Cloning, sequencing, and expression of the N-acyl-D-mannosamine dehydrogenase gene from Flavobacterium sp. strain 141-8 in Escherichia coli.
The gene coding for N-acyl-D-mannosamine dehydrogenase (NAM-DH) from Flavobacterium sp. strain 141-8 was cloned and expressed under the control of a lac promoter in Escherichia coli JM109. The DNA sequence of the gene was determined, and an open reading frame encoding a polypeptide composed of 272 amino acid residues (Mr, 27,473) was identified. The E. coli transformants which showed over 200-fold higher NAM-DH activity than did the Flavobacterium strain produced the enzyme as a protein fused with beta-galactosidase. Despite being a fusion, NAM-DH produced by E. coli transformants appeared unchanged in pH optimum, Km, and substrate specificity from Flavobacterium sp. strain 141-8. This newly recombinant enzyme may be applicable to the quantitative determination of sialic acid in serum.
Yamamoto-Otake
H
H
Research and Development Division, Kikkoman Corporation, Chiba, Japan.
Koyama
Y
Y
Horiuchi
T
T
Nakano
E
E
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005417:Flavobacterium / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids; D011995:Recombination, Genetic; D013401:Sugar Alcohol Dehydrogenases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014170:Transformation, Genetic
D013401:Sugar Alcohol Dehydrogenases; C059050:N-acyl-D-mannosamine dehydrogenase