摘要
Pancreatic α-amylase(α-1, 4-glucan-4-glucanohydrolase, EC.3.2.1.1) plays a primary role in the intestinal digestion of feed starch and is often deficient in weanling pigs.The objective of this study was to clone,express, and characterize porcine pancreatic α-amylase(PPA).The full-length c DNA encoding the PPA was isolated from pig pancreas by RT-PCR and cloned into the pPICZαA vector.After the resultant pPICZαА-PPA plasmid was transferred into Pichia pastoris, Ni Sepharose affinity column was used to purify the over-expressed extracellular recombinant PPA protein(re PPA) that contains a His-tag to the C terminus and was characterized against the natural enzyme(α-amylase from porcine pancreas).The re PPA exhibited a molecular mass of approximately 58 kDa and showed optimal temperature(50℃),optimal pH(7.5), K_m(47.8 mg/mL), and V_(max)(2,783 U/mg) similar to those of the natural enzyme.The recombinant enzyme was stable at 40℃ but lost 60% to 90%(P < 0.05) after exposure to heating at≥50℃ for 30 min.The enzyme activity was little affected by Cu^(2+)or Fe^(3+), but might be inhibited(40% to 50%) by Zn^(2+)at concentrations in pig digesta.However, Ca^(2+)exhibited a dose-dependent stimulation of the enzyme activity.In conclusion, the present study successfully cloned the porcine pancreatic aamylase gene and over-expressed the gene in P.pastoris as an extracellular, functional enzyme.The biochemical characterization of the over-produced enzyme depicts its potential and future improvement as an animal feed additive.
Pancreatic α-amylase(α-1, 4-glucan-4-glucanohydrolase, EC.3.2.1.1) plays a primary role in the intestinal digestion of feed starch and is often deficient in weanling pigs.The objective of this study was to clone,express, and characterize porcine pancreatic α-amylase(PPA).The full-length c DNA encoding the PPA was isolated from pig pancreas by RT-PCR and cloned into the pPICZαA vector.After the resultant pPICZαА-PPA plasmid was transferred into Pichia pastoris, Ni Sepharose affinity column was used to purify the over-expressed extracellular recombinant PPA protein(re PPA) that contains a His-tag to the C terminus and was characterized against the natural enzyme(α-amylase from porcine pancreas).The re PPA exhibited a molecular mass of approximately 58 kDa and showed optimal temperature(50℃),optimal pH(7.5), K_m(47.8 mg/mL), and V_(max)(2,783 U/mg) similar to those of the natural enzyme.The recombinant enzyme was stable at 40℃ but lost 60% to 90%(P < 0.05) after exposure to heating at≥50℃ for 30 min.The enzyme activity was little affected by Cu^(2+)or Fe^(3+), but might be inhibited(40% to 50%) by Zn^(2+)at concentrations in pig digesta.However, Ca^(2+)exhibited a dose-dependent stimulation of the enzyme activity.In conclusion, the present study successfully cloned the porcine pancreatic aamylase gene and over-expressed the gene in P.pastoris as an extracellular, functional enzyme.The biochemical characterization of the over-produced enzyme depicts its potential and future improvement as an animal feed additive.
基金
supported by the 863 program or State High-Tech Development Plan
funded and administered by the Government of the People's Republic of China (2007AA100602 and 2007AA100601-6)
by the Chang Jiang Scholars Program of the Chinese Ministry of Education (to X.G.Lei)
