Cloning and sequencing of the gene for cellobiose 2-epimerase from a ruminal strain of Eubacterium cellulosolvens.
Cellobiose 2-epimerase (CE; EC 5.1.3.11) is known to catalyze the reversible epimerization of cellobiose to 4-O-beta-D-glucopyranosyl-D-mannose in Ruminococcus albus cells. Here, we report a CE in a ruminal strain of Eubacterium cellulosolvens for the first time. The nucleotide sequence of the CE had an ORF of 1218 bp (405 amino acids; 46 963.3 Da). The CE from E. cellulosolvens showed 44-54% identity to N-acyl-D-glucosamine 2-epimerase-like hypothetical proteins in the genomes of Coprococcus eutactus, Faecalibacterium prausnitzii, Clostridium phytofermentans, Caldicellulosiruptor saccharolyticus, and Eubacterium siraeum. Surprisingly, it exhibited only 46% identity to a CE from R. albus. The recombinant enzyme expressed in Escherichia coli was purified by two-step chromatography. The purified enzyme had a molecular mass of 46.7 kDa and exhibited optimal activity at around 35 degrees C and pH 7.0-8.5. In addition to cello-oligosaccharides, it converted lactose to epilactose (4-O-beta-D-galactopyranosyl-D-mannose).
Taguchi
Hidenori
H
Creative Research Initiative 'Sousei' (CRIS), Hokkaido University, Sapporo, Japan. hid-tag@cris.hokudai.ac.jp
Senoura
Takeshi
T
Hamada
Shigeki
S
Matsui
Hirokazu
H
Kobayashi
Yasuo
Y
Watanabe
Jun
J
Wasaki
Jun
J
Ito
Susumu
S
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002475:Cellobiose / Q000378:metabolism*; D003001:Cloning, Molecular; D005051:Eubacterium / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D008969:Molecular Sequence Data; D019998:Racemases and Epimerases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012417:Rumen / Q000382:microbiology*; D016415:Sequence Alignment; D012756:Sheep / Q000382:microbiology; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002475:Cellobiose; D019998:Racemases and Epimerases; D007785:Lactose