Cloning, expression, and characterization of a porcine pancreatic α-amylase in Pichia pastoris

Animal Nutrition
Lv-Hui SunXingen Lei

Abstract

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 cDNA encoding the PPA was isolated from pig pancreas by RT-PCR and cloned into the pPICZαA vector. After the resultant pPICZαΑ-PPA plasmid was transferred into Pichia pastoris, Ni Sepharose affinity column was used to purify the over-expressed extracellular recombinant PPA protein (rePPA) that contains a His-tag to the C terminus and was characterized against the natural enzyme (α-amylase from porcine pancreas). The rePPA exhibited a molecular mass of approximately 58 kDa and showed optimal temperature (50 °C), optimal pH (7.5), K m (47.8 mg/mL), and Vmax (2,783 U/mg) similar to those of the natural enzyme. The recombinant enzyme was stable at 40 °C but lost 60% to 90% (P < 0.05) after exposure to heating at ≥50 °C for 30 min. The enzyme activity was little affected by Cu2+ or Fe3+, but might be inhibited (40% to 50%) by Zn2+ at concentrations in pig digesta. However, Ca2+ exhibited a dose-dependent stimulation of the enzyme activity. In c...Continue Reading

Datasets Mentioned

BETA
AF064742.1

Methods Mentioned

BETA
PCR
electrophoresis

Software Mentioned

SAS

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