Fusarium solani is implicated in plant disease as well as human disease notably infection of the cornea of the eye. Induction of a biopolyester hydrolase cutinase by low. The parameters, chain length of alcohols and acids, alcohol and acid concentration, and the response esterification yield and initial rate were. Catalyzes the hydrolysis of cutin, a polyester that forms the structure of plant cuticle. The fungal plant pathogen fusarium solani produces an extracellular enzyme, cutinase, which catalyzes the degradation of the bipolymer, cutin, in the plant cuticle. Induction of a biopolyester hydrolase cutinase by low levels of cutin monomers in fusarium solani f. Cutinase has been purified and characterized from several different sources, mainly fungi and pollen, but also bacteria 8.
The differences between the pbs residues degraded over time were investigated with respect to their morphology, crystallinity and chemical structures. Diversity of cutinases from plant pathogenic fungi. The three dimensional structure of this cutinase is also analysed. Rabbit antibody to cutinase i, isolated from fusarium solani f. Purification and characterization of cutinase from venturia inaequalis. The anisotropic treatment of thermal motion led to a fivefold increase in accuracy and to a conside. Molecular cloning, characterization, and expression. The effectiveness of fusarium solani pisi cutinase. Pdf the thermal stability of the fusarium solani pisi cutinase as. Fusarium solani pisi cutinasecatalyzed synthesis of. Experiments with several compounds structurally related to these. A homologous sp library 150 sp for recombinant cutinase secretion in b. Stability of cutinase in the system under study is lower than in aqueous solution and decreases with. We compared wildtype strains and a cutinase negative gene replacement mutant strain of fusarium solani f.
The three dimensional structure of fusarium solani f. Fusarium solani pisi cutinase catalyzed synthesis of polyamides e. Kolattukudy ohio state biotechnology center, rightmire hall, 1060 carmack road, the ohio state university, columbus, ohio. Engineering cysteine mutants to obtain crystallographic. Proof for the production of cutinase byfusarium solani f.
Cutinase structure, function and biocatalytic applications bioline. Cutinase gene disruption in fusarium solani f sp pisi. Its highest enzymatic activity coincides with the phrange at which it displays its highest thermal stability. Spores of the phytopathogenic fungus fusarium solani f. Cutinase structure, function and biocatalytic applications.
Cutin monomers released from the cuticle by small amounts of cutinase on fungal spore surfaces can greatly increase the amount of cutinase secreted by the spore, the mechanism for which is as yet unknown. Isolation of a fusarium solani mutant reduced in cutinase. Engineering cysteine mutants to obtain crystallographic phases with a cutinase from fusarium solani pisi. Production and purification of cutinase from fusarium oxysporum using modified growth media and a specific cutinase substrate. Pdf we have investigated the thermal stability of the fusarium solani pisi cutinase as a function of ph, in the range from ph 212. Cutinase activity can be measured by the hydrolysis of either the artifical substrate, pnitrophenylbutyrate pnb, or radioactive cutin containing 14cpalmitic acid. The effectiveness of fusarium solani pisi cutinase mediated synthesis of shortchain alkyl esters in organic solvent isooctane media was evaluated. Request pdf fusarium solani pisi cutinase cutinase from fusarium solani pisi has been studied extensively with respect to its structural and functional properties. Induction of a biopolyester hydrolase cutinase by low levels of. Cutinase, the enzyme postulated to be involved, was first purified and characterized from fusarium solani f sp pisi nectria haematococca grown with cutin as the sole source of carbon purdy and kolattukudy, 1975a, 1975b. This result constitutes the most specific and strongest evidence for an enzymic penetration of a plant cuticle by a pathogen during. Allows pathogenic fungi to penetrate through the cuticular barrier into the host plant during the.
That a cutinase, which immunologically crossreacts with. The rate of production depended on the amount of cutin hydrolysate added up to 80. A radial immunodiffusion assay for cutinase was developed, and the induction of. In the present paper, wedemonstrate that cutinase production by. Pdf photophysics of the single tryptophan residue in. The thermal stability of the fusarium solani pisi cutinase as a. Dihydroxyc16 acid and trihydroxyc18 acid, which are unique cutin monomers, showed the greatest cutinase inducing activity. With this ferritinconjugated antibody it was shown that germinating spores of this fungus excreted cutinase during the penetration of the host pisum sativum. The cutinase isolated from the fungus fusarium solani pisi, is assumed to be involved in the first steps of fungal attack on plants.
Pdf production and purification of cutinase from fusarium. Physicochemical parameters of the system were optimized relative to triolein hydrolysis. The differences between the pbs residues degraded over time were investigated with respect to their. Glucose was found to be a repressor of cutinase production.
Fusarium solani cutinase is a lipolytic enzyme with a. The production of cutinase seems to be highly regulated by growth conditions. Effect of immobilization support, water activity, and. Photophysics of the single tryptophan residue in fusarium solani cutinase. A synthetic copy of the cutinase cdna was constructed and expressed under the control of the endoxylanase ii expression signals from a. Fusarium solani pisi cutinase request pdf researchgate. Four different constructs were used to test the effect of different pre and prosequences.
