Purification and characterization of a novel cutinase from nasturtium (Tropaeolum majus) pollen

Maiti, Indu B.; Kolattukudy, P. E.; Shaykh, Mashouf

doi:10.1016/0003-9861(79)90292-3

Cited by 40 publications

(18 citation statements)

References 17 publications

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“…The pH dependence of cutin hydrolysis is quite similar to that observed with fungal cutinases (15) but quite different from that of pollen cutinase, which showed maximal activity at pH 6.5 (12 (3,15) and differs from pollen cutinase, which has a thiol group at the active site (12).…”

Section: Discussionsupporting

confidence: 69%

Discovery of a cutinase-producing Pseudomonas sp. cohabiting with an apparently nitrogen-fixing Corynebacterium sp. in the phyllosphere

Strzalka¹,

Chandra²,

Kolattukudy³

1987

J Bacteriol

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A phyllospheric bacterial culture, previously reported to partially replace nitrogen fertilizer (B. R. Patti and A. K. Chandra, Plant Soil 61:419-427, 1981) was found to contain a fluorescent pseudomonas which was identified as Pseudomonas putida and a Corynebacterium sp. The P. putida isolate was found to produce an extraceliular cutinase when grown in a medium containing cutin, the polyester structural component of plant cuticle. The Corynebacterium sp. grew on nitrogen-free medium but could not produce cutinase under any induction conditions tested, whereas P. putida could not grow on nitrogen-free medium. When cocultured with the nitrogen-fixing Corynebacterium sp., the P. putida isolate grew in a nitrogen-free medium, suggesting that the former provided fixed N2 for the latter. These results suggest that the two species coexist on the plant surface, with one providing carbon and the other providing reduced nitrogen for their growth. The presence of cutin in the medium induced cutinase production by P. putida. However, unlike the previously studied fungal systems, cutin hydrolysate did not induce cutinase. Thin-layer chromatographic analysis of the products released from labeled apple fruit cutin showed that the extracellular enzyme released all classes of cutin monomers. This enzyme also catalyzed hydrolysis of the model ester substrates, p-nitrophenyl esters of fatty acids, and optimal conditions were determined for a spectrophotometric assay with p-nitrophenyl butyrate as the substrate. It did not hydrolyze triacyl glycerols, indicating that the cutinase activity was not due to a nonspecific lipase. It showed a broad pH optimum between 8.0 and 10.5 with 3H-labeled apple cutin as the substrate. Diisopropylfluorophosphate severely inhibited the enzyme, whereas thiol-directed reagents did not inhibit it, suggesting that catalysis by this bacterial cutinase involves active serine.It has been suggested that a well-established and mixed population of microorganisms in the phyllosphere could make a substantial contribution to the nitrogen requirements of field vegetation (16). Some nitrogen-fixing organisms have been isolated from the leaf surface (8, 13, 18). These nitrogen fixers, when sprayed on the crop plants, increased crop yield when compared with crops which received no nitrogen fertilizers (8). The carbon source for these organisms on the leaf surface is not known. Proposed sources of nutrients include leaf exudates or organic deposits on the leaf surface. Whether these nitrogen fixers are capable of utilizing any of the leaf surface components has not been determined. If the microorganisms are to utilize the insoluble polymer cutin on the leaf surface, these organisms would have to produce an extracellular cutinase. Although cutinases have been purified and characterized from fungi and pollen (2, 3), little is known about bacterial cutinase. In this paper we report that a phyllospheric fluorescent Pseudomonas putida strain, coisolated with a nitrogen-fixing bacterium, may be induced to produce cutina...

