A novel beta-galactosidase capable of glycosyl transfer from Enterobacter agglomerans B1.
A novel transglycosylating beta-galactosidase was purified from Enterobacter agglomerans B1. It was a homodimer of approximately 248 kDa. The optimal pH and temperature for oNPGal hydrolysis were 7.5-8.0 and 37-40 degrees C, respectively. The K(m) values for oNPGal and lactose were 0.06 and 114 mM, respectively. The enzyme produced galacto-oligosaccharides in a 38% yield at the lactose concentration of 12.5% (w/v). When using oNPGal as donor, the enzyme was able to catalyze glycosyl transfer to a series of acceptors, including hexose, pentose, beta- or alpha-disaccharides, hexahydroxy alcohol, cyclitol, and aromatic glycosides. This suggested the enzyme to be a potential synthetic tool for preparing galactose-containing chemicals. The gene encoding this enzyme was cloned by degenerate PCR and TAIL-PCR. It revealed an ORF of 3090 nucleotides encoding a 1029 amino-acid protein, which had been expressed in Escherichia coli. Transferase activities in both recombinant and natural enzymes were similar.
Lu
Lili
L
State Key Lab of Microbial Technology, Shandong University, Jinan 250100, China.
Xiao
Min
M
Xu
Xiaodong
X
Li
Zhengyi
Z
Li
Yumei
Y
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004754:Enterobacter / Q000201:enzymology* / Q000235:genetics; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D009005:Monosaccharides / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013012:Sorbitol / Q000737:chemistry / Q000378:metabolism; D013402:Sugar Alcohols / Q000737:chemistry / Q000378:metabolism; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004269:DNA, Bacterial; D004187:Disaccharides; D006027:Glycosides; D009005:Monosaccharides; D013402:Sugar Alcohols; D013012:Sorbitol; D001616:beta-Galactosidase