In characterizing mutants and covalently inhibited complexes of fusarium solani cutinase, which is a 197residue lipolytic enzyme, 34 variant structures, crystallizing in 8 different crystal forms, have been determined, mostly at high resolution. Fusarium graminearum fg and span at least 330 million yr. With this fermtinconjugated antibody it was shown that germinating spores of this fungus excreted cutinase during the penetration of the host pisum sativum. Evidence for the occurrence of conformational substates with unusual fluorescence behaviour. Polybutylene succinate pbs was hydrolyzed by two different enzymes. The choice of 2phenyl1propanol was based on modeling studies that suggested moderate cutinase enantioselectivity towards this substrate. Comparison of polybutylene succinate biodegradation by.
It is a common soil fungus and colonist of plant materials. The enzyme was repressed when the microorganism was grown on a medium containing glucose and induced to high levels by cutin or its hydrolysis products, the true inducers. These reactions were catalyzed by immobilized cutinase from fusarium solani pisi on lewatit beads, cutinase in the form of crosslinked enzyme aggregates clea, or by immobilized lipase b from candida antarctica n435. We studied the reaction between vinyl butyrate and 2phenyl1propanol in acetonitrile catalyzed by fusarium solani pisi cutinase immobilized on zeolites naa and nay and on accurel pa6. A cutinase found in the fungus aspergillus oryzae, used in the soy bean fermentation industry, has been isolated and appears to be responsible for the flavor formation in. Cutin hydrolysate induced the production of an extracellular cutinase by glucosegrown fusarium solani f. We have investigated the thermal stability of the fusarium solani pisi cutinase as a function of ph, in the range from ph 212. This item appears in the following collections academic publications 176382 academic output radboud university. Heterologous expression ofthe fusarium solani pisi. Pdf genetic engineering of the fusarium solani pisi. A recombinant cutinase fromfusarium solani was encapsulated in aot reversed micelles. By contrast, knockout of the cutinase gene cut1 in f. Polymer chemistry department, zernike institute for advanced materials, university of groningen, nijenborgh 4, 9747 ag groningen, the netherlands.
The fusarium solani pisi lipase cutinase has been genetically engineered to investigate the influence of cterminal peptide extensions on the partitioning of the enzyme in pegsalt based aqueous twophase bioseparation systems. Fusarium solani f sp pisi nectria haematococca isolate 7723 with one cutinase gene produced 10 to 20% of the cutinase produced by isolate t8 that has multiple cutinase genes, whereas cutinase genedisrupted mutant 77102 of isolate 7723 did not produce cutinase. Fusarium solani pisi cutinase belongs to a group of homologous enzymes of relative molecular mass 2225k ref. Prooffor production cutinase byfusarium during penetration. Amino acid sequence deduced from the nucleotide sequence of cut1 and cut23 matched with that of the peptides from cutinase 1 and cutinase 2, respectively, isolated from f. Fusarium polycaprolactone depolymerase is cutinase. Proof for the production of cutinase by fusarium solani f. Three structural features were noticed during the assignment. Cutinase from fusarium solani pisi has been studied extensively with respect to its structural and functional properties. Electronic publications 80624 freely accessible full text publications plus those not yet available due to embargo. Weattempted toshowdirectlythatthe24kdaproteinhadpcldepoly. Materials and methods cutinase i was isolated in homogeneous form from the extracellular fluid of f. Kinetic studies of triglyceride hydrolysis showed a decrease in specificity with increase of the acyl chain length.
Cutinase is a serine esterase containing the classical ser, his, asp triad of serine hydrolases. Fusarium solani isolate t8 produces an extracellular enzyme, cutinase, which catalyzes the degradation of cutin in the plant cuticle. Ctf1, a transcriptional activator of cutinase and lipase genes in. Soliday cl, pe kolatrukudy 1976 isolation and characterization of a cutinase from fusarium roseum culmorum and its immunological comparison with cutinases from f. The cutinase isolated from the fungus fusarium solani pisi, is assumed to be involved in the first steps of fungal attack on plants 2. The j b c 2002 by the american society for biochemistry. Mechanism by which contact with plant cuticle triggers. Properties of a cutinasedefective mutant of fusarium solani. Production and purification of cutinase from fusarium. Cutins from fruit of cucurbita maxima and cucurbita moschata cultivars, apple and a c 16 alcohol hexadecanol were used to induce cutinolytic esterase activity during saprophytic growth of strains of the two cucurbit pathogens, fusarium solani f. Christian cambillau, engineering cysteine mutants to obtain crystallographic phases with a cutinase from fusarium solani pisi, protein engineering, design and selection, volume 6. Optimization of flavor esters synthesis by fusarium solani.
The thermal stability of the fusarium solani pisi cutinase. The majority of the work has been done with a fungal pathogen of peas, fusarium solani. Evolutionary history of the ancient cutinase family in five filamentous. Fusarium solani is a species complex of at least 26 closely related filamentous fungi in the division ascomycota, family nectriaceae. The crystal structure of the enzyme was solved to high atomic resolution 1 angstrom, while data on structural dynamics have been obtained from detailed nmr studies. Fusarium venenatum a35 was transformed using the aspergillus niger expression plasmid, pigf, in which the coding sequence for the f. In the present study, the culture filtrate contained basal levels of cutinase when t8 was. Use of a sec signal peptide library from bacillus subtilis. The cutinase protein from fusarium solani pisi is used as a model for the production of heterologous proteins. Whether the germinating spores synthesize cutinase during the early phaseofpathogenesisis notknownand,ifso,howthespores sense the contact with the plant is also not known.
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