Characterization and cloning of cutinase from Ascochyta rabiei.
Ascochyta rabiei, the causal agent of Ascochyta blight on chickpea plants, secretes a cutinase in the culture filtrate when it is induced by cutin or hydroxylated fatty acids. This cutinase is the main esterase in the culture fluids. The enzyme was purified to homogeneity by three successive chromatographic steps. It showed an apparent molecular weight of 22 kD in SDS-PAGE and cleaved ester bonds of 3H-labelled cutin or p-nitrophenylbutyrate with maximal activities around pH 8. As a serine esterase, cutinase is strongly inhibited by organophosphorous compounds and the most effective inhibitor 2,3,5-trichloropyridine-6-(O-methyl-O-n-butyl)-phosphateester++ + (MAT 9564) shows a Ki value of 0.8 nM. The cutinase gene was cloned from a genomic cosmid library by screening with two oligonucleotides directed against cutinase consensus peptides. The gene was subcloned to a 1.7 Kb SaII/HindIII-insert and sequenced. The cutinase gene codes for a 223 amino acid protein with strong homology to other fungal cutinase sequences. The purified cutinase is encoded by a single copy gene.
Tenhaken
R
R
Institut für Biochemie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Bundesrepublik Deutschland.
Arnemann
M
M
Köhler
G
G
Barz
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D003001:Cloning, Molecular; D007887:Fabaceae / Q000382:microbiology; D003904:Mitosporic Fungi / Q000201:enzymology* / Q000472:pathogenicity; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000382:microbiology; D010946:Plants, Medicinal; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C019952:cutinase