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 homogene ity from P. fluorescens DSM50106 and the N-terminal amino acid sequenc e was determined. An oligonucleotide deduced from this peptide sequenc e was used as a probe to isolate the mannitol dehydrogenase gene (mtlD ) from a genomic library of P. fluorescens. Nucleotide sequence analys is 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 wit h a mannitol dehydrogenase from Rhodobacter sphaeroides and a putative mannitol dehydrogenase of Saccharomyces cerevisae with an overall ide ntity in amino acid sequence of 44% and 42%, respectively, whereas the similarity to mannitol-l-phosphate dehydrogenases of Escherichia coli or Enterococcus faecalis was only about 23% of identical amino acids. By construction of inducible expression plasmids the specific activit y of the mannitol dehydrogenase synthesized in E. coli was increased f rom 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. c oli active mannitol dehydrogenase could be purified in a two-step proc edure by affinity chromatography using a Ni2+ matrix column. The purif ied 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.