Cell surface display of Chi92 on Escherichia coli using ice nucleation protein for improved catalytic and antifungal activity.
The gene encoding chitinase 92 (Chi92) from Aeromonas hydrophila JP10 has been displayed on the cell surface of Escherichia coli using the N-terminal region of ice nucleation proteins (INPN) as an anchoring motif. Immunofluorescence microscopy confirmed that Chi92 was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the N-terminal domain INPN-Chi92 fusion protein of the expected size (112 kDa). Whole cell enzyme assay indicated that the displayed Chi92 showed enhanced catalytic activity toward colloidal chitin. In addition, the Chi92-displayed cells exhibited inhibitory effects on the mycelial growth of phytopathogenic fungi, including Fusarium decemcellulare, Sclerotium rolfsii, Rhizoctonia solani kuhn, and Fusarium oxysporum f.sp. melonis. This study suggested that the INP-based display systems can be used to express a large protein (90 kDa Chi92) on the cell surface of E. coli without growth inhibition. In addition, the display of chitinase on the cell surface may provide an attractive method for the development of biocontrol agents against phytopathogenic fungi.
Wu
Mei Li
ML
Department of Food Science, National Pingtung University of Science and Technology, Pingtung, Taiwan. mlwu@mail.npust.edu.tw
Tsai
Chun Yung
CY
Chen
Tsai Hsia
TH
D016980:Aeromonas hydrophila / Q000201:enzymology / Q000235:genetics*; D000935:Antifungal Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000502:physiology*; D015153:Blotting, Western / Q000379:methods; D002384:Catalysis / Q000187:drug effects; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000254:growth & development / Q000502:physiology*; D005658:Fungi / Q000187:drug effects; D005670:Fusarium / Q000187:drug effects; D008565:Membrane Proteins / Q000502:physiology*; D008856:Microscopy, Fluorescence / Q000379:methods; D010957:Plasmids; D011994:Recombinant Proteins / Q000494:pharmacology; D012232:Rhizoctonia / Q000187:drug effects
D000935:Antifungal Agents; D001425:Bacterial Outer Membrane Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C063113:ice nucleation protein; D002686:Chitin; D002688:Chitinases