Open AccessCloning, expression, and characterization of a porcine pancreatic α-amylase in Pichia pastoris
Author(s) -
Luo Sun,
Tao Qin,
Yan Liu,
Hua Zhao,
X. Y. Xia,
Xin Gen Lei
Publication year - 2018
Publication title -
animal nutrition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.356
H-Index - 28
eISSN - 2405-6545
pISSN - 2405-6383
DOI - 10.1016/j.aninu.2017.11.004
Subject(s) - pichia pastoris , amylase , recombinant dna , enzyme , microbiology and biotechnology , complementary dna , extracellular , biology , expression vector , molecular mass , biochemistry , molecular cloning , affinity chromatography , enzyme assay , chemistry , gene
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 V max (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 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 α-amylase 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.