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Section: Discussionsupporting

confidence: 69%

Discovery of a cutinase-producing Pseudomonas sp. cohabiting with an apparently nitrogen-fixing Corynebacterium sp. in the phyllosphere

Strzalka¹,

Chandra²,

Kolattukudy³

1987

J Bacteriol

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“…The enzyme hydrolyzed the stigmatic cuticle very rapidly, suggesting it may be located in the pollen wall (SHAYK and KOLATTUKUDY 1977). The cutinase has been isolated, and shown to be a glycoprotein of molecular weight 40,000 containing 7% carbohydrate and having an isoelectric point of pH 5.5 (MAITI et al 1979). With apple cutin as substrate, the isolated pollen enzyme is capable of catalyzing the hydrolysis of p-nitrophenyl esters of C r C 18 fatty acids and has a pH optimum of 6.8.…”

Section: Wall-associated Enzymesmentioning

confidence: 98%

“…The pollen enzyme showed no antigenic cross-reactivity with fungal cutinase, nor was its activity inhibited by antisera to the fungal cutinase of Fusarium solani pisi. MAITI et al (1979) were able to differentiate their cutinase fraction from a non-specific esterase in nasturtium pollen which hydrolyzed p-nitrophenyl acetate preferentially and had an isoelectric point of pH 5.6.…”

Section: Wall-associated Enzymesmentioning

confidence: 98%

Pollen-Pistil Interactions

Knox¹

1984

Cellular Interactions

185

134

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“…Cutin was purified from Red Delicious apples (29); hydrolyzed cutin was prepared as described by Lin and Kolattukudy (15). Radioactively labeled cutin was prepared from ['4C]palmitic acid (60 mCi/mmol) as reported by Maiti et al (19).…”

mentioning

confidence: 99%

“…For each assay, 4 mg of finely ground [14C]cutin was suspended in a total volume of 1 ml of 0.07 M phosphate buffer (pH 8.0) containing the enzyme and assayed for 2 h at 30°C in a shaking water bath. The released [14C]palmitic acid was measured as described previously (19). Under these assay conditions, the release of radioactivity was found to be linear over the range of 5.27 to 52.7 ,umol/min per mg of PNBase activity.…”

mentioning

confidence: 99%

Isolation of a Fusarium solani mutant reduced in cutinase activity and virulence

Dantzig¹,

Zuckerman²,

Andonov-Roland³

1986

J Bacteriol

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Fusarium solani isolate T-8 produces an extracellular enzyme, cutinase, which catalyzes the degradation of cutin in the plant cuticle. Cutinase activity can be measured by the hydrolysis of either the artifical substrate, p-nitrophenylbutyrate (PNB), or radioactive cutin containing [14C]palmitic acid. In the present study, the culture filtrate contained basal levels of cutinase when T-8 was grown on acetate as a sole source of carbon. After mutagenesis, a cutinase-defective mutant (PNB-1) was identified by screening acetate-grown colonies for a loss of PNBase activity. The mutant possessed an 80 to 90% reduction in cutinase activity when grown for 3 to 5 days on acetate-or cutin-containing medium. Induction of cutinase by cutin or hydrolyzed cutin after growth on glucose medium was similarly reduced. Kinetic analysis indicated that cutinase from the mutant possessed a near normal Km for PNB and a 92% reduction in V... Fluorography and Western blotting of 15% sodium dodecyl sulfate-polyacrylamide gels of separated 35S-labeled proteins from cutin induction medium revealed that in the mutant the 22,000-molecular-weight band corresponding to cutinase was reduced approximately 85%. The virulence of the mutant in a pea stem bioassay was decreased by 55% and was restored to nearly the parental level by the addition of purified cutinase. The data suggest that the mutant synthesizes reduced quantities of a functional and immunoreactive cutinase enzyme and that cutinase plays a critical role in infection. The PNB1 mutation may be within a regulatory gene or a promoter for cutinase.

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Purification and characterization of a novel cutinase from nasturtium (Tropaeolum majus) pollen

Cited by 40 publications

References 17 publications

Discovery of a cutinase-producing Pseudomonas sp. cohabiting with an apparently nitrogen-fixing Corynebacterium sp. in the phyllosphere

Discovery of a cutinase-producing Pseudomonas sp. cohabiting with an apparently nitrogen-fixing Corynebacterium sp. in the phyllosphere

Pollen-Pistil Interactions

Isolation of a Fusarium solani mutant reduced in cutinase activity and virulence

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