Heterologous expression and biochemical characterization of alpha-glucosidase from Aspergillus niger by Pichia pastroris.
The aglu of Aspergillus niger encodes the pro-protein of alpha-glucosidase, and the mature form of wild-type enzyme is a heterosubunit protein. In the present study, the cDNA of alpha-glucosidase was cloned and expressed in Pichia pastoris strain KM71. The activity of recombinant enzyme in a 3 L fermentor reached 2.07 U/mL after 96 h of induction. The recombinant alpha-glucosidase was able to produce oligoisomaltose. The molecular weight of the recombinant enzyme was estimated to be about 145 kDa by SDS-PAGE, and it reduced to 106 kDa after deglycosylation. The enzymatic activity of recombinant alpha-glucosidase was not significantly affected by a range of metal ions. The optimum temperature of the enzyme was 60 degrees C, and it was stable below 50 degrees C. The enzyme was active over the range of pH 3.0-7.0 with maximal activity at pH 4.5. Using pNPG as substrate, the K(m) and V(max) values were 0.446 mM and 43.48 U/mg, respectively. These studies provided the basis for the application of recombinant alpha-glucosidase in the industry of functional oligosaccharides.
Chen
Dong-Li
DL
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenve, Wuxi 214122, People's Republic of China.
Tong
Xing
X
Chen
Shang-Wei
SW
Chen
Sheng
S
Wu
Dan
D
Fang
Shu-Guang
SG
Wu
Jing
J
Chen
Jian
J
D001234:Aspergillus niger / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D010843:Pichia / Q000235:genetics*; D000520:alpha-Glucosidases / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D000520:alpha-Glucosidases