Production of a recombinant chitin deacetylase in the culture medium of Escherichia coli cells.
With the aid of a signal sequence of a chitinase from Streptomyces lividans, a recombinant chitin deacetylase, whose gene originated from a Deuteromycete, Colletotrichum lindemuthianum, was produced in the culture medium of Escherichia coli cells, existing as a highly active form without the signal peptide. During the production of the recombinant chitin deacetylase, both a slight increase in the value of OD600 nm in the culture medium and a drastic decrease in viable cell number were observed. When penta-N-acetyl-chitopentaose was used as the substrate, the recombinant chitin deacetylase had comparable kinetic parameters to those of the original enzyme from the fungus. The addition of a C-terminal six histidine sequence to the recombinant enzyme caused a slight decrease in the kcat value, and the further addition of a 12 amino acid sequence at its N-terminus caused a further decrease in the value. This production system allowed us to easily produce in the culture media the recombinant chitin deacetylases possessing as good properties as the original enzyme, without any disruption steps of the E. coli cells.
Tokuyasu
K
K
National Food Research Institute, Tsukuba, Japan. tokuyasu@nfri.affrc.go.jp
Kaneko
S
S
Hayashi
K
K
Mori
Y
Y
D000581:Amidohydrolases / Q000378:metabolism*; D001483:Base Sequence; D018929:Cell Culture Techniques; D002688:Chitinases / Q000235:genetics; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology; D013997:Time Factors
D003470:Culture Media; D010940:Plant Proteins; D011994:Recombinant Proteins; D002688:Chitinases; C119825:chitinase C; D000581:Amidohydrolases; C019866:chitin deacetylase