Christian cambillau, engineering cysteine mutants to obtain crystallographic phases with a cutinase from fusarium solani pisi, protein engineering, design and selection, volume 6. Polymer chemistry department, zernike institute for advanced materials, university of groningen, nijenborgh 4, 9747 ag groningen, the netherlands. Proof for the production of cutinase byfusarium 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.
In the present study, the culture filtrate contained basal levels of cutinase when t8 was. With this ferritinconjugated antibody it was shown that germinating spores of this fungus excreted cutinase during the penetration of the host pisum sativum. Cutinase structure, function and biocatalytic applications. The differences between the pbs residues degraded over time were investigated with respect to their. 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. Spores of the phytopathogenic fungus fusarium solani f. Fusarium solani cutinase is a lipolytic enzyme with a. A recombinant cutinase fromfusarium solani was encapsulated in aot reversed micelles. Mechanism by which contact with plant cuticle triggers. This item appears in the following collections academic publications 176382 academic output radboud university. Evolutionary history of the ancient cutinase family in five filamentous. Fusarium solani pisi cutinase catalyzed synthesis of polyamides e.
A synthetic copy of the cutinase cdna was constructed and expressed under the control of the endoxylanase ii expression signals from a. Request pdf fusarium solani pisi cutinase cutinase from fusarium solani pisi has been studied extensively with respect to its structural and functional properties. Pdf production and purification of cutinase from fusarium. 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. Stability of cutinase in the system under study is lower than in aqueous solution and decreases with. Cutinase gene disruption in fusarium solani f sp pisi. The rate of production depended on the amount of cutin hydrolysate added up to 80.
Polybutylene succinate pbs was hydrolyzed by two different enzymes. Kinetic studies of triglyceride hydrolysis showed a decrease in specificity with increase of the acyl chain length. Purification and characterization of cutinase from venturia inaequalis. The majority of the work has been done with a fungal pathogen of peas, fusarium solani. It is a common soil fungus and colonist of plant materials. Isolation of a fusarium solani mutant reduced in cutinase. The production of cutinase seems to be highly regulated by growth conditions. Ctf1, a transcriptional activator of cutinase and lipase genes in. Fusarium solani pisi cutinase belongs to a group of homologous enzymes of relative molecular mass 2225k ref.
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. The anisotropic treatment of thermal motion led to a fivefold increase in accuracy and to a conside. Physicochemical parameters of the system were optimized relative to triolein hydrolysis. A radial immunodiffusion assay for cutinase was developed, and the induction of. Fusarium solani pisi cutinasecatalyzed synthesis of. Rabbit antibody to cutinase i, isolated from fusarium solani f. Pdf the thermal stability of the fusarium solani pisi cutinase as. Fusarium venenatum a35 was transformed using the aspergillus niger expression plasmid, pigf, in which the coding sequence for the f. 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. Cutinase activity can be measured by the hydrolysis of either the artifical substrate, pnitrophenylbutyrate pnb, or radioactive cutin containing 14cpalmitic acid. The fungal plant pathogen fusarium solani produces an extracellular enzyme, cutinase, which catalyzes the degradation of the bipolymer, cutin, in the plant cuticle. Evidence for the occurrence of conformational substates with unusual fluorescence behaviour. 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. Diversity of cutinases from plant pathogenic fungi. Fusarium graminearum fg and span at least 330 million yr. 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. Proof for the production of cutinase by fusarium solani f. A homologous sp library 150 sp for recombinant cutinase secretion in b. Weattempted toshowdirectlythatthe24kdaproteinhadpcldepoly. Soliday cl, pe kolatrukudy 1976 isolation and characterization of a cutinase from fusarium roseum culmorum and its immunological comparison with cutinases from f.
The choice of 2phenyl1propanol was based on modeling studies that suggested moderate cutinase enantioselectivity towards this substrate. Photophysics of the single tryptophan residue in fusarium solani cutinase. Fusarium solani is a species complex of at least 26 closely related filamentous fungi in the division ascomycota, family nectriaceae. Fusarium solani isolate t8 produces an extracellular enzyme, cutinase, which catalyzes the degradation of cutin in the plant cuticle.
Comparison of polybutylene succinate biodegradation by. Cutinase has been purified and characterized from several different sources, mainly fungi and pollen, but also bacteria 8. Pdf photophysics of the single tryptophan residue in. 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. Production and purification of cutinase from fusarium oxysporum using modified growth media and a specific cutinase substrate. Induction of a biopolyester hydrolase cutinase by low. By contrast, knockout of the cutinase gene cut1 in f. Properties of a cutinasedefective mutant of fusarium solani. Engineering cysteine mutants to obtain crystallographic phases with a cutinase from fusarium solani pisi. Fusarium solani is implicated in plant disease as well as human disease notably infection of the cornea of the eye. Cutinase is a serine esterase containing the classical ser, his, asp triad of serine hydrolases. In the present paper, wedemonstrate that cutinase production by.
Four different constructs were used to test the effect of different pre and prosequences. This result constitutes the most specific and strongest evidence for an enzymic penetration of a plant cuticle by a pathogen during. Engineering cysteine mutants to obtain crystallographic. 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. Catalyzes the hydrolysis of cutin, a polyester that forms the structure of plant cuticle. 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. The cutinase isolated from the fungus fusarium solani pisi, is assumed to be involved in the first steps of fungal attack on plants 2. Its highest enzymatic activity coincides with the phrange at which it displays its highest thermal stability. Prooffor production cutinase byfusarium during penetration. The differences between the pbs residues degraded over time were investigated with respect to their morphology, crystallinity and chemical structures. Induction of a biopolyester hydrolase cutinase by low levels of. The thermal stability of the fusarium solani pisi cutinase as a. Optimization of flavor esters synthesis by fusarium solani.
Fusarium solani pisi cutinase request pdf researchgate. Production and purification of cutinase from fusarium. The j b c 2002 by the american society for biochemistry. The cutinase isolated from the fungus fusarium solani pisi, is assumed to be involved in the first steps of fungal attack on plants. Kolattukudy ohio state biotechnology center, rightmire hall, 1060 carmack road, the ohio state university, columbus, ohio. The parameters, chain length of alcohols and acids, alcohol and acid concentration, and the response esterification yield and initial rate were. 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. With this fermtinconjugated antibody it was shown that germinating spores of this fungus excreted cutinase during the penetration of the host pisum sativum. Effect of immobilization support, water activity, and.
Cutinase structure, function and biocatalytic applications bioline. 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. The three dimensional structure of this cutinase is also analysed. We compared wildtype strains and a cutinase negative gene replacement mutant strain of fusarium solani f. Fusarium polycaprolactone depolymerase is cutinase. Electronic publications 80624 freely accessible full text publications plus those not yet available due to embargo. Materials and methods cutinase i was isolated in homogeneous form from the extracellular fluid of f. The effectiveness of fusarium solani pisi cutinase mediated synthesis of shortchain alkyl esters in organic solvent isooctane media was evaluated. Glucose was found to be a repressor of cutinase production.
Dihydroxyc16 acid and trihydroxyc18 acid, which are unique cutin monomers, showed the greatest cutinase inducing activity. Allows pathogenic fungi to penetrate through the cuticular barrier into the host plant during the. Cutin hydrolysate induced the production of an extracellular cutinase by glucosegrown fusarium solani f. Pdf we have investigated the thermal stability of the fusarium solani pisi cutinase as a function of ph, in the range from ph 212. 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. Experiments with several compounds structurally related to these. Molecular cloning, characterization, and expression.
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