2015 ; Vol.25-7: 999~1006
|Author||Huoxiang Zhou, Xi Li, Mingyue Guo, Qingrui Xu, Yu Cao, Dairong Qiao, Yi Cao, Hui Xu|
|Place of duty||Microbiology and Metabolic Engineering of Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu 610065, P.R. China|
|Title||Secretory Expression and Characterization of an Acidic Endo-Polygalacturonase from Aspergillus niger SC323 in Saccharomyces cerevisiae|
J. Microbiol. Biotechnol.2015 ;
|Abstract||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 fulllength
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 Ca2+, Cu2+, and Na+,
and strongly inhibited by Pb2+ and Mn2+. 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.|
|Key_word||Aspergillus niger, endo-polygalacturonase, Saccharomyces cerevisiae, secretory expression, characterization|
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