Gene cloning and characterization of a novel recombinant antifungal chitinase from papaya (Carica papaya).
A chitinase cDNA clone (CpCHI, 1002 bp) was isolated from papaya fruit, which encoded a 275 amino acid protein containing a 28 amino acid signal peptide in the N-terminal end. The predicted molecular mass of the mature protein was 26.2 kDa, and its pI value was 6.32. On the basis of its amino acid sequence homology with other plant chitinases, it was classified as a class IV chitinase. An active recombinant CpCHI enzyme was overexpressed in Escherichia coli. The purified recombinant papaya chitinase showed an optimal reaction temperature at 30 degrees C and a broad optimal pH ranging from 5.0 to 9.0. The recombinant enzyme was quite stable, retaining >64% activity for 3 weeks at 30 degrees C. The spore germination of Alternaria brassicicola could be completely inhibited by a 76 nM level of recombinant CpCHI. Recombinant CpCHI also showed antibacterial activity in which 50% of E. coli was inhibited by a 2.5 microM concentration of the enzyme.
Chen
Yu-Ting
YT
Biotechnology Center and Department of Food Science and Biotechnology, National Chung-Hsing University, 250 Kuo Kuang Road, Taichung, Taiwan 402.
Hsu
Lien-Hua
LH
Huang
I-Ping
IP
Tsai
Tsun-Chuang
TC
Lee
Guan-Chiun
GC
Shaw
Jei-Fu
JF
D000528:Alternaria / Q000187:drug effects; D000595:Amino Acid Sequence; D001483:Base Sequence; D029441:Carica / Q000201:enzymology*; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005638:Fruit / Q000201:enzymology*; D005659:Fungicides, Industrial / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
D005659:Fungicides, Industrial; D011994:Recombinant Proteins; D002688:Chitinases