzyme activity increased about 5 folds (1900 IU.L−1 vs 367 IU.L−1) compared with those native naPPL/pPICZαΑ tranformant. Comparison of gene copies and mRNA profiles between the two strains indicated the increased rePPL yields may partly be ascribed to the increased protein translational efficiency after codon optimization. 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. The recombination enzyme demonstrates a potential for future use as an animal feed additive for animal improvement.
Highlights
As a family member of serine hydrolases, Lipases (EC 3.1.1.3) are a class of hydrolases that catalyse a wide range of reactions including hydrolysis, interesterification, alcholysis, acidolysis, esterification and aminolysis
It is reported that pancreatic-like microbial enzyme supplementation improved the growth of the exocrine pancreatic insufficiency (EPI) in pigs [9], increased piglet activity [10] and enhance piglet gastrointestinal tract development [11]
After the cloned expression vector optimized porcine pancreatic lipase (opPPL)/pPICZaA was digested with Xho I and Xba I, a ca 1,500 bp target gene band and a 3,600 bp expression vector band were shown on the 1% agarose gel (Fig. 2, lane2)
Summary
As a family member of serine hydrolases, Lipases (EC 3.1.1.3) are a class of hydrolases that catalyse a wide range of reactions including hydrolysis, interesterification, alcholysis, acidolysis, esterification and aminolysis. Porcine pancreatic lipase (PPL) is a secreted glycoprotein composed of a single chain of 449 amino acids, with a molecular weight of 50–52 Kd [4] It is an endolipase, and has high efficiency in catalyzing the hydrolysis of triglycerides and release free fatty acids. It is reported that pancreatic-like microbial enzyme (including lipase) supplementation improved the growth of the exocrine pancreatic insufficiency (EPI) in pigs [9], increased piglet activity [10] and enhance piglet gastrointestinal tract development [11]. 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. Our findings suggest a potential approach for producing rePPL for the animal feed industry
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