Purification and antifungal activity of recombinant chitinase from Escherichia coli carrying the family 19 chitinase gene of Streptomyces sp. J-13-3.
A recombinant chitinase was purified from the cell extract of Escherichia coli JM109 transformed by plasmid pUC19 carrying the gene encoding family 19 chitinase of Streptomyces sp. J-13-3 by column chromatography on DEAE-Sepharose, CM-Sepharose, and Bio-Gel P-100. The final preparation was homogenous in polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be 32,000. The recombinant chitinase hydrolyzed the trimer to hexamer of N-acetylglucosamine and had the identical N-terminal amino acid sequence of the mature protein, indicating removal of the signal sequence by E. coli signal peptidase. The fungal growth in well (200 microl of medium) of microplate by measurement of absorbance at 595 nm indicated that the chitinase (10 microg) completely and half inhibited growth of Trichoderma reesei and Aspergillus niger respectively.
Yamashita
Yousuke
Y
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan.
Okazaki
Katsuichiro
K
D000117:Acetylglucosamine / Q000378:metabolism; D001234:Aspergillus niger / Q000187:drug effects* / Q000254:growth & development; D049109:Cell Proliferation / Q000187:drug effects*; D002688:Chitinases / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008565:Membrane Proteins / Q000378:metabolism; D021382:Protein Sorting Signals / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*; D012697:Serine Endopeptidases / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics; D014242:Trichoderma / Q000187:drug effects* / Q000254:growth & development
D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D002688:Chitinases; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D000117:Acetylglucosamine