Heterologous expression of a gene encoding a thermostable beta-galactosidase from Alicyclobacillus acidocaldarius.
A genomic DNA library screen yielded the nucleotide sequence of a 12 kb fragment containing a gene (2067 bp) coding a thermostable beta-galactosidase from Alicyclobacillus acidocaldarius ATCC 27009. The beta-galactosidase gene was expressed in Pichia pastoris, and up to 90 mg recombinant beta-galactosidase/l accumulated in shake flask cultures. Using o-nitrophenyl-beta-D: -galactopyranoside as a substrate, the optimum pH and temperature of the purified recombinant beta-galactosidase were 5.8-6.0 and 70 degrees C, respectively. The enzyme retained 90% of its activity when heated at 70 degrees C for 30 min. Approximately 48% of lactose in milk was hydrolyzed following treatment with the recombinant enzyme over 60 min at 65 degrees C.
Yuan
Tiezheng
T
Microbial Engineering Department, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Yang
Peilong
P
Wang
Yaru
Y
Meng
Kun
K
Luo
Huiying
H
Zhang
Wei
W
Wu
Ningfeng
N
Fan
Yunliu
Y
Yao
Bin
B
D003001:Cloning, Molecular; D004795:Enzyme Stability; D016937:Gram-Positive Endospore-Forming Rods / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D001616:beta-Galactosidase; D007785:Lactose