Cloning And Expression In Pichia Pastoris Of A CDNA For Porcine Pancreatic A-amylase (PPA)

Abstract: a-Amylase (a-1,4-glucan-4-glucanohydrolase, EC.3.2.1.1) catalyzes the hydrolysis of a-(1,4) glycosidic linkages in starch, glycogen and various malto-oligosaccharides, releasing a-anomeric products. This enzyme is widely distributed in all the three domains of life: Bacteria, Archaea, and Eucarya. For its special enzymatic characteristic a-amylase has broad application in research, medical therapy, food processing, feed industrial and so on. Though a-amylase purified from microorganisms is already commercially-available, but it remains many problems, such as food security for feed. With the help of biotechnologies to produce a-amylase from microorganisms is promising for its advantage in high productivity, simple process security for feed and low cost.The objective of this study was to develop an efficient expression system to produce porcine pancreatic a-amylase (PPA). The full-length cDNA of encoding the PPA was isolated from porcine pancreas by RT-PCR and cloned into the pPICZaA (Invitrogen) expression vector under the control of AOX1 promoter. The pPICZaA-PPA vector was delivered into Pichia pastoris (X-33) cells. The transformants were screened by SYBR-green quantitative real-time PCR (ABI 7900HT, Applied Biosystems). After 3 days of 0.5% methanol induction, the extracellular PPA protein containing a histidine tag appended to the C terminus was purified using Ni Sepharose High Performance affinity column (GE HealthcareJ). The purified protein showed a molecular mass of approximately 58 kDa as determined by SDS-PAGE analysis. The recombinant PPA (rePPA) exhibited an optimum temperature of 50 and pH of 7.5, and the Km and Vmax values for rePPA were 48.8mg/ml and 1.3mg/min, respectively. The stability of rePPA will be greatly declined after warm-bathe for 30 minutes at 50℃, As observed, the enzymatic activity was inhibited by metal ions. Ion Cu2+(0.08 mMol/1) exhibited a sharper inhibition than Fe3+(24m Mol/1), Ca2+(15m Mol/1), Zn2+(12m Mol/1). The obtained results showed that the enzymatic properties of rePPA were similar to those of the native PPA (sigma). In conclusion, the recombinant PPA was successfully produced at a relative high level in P. pastoris.