Hydrolysis Of Cutin Research Articles

Development of the infection strategy of the hemibiotrophic plant pathogen, Colletotrichum orbiculare, and plant immunity

The hemibiotrophic fungus Colletotrichum orbiculare forms appressoria as infection structures and primarily establishes biotrophic infection in cucumber epidermal cells. Subsequently, it develops necrotrophic infection. In the pre-invasion stage, morphogenesis of appressoria of C. orbiculare is triggered by signals from the plant surface. We found that C. orbiculare PAG1 (Perish-in-the-Absence-of-GYP1), a component of MOR [morphogenesis-related NDR (nuclear Dbf2-related) kinase network] plays an essential role as a key component of the plant-specific signaling pathway and that hydrolysis of cutin by a spore surface esterase creates a cutin monomer that constitutes a key plant-derived signal. Development of the infection structure of C. orbiculare is strictly regulated by the cell cycle and we found that proper regulation of G1/S progression via two-component GAP genes, consisting of BUB2 (Budding-Uninhibited-by-Benomyl-2) and BFA1 (Byr-Four-Alike-1) is essential for the establishment of successful infection. In the post-invasion stage, the establishment of the biotrophic phase of hemibiotrophic fungi is crucial for successful infection. We found that C. orbiculare WHI2 (WHIsky-2), an Saccharomyces cerevisiae stress regulator homolog, is involved in the phase transition from biotrophy to necrotrophy through TOR (Target of Rapamycin) signaling, and is thus essential for full pathogenesis.

Hydrolysis of Cutin by PET‐Hydrolases

AbstractFunctionalisation of synthetic polymers by using enzymes has been recently demonstrated. The major advantage of enzymes over chemical processes lies in their surface specific and endo‐wise mode of action. Surface hydrophilisation of PET with lipases and cutinases leads to a dramatic increase of the surfacial acid and hydroxyl group content while conventional chemical treatment does not cause any change. However, this PET‐hydrolysing activity by enzymes from distinct classes has not yet been correlated to activity on natural polyesters. Here, we show that lipases, cutinases and a PHA‐depolymerase are all capable of hydrolysing PET, while only lipases and cutinases also hydrolysed cutin to various degrees. Lipases showed a higher specificity for terminal fatty acids while the cutinases preferred hydroxy fatty acids during cutin hydrolysis.

A rapid screening method for cutinase producing microorganisms

Both cutinase and lipase belong to the esterase group of enzymes (EC 3.1.1.X), which are capable of catalyzing the hydrolysis of ester bonds. Cutinase catalyzes the hydrolysis of cutin, an insoluble biopolyester which is the structural component of plant cuticles. As cutinase is an efficient catalyst in watery or organic media, it is potentially appropriate for the detergent, food and cosmetic industries. The objective of this work was to isolate microorganisms from plants and perform a pre-selection of molds showing esterase producing ability. The selected strains were then submitted to fermentation in media containing cutin. The lipolytic and cutinolytic activities of the supernatant were determined in order to differentiate lipase producers from cutinase-producing strains. The mold strain selected as the best cutinase producer was identified as being Fusarium oxysporium.

Method for the Production and Characterization of Tomato Cutin Oligomers

A series of oligomeric fragments can be obtained by partial alkaline hydrolysis of cutin. The oligomers obtained from tomato cutin were analyzed by high-performance liquid chromatographymass spectrometry and the major products were characterized as linear dimeric esters of w,8-, w,9-, and w,lO-dihydroxyhexadecanoic acids, the major monomers of tomato cutin. Analysis of the products obtained from tomato cutin by treatment with cutinase suggests an exo mode of depolymerization for this enzyme.

Diversity of cutinases from plant pathogenic fungi: Evidence for a relationship between enzyme properties and tissue specificity

