Secretory Expression and Characterization of an Acidic Endo-Polygalacturonase Gene from Aspergillus niger SC323 in Saccharomyces cerevisiae.
The endo-polygalacturonase gene (endo-pgaA) was cloned from DNA of Aspergillus niger SC323 using the cDNA synthesized by overlapping PCR, and successfully expressed in Saccharomyces cerevisiae EBY100 through fusing the α-factor signal peptide of yeast. The full-length cDNA consists of 1,113 bp and encodes a protein of 370 amino acids with a calculated molecular mass of 38.8 kDa. After induction by galactose for 48 h, the activity of recombinant endo-PgaA in the culture supernatant can reach up to 1,448.48 U/mg. The recombinant protein was purified to homogeneity by ammonium sulfate precipitation and gel filtration column chromatography and subsequently characterized. The optimal pH and temperature of the purified recombinant enzyme were 5.0 and 50°C, respectively. The Michaelis-Menten constant (Km) and maximal velocity (Vmax) of the enzyme for pectin were 88.54 μmol/ml and 175.44 μmol/mg/min, respectively. The enzyme activity was enhanced by Ca(2+), Cu(2+), and Na(+), and strongly inhibited by Pb(2+) and Mn(2+). The pectin hydrolysates were mainly galacturonic acid and other oligo-galacturonates. Therefore, these characteristics suggest that the recombinant endo-PgaA may be of potential use in the food and feed industries.
Microbiology and Metabolic Engineering of Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu 610065, P.R. China.
Huoxiang
H
Zhou
Microbiology and Metabolic Engineering of Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu 610065, P.R. China.
Xi
X
Li
Microbiology and Metabolic Engineering of Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu 610065, P.R. China.
Mingyue
M
Guo
Microbiology and Metabolic Engineering of Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu 610065, P.R. China.
Qingrui
Q
Xu
Microbiology and Metabolic Engineering of Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu 610065, P.R. China.
Yu
Y
Cao
Microbiology and Metabolic Engineering of Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu 610065, P.R. China.
Dairong
D
Qiao
Microbiology and Metabolic Engineering of Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu 610065, P.R. China.
Yi
Y
Cao
Microbiology and Metabolic Engineering of Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu 610065, P.R. China.
Hui
H
Xu
D001234:Aspergillus niger / Q000201:enzymology*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D011096:Polygalacturonase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D011993:Recombinant Fusion Proteins; D011096:Polygalacturonase
Aspergillus niger; Saccharomyces cerevisiae; characterization; endo-polygalacturonase; secretory expression