Production of a recombinant chitin oligosaccharide deacetylase from Vibrio parahaemolyticus in the culture medium of Escherichia coli cells.
An open reading frame (ORF) encoding chitin oligosaccharide deacetylase (Pa-COD) gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome and its sequence was analyzed. The ORF encoded a 427 amino acid protein, including the 22 amino acid signal sequence. The deduced amino acid sequence was highly similar to several bacterial chitin oligosaccharide deacetylases in carbohydrate esterase family 4. An expression plasmid containing the gene was constructed and inserted into Escherichia coli cells and the recombinant enzyme was secreted into the culture medium with the aid of the signal peptide. The concentration of the recombinant enzyme in the E. coli culture medium was 150 times larger than that of wild-type enzyme produced in the culture medium by V. parahaemolyticus KN1699. The recombinant enzyme was purified to homogeneity from culture supernatant in an overall yield of 16%. Substrate specificities of the wild-type and the recombinant enzymes were comparable.
Kadokura
Kazunari
K
Department of Biological Chemistry, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa, 252-8510, Japan.
Sakamoto
Yusuke
Y
Saito
Kaori
K
Ikegami
Takanori
T
Hirano
Takako
T
Hakamata
Wataru
W
Oku
Tadatake
T
Nishio
Toshiyuki
T
D000581:Amidohydrolases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D002686:Chitin / Q000737:chemistry*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D016681:Genome, Fungal; D008954:Models, Biological; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000737:chemistry*; D013379:Substrate Specificity; D014736:Vibrio parahaemolyticus / Q000201:enzymology*
D009844:Oligosaccharides; D011994:Recombinant Proteins; D002686:Chitin; D000581:Amidohydrolases; C019866:chitin deacetylase