Cloning And Expression In Pichia Pastoris Of A CDNA For Porcine Pancreatic Lipase (PPL) And The Enzyme Activity Analysis

Lipases catalyze the hydrolysis of ester bonds of triacylglycerols at the interface between the aqueous and non-aqueous phases, which are widespread enzymes. Lipases are a versatile group of enzymes and often show other activities. Because of their versatile and superiority as biocatalyst, lipases are widely used in many fields.In the body of animals, lipases are secreted by pancreas and digest the fat in the food. Lipases have an expansion prospect as feed additive. The reasons are lipases can reparation the deficiency of lipase in young domestic animals and birds and remove lipid anti-nutriton factors in plant feed resources. At present, the industrial lipases are mainly obtained by microbial fermentation. There are some defects in microbial lipases, such as the wide sources of lipases cause the difficulty of screening, the obtained strains produce low levels of enzymes and the enzymatic activity is low, the stability is poor. The mechanisms of gene processing, secretion and regularization are not clear. The strains used to produce lipase products may exist some pathogenicity and toxicity. Those features may have potential harm to animals.We wanted to develop an efficient expression system in Pichia pastoris to produce a recombinant porcine pancreatic lipase with high yield and activity in low cost by genetic engineering. It overcomes the shortcomings of microbial lipase and has a potential feed safety and broad application prospects.In this experiment, the porcine pancreas was used for RNA preparation. After extraction of the total RNA from porcine pancreas using the TRIzol method, the cDNA of PPL gene was amplified using RT-PCR method and subcloned into the expression vector pPICZaA. The constructed expression vector pPICZaA-PPL was sequenced for the cDNA insert. The pPICZaA-PPL digested by Pmel. The linearization pPICZaA-PPL plasmid was delivered into Pichia pastoris X-33 cells. Transformants with high levels of the PPL gene RNA were identified by Syber-Green quantitative real-time RT-PCR and selected for the protein production. The selected transformant was first cultured in BMGY and then induced for the production of the pocrcine pancreatic lipase in BMMY (pH6.0). The data showed that the recombinant PPL gene was successful expressed under the control of AOX1, and PPL was secreted into the culture medium as expected. The extracellular PPL protein containing a histidine tag appended to the C terminus was purified using Ni Sepharose High Performance affinity column. The purified protein showed a molecular mass of approximately 60 kDa as determined by SDS-PAGE analysis which has the similar molecular mass to the theoretical value. The content of PPL reached about 42mg/L in the liquid culture medium. The enzymatic activity of PPL was detected by using plate qualitative detection method. In conclusion, the recombinant PPL gene was cloned and successfully expressed in Pichia pastoris. The PPL was produced at a relative high level and showed its enzymatic activity.