Journal of Microbiology and Biotechnology
The Korean Society for Microbiology and Biotechnology publishes the Journal of Microbiology and Biotechnology.

2015 ; Vol.25-7: 999~1006

AuthorHuoxiang Zhou, Xi Li, Mingyue Guo, Qingrui Xu, Yu Cao, Dairong Qiao, Yi Cao, Hui Xu
Place of dutyMicrobiology and Metabolic Engineering of Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu 610065, P.R. China
TitleSecretory Expression and Characterization of an Acidic Endo-Polygalacturonase from Aspergillus niger SC323 in Saccharomyces cerevisiae
PublicationInfo J. Microbiol. Biotechnol.2015 ; Vol.25-7
AbstractThe 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_wordAspergillus niger, endo-polygalacturonase, Saccharomyces cerevisiae, secretory expression, characterization
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