Cloning, nucleotide sequence and expression of a mannitol dehydrogenase gene from Pseudomonas fluorescens DSM 50106 in Escherichia coli.
A NAD-dependent mannitol dehydrogenase (MtlD) was purified to homogeneity from P. fluorescens DSM50106 and the N-terminal amino acid sequence was determined. An oligonucleotide deduced from this peptide sequence was used as a probe to isolate the mannitol dehydrogenase gene (mtlD) from a genomic library of P. fluorescens. Nucleotide sequence analysis of a 1.8 kb NruI fragment containing the entire mtlD gene revealed an open reading frame of 1482 bp encoding a protein with a calculated molecular weight of 54.49 kDa. The enzyme shared a high similarity with a mannitol dehydrogenase from Rhodobacter sphaeroides and a putative mannitol dehydrogenase of Saccharomyces cerevisae with an overall identity in amino acid sequence of 44% and 42%, respectively, whereas the similarity to mannitol-1-phosphate dehydrogenases of Escherichia coli or Enterococcus faecalis was only about 23% of identical amino acids. By construction of inducible expression plasmids the specific activity of the mannitol dehydrogenase synthesized in E. coli was increased from 0.02 U (mg protein)(-1) to 10 U (mg protein)(-1). After fusion of six histidine codons to the 3' end of mtlD gene and expression in E. coli active mannitol dehydrogenase could be purified in a two-step procedure by affinity chromatography using a Ni2+ matrix column. The purified enzyme exhibited a specific activity of 46 U (mg protein)(-1) and was shown to be a polyol dehydrogenase with a broad substrate spectrum oxidizing efficiently mannitol, sorbitol and arabitol.
Brünker
P
P
Institut für Biotechnologie, ETH Zürich, Switzerland.
Altenbuchner
J
J
Kulbe
K D
KD
Mattes
R
R
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D015698:Genomic Library; D006639:Histidine; D008354:Mannitol Dehydrogenases / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010455:Peptides / Q000235:genetics; D015202:Protein Engineering; D011551:Pseudomonas fluorescens / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D010455:Peptides; D011994:Recombinant Proteins; D009243:NAD; C033223:polyhistidine; D006639:Histidine; D009249:NADP; D008354:Mannitol Dehydrogenases