Three fungal pathogens with different tissue specificities were cultivated on cutin as the sole carbon source and assayed for cutinase activity over a wide range of pH values. Cutin hydrolysis was optimal at pH 6·5 for the leaf pathogen Cochliobolus heterostrophus, and at pH 8·5 for the stem-base pathogen Rhizoctonia solani (AG 2-2). The pH-profile observed for Altemaria brassicicola, a pathogen that infects both leaves and stems, exhibited two distinct optima. Two cutinases from this organism were separated by chromatofocusing, one with a pH optimum at 7·0 and the other with an optimum at pH 9·0. The presence of different types of cutinase secreted by these pathogens was substantiated by treatment of the culture filtrate with [ 3H] diisopropyl fluorophosphate, an active-site probe for serine esterases. Electrophoresis of labelled proteins under denaturing conditions revealed two types of serine esterases in the molecular weight range of cutinase. Serine esterases with cutinolytic pH optima in the slightly acidic range had molecular weights of 23·0 kDa ( A. brassicicola) and 22·0 kDa ( C. heterostrophus). Esterases with cutinolytic pH optima in the alkaline range appeared as doublets, as previously observed in Fusarium spp. The esterase doublet from A. brassicicola had molecular weights of 19·0 and 21·0 kDa, and that from R. solani had molecular weights of 17·0 and 18·5 kDa. These results, together with evidence from the literature, indicated a relationship between cutinase properties and the expression of tissue specificity by directly penetrating fungi. To substantiate this hypothesis, inoculum from a stem specific isolate of R. solani nonpathogenic on leaves was amended with cutinases purified from either Venturia inaequalis or Fusarium solani f. sp pisi. Inoculum amended with cutinase from the leaf-specific pathogen penetrated the bean leaf cuticle and produced disease symptoms, whereas inoculum amended with cutinase from the stem-base pathogen remained nonpathogenic on bean leaves. These results are the first evidence for a role of cutinase in the expression of tissue specificity by fungal pathogens.

Purification and Characterization of Cutinase fromVenturia inaequalis

K61ler, W., and Parker, D. M. 1989. Purification and characterization of cutinase from Venturia inaequalis. Phytopathology 79:278-283. Venturia inaequalis was grown in a culture medium containing purified cutinase from V. inaequalis is optimal at a pH of 6 and thus different from apple cutin as the sole carbon source. After 8 wk of growth an esterase was the alkaline pH-optimum reported for other purified cutinases. The isolated from the culture fluid and purified to apparent homogeneity. The hydrolysis of the model esterp-nitrophenyl butyrate was less affected by the enzyme hydrolyzed tritiated cutin and thus was identified as cutinase. The pH. The esterase activity was strongly inhibited by diisopropyl purified cutinase is a glycoprotein with a molecular mass of 21-23 kg/ mol, fluorophosphate, and the phosphorylation of one serine was sufficient for as determined by various procedures. Remarkable structural features are a complete inhibition. Thus, cutinase from V. inaequalis belongs to the class high content of glycine, a high content of nonpolar amino acids, two of serine hydrolases, a characterisitic shared with other fungal cutinases. disulfide bridges, and a high degree of hydrophobicity. Cutin hydrolysis by

Purification and characterization of cutinase from a fluorescent Pseudomonas putida bacterial strain isolated from phyllosphere

Cutinase, an extracellular enzyme, was induced by cutin in a fluorescent Pseudomonas putida strain that was found to be cohabiting with an apparently nitrogen-fixing Corynebacterium. This enzyme was purified from the culture fluid by acetone precipitation followed by chromatography on DEAE-cellulose, QAE-Sephadex, Sepharose 6B, and Sephadex G-100. The purified enzyme showed a single band when subjected to polyacrylamide electrophoresis and the enzymatic activity coincided with the protein band. Sodium dodecyl sulfate-polyacrylamide electrophoresis showed a single band at a molecular weight of 30,000 and gel filtration of the native enzyme through a calibrated Sephadex G-100 column indicated a molecular weight of 30,000, showing that the enzyme is a monomer. The amino acid composition of bacterial cutinase is distinctly different from that of fungal or plant cutinases. This bacterial cutinase showed a broad pH optimum between 8.5 and 10.5 with 3H-labeled apple cutin as the substrate. Linear rates of cutin hydrolysis were measured up to 20 min of incubation time and 4 mg/ml of cutin gave the maximum hydrolysis rate. This cutinase catalyzed hydrolysis of p-nitrophenyl esters of C 4 to C 16 fatty acids with decreasing V and increasing K m for the longer chain esters. It did not hydrolyze tripalmitoyl glycerol or trioleyl glycerol, indicating that this is not a general lipase. Active serine-directed reagents such as organophosphates and organoboronic acids severely inhibited the enzyme, suggesting that bacterial cutinase is an “active serine” enzyme. Neither thiol-directed reagents nor metal ion chelators had any effect on this enzyme. Antibody raised against purified enzyme gave a single precipitin line on Ouchterlony double diffusion analysis. Western blot analysis of the extracellular fluid of induced Ps. putida showed a single band at 30 kDa. No immunological cross-reactivity was detected between the present bacterial enzyme and the fungal enzyme from Fusarium solani pisi when rabbit antibodies against either enzyme was used.

