Hydrolysis of plant cuticle by plant pathogens. Purification, amino acid composition, and molecular weight of two isoenzymes of cutinase and a nonspecific esterase from Fusarium solani f. pisi

Abstract

The extracellular fluid of the plant pathogen, Fusarium solani f. pisi, grown on the plant cuticular polymer, cutin, was shown to contain cutinase and p-nitrophenyl palmitate hydrolase activities (R.E. Purdy and P.E. Kolattukudy (1973), Arch. Biochem. Biophys. 159, 61). From this extracellular fluid two isozymes of cutinase and a nonspecific esterase (p-nitrophenyl palmitate hydrolase) were isolated using Sephedex G-100 gel filtration, QAE-Sephadex chromatography, and SE-Sephedex chromatography. Phenolics contained in the extracellular fluid were found to be associated with the cutinase but not with the nonspecific esterase, and the phenolic materials were removed from cutinase at the QAE-Sephedex step. A 34-fold purification of the nonspecific esterase and a 6.5-fold purification of cutinase were achieved by the procedure described. The two isozymes of cutinase (I and II) and the nonspecific esterase were homogeneous as judged by polyacrylamide disc gel electrophoresis and sedimentation equilibrium centrifugation. Molecular weights of cutinase I, cutinase II, and the nonspecific esterase were determined by Sephedex G-100 gel filtration, sedimentation equilibrium centrifugation, amino acid composition, and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. The values obtained with these techniques agreed with each other and were about 22,000 for both cutinases and 52,000 for the nonspecific esterase. The dodecyl sulfate gel electrophoresis indicated that a small portion of cutinase II contained proteolylic clips, near the middle of the polypeptide chain, and that the nonspecific esterase might also have undergone some proteolylic modification. The amino acid composition of cutinase I was similar to that of cutinase II except for the presence of a larger number of tryptophan residues in the latter, while the amino acid composition of the nonspecific esterase showed more differences from that of either cutinase.

Regulation of Constitutively Expressed and Induced Cutinase Genes by Different Zinc Finger Transcription Factors inFusarium solani f. sp. pisi(Nectria haematococca)

“…The first purification of fungal cutinase from the extracellular fluid of cutin-grown F. solani pisi yielded two enzymes of similar size (8). Amino acid sequence of some peptides obtained from one of these enzymes (cutinase 1) matched with that predicted from the cDNA of the induced cutinase transcript (10).…”

“…Amino Acid Sequence of Cutinase 1 and Cutinase 2-Cutinase 1 and cutinase 2 were purified from the culture filtrates of cutin-grown F. solani pisi (8). Samples of each cutinase were reduced, and SH groups were derivatized with 14 C-labeled iodoacetamide (2.8 Ci/mol, PerkinElmer Life Sciences) and digested with trypsin as described before (10).…”

“…It is possible that the gene that encodes the constitutively expressed enzyme is different from that which encodes the inducible one. The presence of the multiple cutinase genes was indicated by Southern analysis of the genome of F. solani pisi (11) and by the production of multiple cutinases (8). However, only the highly inducible cutinase gene had been cloned (10).…”

“…Whether the constitutively expressed cutinase and the inducible cutinase are encoded by the same or different genes is not known, and the nature of the transcription factors that are involved in the constitutive expression of cutinase is unknown. Two different but very similar cutinases had been isolated from F. solani pisi (8,9), and the gene previously cloned matched the amino acid sequence of one of these proteins (10), suggesting that another gene encodes the other. Such a gene had not been previously cloned, although Southern analysis had indicated the presence of multiple cutinase genes in the F. solani pisi genome (11).…”

Cutinase and pectinase in cotton bioscouring: an innovative and fast bioscouring process

“…The cuticle of the cotton fibre is cross-linked to the primary cell wall by esterified pectic substances, which hinder pectinase action on the pectin backbone. Cutin forms a three-dimensional network structure in which other amorphous waxy materials are embedded (Purdy et al 1975;Kolattukudy 2001). Pre-rinsing in hot water (!908C) with a surfactant (Hartzell-Lawson & Hsieh 1998) or extraction with boiled n-hexane helps to reduce wax impurities and, subsequently, results in better pectinase performance towards primary wall destabilization (Agrawal et al 2007).…”