Characterization of cutinase from Colletotrichum capsici and comparison of the gene to cutinase genes from Colletotrichum gloeosporioides and Fusarium solani f. sp. pisi.

摘要

Two studies were made on an extracellular cutinase from Colletotrichum capsici, a broad host range fungal plant pathogen.;I. Colletotrichum capsici was found to grow vigorously in basal mineral media with cutin as the sole source of carbon. Cutinase was purified from the extracellular fluid of the cultures. The enzyme hydrolyzed the model substrate p-nitrophenyl butyrate at a rate similar to that of cutinases from Colletotrichum gloeosporioides and Fusarium solani f. sp. pisi. However, the enzymes from Colletotrichum hydrolyzed the model substrate p-nitrophenyl palmitate at much higher rates than the enzyme from Fusarium. The enzyme from C. capsici is inducible, addition of cutin-hydrolysate to glucose grown cultures greatly increases the production of cutinase. An in vitro translation product from induced C. capsici mRNA, precipitated with antiserum raised against the enzyme and protein A-Sepharose, was found to be 3000 Da larger than the extracellular enzyme; indicating the existence of a percursor form of the protein.;II. The primary structure of cutinase from C. capsici was deduced from the nucleotide sequence of the cDNA. The cDNA clone was used to screen C. capsici and C. gloeosporioides genomic libraries. The nucleotide sequences of the cutinase structural genes from the Colletotrichum species were determined. S1 mapping revealed the transcriptional start sites and polyadenylation site of the mRNA from C. capsici. The primary sequences and gene structure of the enzymes from the Colletotrichum species were compared with the primary structure and gene structure of the cutinase from Fusarium. Only 43% of the amino acid residues are conserved between all three enzymes. The transcriptional start site of the C. capsici gene was centered on the sequence TCCAGACCA, the core of which is found repeated after 21 nucleotides. The same repeated core sequence was also identified in the 5