Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli

The present work aims to study a new NADH-cytochrome b(5) reductase (cb(5)r) front Mucor racemosus PTCC 5305. A cDNA coding for cb(5)r was isolated front a Mucor racemosus PTCC 5305 cDNA library. The nucleotide Sequence of the cDNA including coding and sequences flanking regions was determined. The open reading frame starting, front ATG and ending with TAG stop codon encoded 228 amino acids and displayed the closest similarity (73%) with Mortierella alpina cb(5)r. Lack Of hydrophobic residues ill file N-terminal sequence was apparent. suggesting that the enzyme is a soluble isoform. The coding sequence was then cloned ill the pET16b transcription Vector Carrying ail N-terminal-linked His-Tag (R) sequence and expressed in Escherichia coli BL21 (DE3). The enzyme was then homogeneously purified by a metal affinity column. The recombinant Mucor enzyme was shown to have its optimal activity at pH and temperature of about 7.5 and 40 degrees C, respectively. The apparent K-m value Was calculated to be 13 mu M for ferricyanide. To our knowledge. this is the first report on cloning and expression of a native fungal Soluble isoform of NADH-cytochrome b(5) reductase in E. coli.