Rumen digestion of untreated and alkali‐treated cereal straws: A study by multiple internal reflectance infrared spectroscopy

AbstractMultiple internal reflectance (MIR) infrared spectroscopy of wheat and barley straw shows fundamental chemical differences between their inner and outer surface layers (1‐3 μm thick). Inner layers were dominated by polysaccharide in which cellulose and hemicellulose characteristics could be identified. Weak absorption arising from small amounts of lignin and acetyl groups was also detectable. In contrast, the outer layers were silicified (SiO2), more highly lignified, and contained ester groups which originate predominantly in cutin and long‐chain waxes in the outermost cuticular layer.MIR spectra of rumen‐incubated straw clearly indicate that little or no degradation occurred at the outer surface but that appreciable polysaccharide had been solubilised from the inner surface after 120 h of digestion. This was concluded from the detection of much higher levels of lignin at the inner surface, suggesting that the concentration of this component may be a limiting factor in the progress of the degradation. Absorption due to acetyl groups was also enhanced in spectra of the digested inner surface, suggesting that these groups also may be indirectly implicated in control of digestion.From changes in MIR spectra it was concluded that alkali pretreatment of straw scarcely alters the chemistry of the inner surfaces but significantly modifies that of the outer layers as a result of hydrolysis of cutin and acetyl ester groups and dissolution of a large proportion of the silica phase. These chemical changes result in surface disruption, probably providing additional routes by which enzymic attack can proceed beyond the point at which digestion of untreated straw ceases. An as yet unidentified organic component presumably of microbial origin accumulated preferentially on the outer straw surfaces during rumen digestion. It is not yet known whether this component exerts any influence on digestibility.

Evidence that pancreatic lipase is responsible for the hydrolysis of cutin, a biopolyester present in mammalian diet, and the role of bile salt and colipase in this hydrolysis

The enzyme, which catalyzes hydrolysis of cutin, an insoluble biopolyester of hydroxy and epoxy fatty acids, was purified from porcine pancreas. With three different purification methods, previously used for the purification of pancreatic lipase, it is shown that cutin hydrolase is pancreatic lipase. This enzyme released oligomers and all types of monomers from the polymer with a pH optimum around 7.5. Taurodeoxycholate inhibited cutin hydrolysis by lipase and colipase reversed this inhibition. Evidence is presented which suggests that bile salt stabilizes the enzyme at the surface of the insoluble substrate and that the interaction of the polymer surface with the lipase-colipase-bile salt system is similar to that previously observed with triglycerides. Diethyl- p-nitrophenyl phosphate inhibited cutin hydrolysis by lipase but the hydrolysis was insensitive to diisopropyl fluorophosphate.

Isolation and characterization of a cutinase from Fusarium roseum culmorum and its immunological comparison with cutinases from F. solani pisi

Fusarium roseum culmorum, grown on apple cutin as the sole source of carbon, was shown to produce a cutin depolymerizing enzyme. From the extracellular fluid of these F. roseum cultures, a cutinase and a nonspecific esterase were isolated utilizing Sephadex G-100, QAE-Sephadex, and SP-Sephadex chromatography. The homogeneity of the cutinase was verified by polyacrylamide disc gel electrophoresis. The molecular weight of the cutinase was estimated to be 24,300 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Electrophoretic mobility of this enzyme was between that of Cutinases I and II from Fusarium solani pisi. The F. roseum cutinase hydrolyzed p-nitrophenyl butyrate and cutin, but not p-nitrophenyl palmitate, while the nonspecific esterase hydrolyzed the long-chain esters. Amino acid composition of F. roseum cutinase was found to be similar to that of F. solani pisi Cutinase I except for differences in the number of serine, valine, and cysteine residues. The time-course, protein concentration dependence, substrate concentration dependence, and pH optimum (10.0 for cutin hydrolysis) of the F. roseum cutinase was similar to the cutinases from F. solani pisi. The F. roseum cutinase was inhibited by diisopropylfluorophosphate and paraoxon, and the [ 3H]diisopropylphosphate group was covalently attached to the enzyme upon treatment with tritiated diisopropylfluorophosphate. Therefore, it is concluded that catalysis by cutinase involves an “active serine.” Immunochemical studies with a rabbit antibody prepared against F. solani pisi Cutinase I demonstrated that Cutinase II from this organism was immunologically very similar to, but not identical to, Cutinase I. On the other hand, the cutinase from F. roseum was immunologically quite different from the cutinases isolated from F. solani pisi in that it did not cross-react with anticutinase I. However, all three cutinases were virtually identical in their sensitivity to inhibition by anticutinase I, and all three enzymes were virtually completely inhibited by the anticutinase I.

Utilization of cutin by a pseudomonad isolated from soil

A Pseudomonas sp., which has been isolated from orchard soil, is able to utilize cutin as a sole source of carbon. Products obtained from the culture filtrate corresponded to that obtained by alkaline hydrolysis of cutin.