Biodegradation of dipropyl phthalate and toxicity of its degradation products: a comparison of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">pisi</Emphasis> cutinase and <Emphasis Type="Italic">Candida cylindracea</Emphasis> esterase

The efficiency of two lypolytic enzymes (fungal cutinase, yeast esterase) in the degradation of dipropyl phthalate (DPrP) was investigated. The DPrP-degradation rate of fungal cutinase was surprisingly high, i.e., almost 70% of the initial DPrP (500 mg/l) was decomposed within 2.5 h and nearly 50% of the degraded DPrP disappeared within the initial 15 min. With the yeast esterase, despite the same concentration, more than 90% of the DPrP remained even after 3 days of treatment. During the enzymatic degradation of DPrP, several DPrP-derived compounds were detected and time-course changes in composition were also monitored. The final chemical composition after 3 days was significantly dependent on the enzyme used. During degradation with fungal cutinase, most DPrP was converted into 1,3-isobenzofurandione (IBF) by diester hydrolysis. However, in the degradation by yeast esterase, propyl methyl phthalate (PrMP) was produced in abundance in addition to IBF. The toxic effects of the final degradation products were investigated using various recombinant bioluminescent bacteria. As a result, the degradation products (including PrMP) from yeast esterase severely caused oxidative stress and damage to protein synthesis in bacterial cells, while in the fungal cutinase processes, DPrP was significantly degraded to non-toxic IBF after the extended period (3 days).     

Accelerated Degradation of Dipentyl Phthalate by Fusarium oxysporum f. sp. pisi Cutinase and Toxicity Evaluation of Its Degradation Products Using Bioluminescent Bacteria

The efficiency of two lipolytic enzymes (fungal cutinase and yeast esterase) in the degradation of dipentyl phthalate (DPeP) was investigated. The DPeP degradation rate of fungal cutinase was surprisingly high, i.e., almost 60% of the initial DPeP (500 mg/L) was decomposed within 2.5 hours, and nearly 40% of the degraded DPeP disappeared within the initial 15 minutes. With the yeast esterase, despite the same concentration, >87% of the DPeP remained even after 3 days of treatment. The final chemical composition after 3 days was significantly dependent on the enzyme used. During degradation with cutinase, most DPeP was converted into 1,3-isobenzofurandione (IBF) by diester hydrolysis. However, in the degradation by esterase, pentyl methyl phthalate, in addition to IBF, was produced in abundance. Toxicity monitoring using various recombinant bioluminescent bacteria showed that the degradation products from yeast esterase contained a toxic hazard, causing oxidative stress and damage to protein synthesis. Ji-Young Ahn, Yang-Hoon Kim are contributed equally to this work     

Degradation of an endocrine disrupting chemical,DEHP [di-(2-ethylhexyl)-phthalate], by<Emphasis Type="Italic"> Fusarium oxysporum</Emphasis> f. sp.<Emphasis Type="Italic"> pisi</Emphasis> cutinase

The efficiency of two lypolytic enzymes (fungal cutinase, yeast esterase) in the degradation of di-(2-ethylhexyl)-phthalate (DEHP) was investigated. The DEHP-degradation rate of fungal cutinase was surprisingly high, i.e. almost 70% of the initial DEHP (500 mg/l) was decomposed within 2.5 h and nearly 50% of the degraded DEHP disappeared within the initial 15 min. With the yeast esterase, despite the same concentration, more than 85% of the DEHP remained even after 3 days of treatment. During the enzymatic degradation of DEHP, several DEHP-derived compounds were detected and time-course changes in composition were also monitored. During degradation with fungal cutinase, most DEHP was converted into 1,3-isobenzofurandione (IBF) by diester hydrolysis. In the degradation by yeast esterase, two organic chemicals were produced from DEHP: IBF and an unidentified compound (X). The final chemical composition after 3 days was significantly dependent on the enzyme used. Fungal cutinase produced IBF as a major degradation compound. However, in the DEHP degradation by yeast esterase, compound X was produced in abundance in addition to IBF. The toxic effects of the final degradation products were investigated, using various recombinant bioluminescent bacteria and, as a result, the degradation products from yeast esterase were shown to contain a toxic hazard, causing oxidative stress and damage to protein synthesis.     

Enzymatic degradation of dibutyl phthalate and toxicity of its degradation products

Dibutyl phthalate (DBP) was more efficiently degraded by cutinase compared to yeast esterase; i.e. almost 80% of initial DBP (500 mg l⁻¹) was decomposed within 7.5 h, and nearly 50% of the degraded DBP disappeared within the initial 30 min. The toxicity of the final DBP degradation products were investigated using various recombinant bioluminescent bacteria. Butyl methyl phthalate, the major product of degradation by the esterase, was an oxidative toxic hazard that damaged protein synthesis.     

Enhanced degradation of an endocrine-disrupting chemical,butyl benzyl phthalate,by Fusarium oxysporum f. sp. pisi cutinase

Compared to yeast esterase, fungal cutinase degraded butyl benzyl phthalate (BBP) far more efficiently; i.e., almost 60% of the BBP disappeared within 7.5 h. Also, the final chemical composition significantly depended on the enzyme used. Toxicity monitoring using bioluminescent bacteria showed that butyl methyl phthalate, a major product of degradation by esterase, was an oxidative toxic hazard.     

Enhanced Degradation of an Endocrine-Disrupting Chemical, Butyl Benzyl Phthalate, by Fusarium oxysporum f. sp. pisi Cutinase

Compared to yeast esterase, fungal cutinase degraded butyl benzyl phthalate (BBP) far more efficiently; i.e., almost 60% of the BBP disappeared within 7.5 h. Also, the final chemical composition significantly depended on the enzyme used. Toxicity monitoring using bioluminescent bacteria showed that butyl methyl phthalate, a major product of degradation by esterase, was an oxidative toxic hazard.     

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

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.     

Fusarium polycaprolactone depolymerase is cutinase.

Polycaprolactone (PCL), a synthetic polyester, is degraded by a variety of microorganisms, including some phytopathogens. Many phytopathogens secrete cutinase, a serine hydrolase that degrades cutin, the structural polymer of the plant cuticle. We compared wild-type strains and a cutinase-negative gene replacement mutant strain of Fusarium solani f. sp. pisi (D. J. Stahl and W. Schäfer, Plant Cell 4:621-629, 1992) and a wild-type strain of Fusarium moniliforme to show that Fusarium cutinase is a PCL depolymerase. The wild-type strains, but not the mutant strain, (i) degraded PCL and used it as a source of carbon and energy, (ii) showed induction of secreted PCL depolymerase and an esterase activity of cutinase when grown in the presence of cutin, and (iii) showed induction of PCL depolymerase and an esterase activity of cutinase when grown in the presence of a hydrolysate of PCL, which contains PCL oligomers that are structurally similar to the natural inducers of cutinase. These results together with other details of regulation and conditions for optimal enzyme activity indicate that the Fusarium PCL depolymerase, required for degradation and utilization of PCL, is cutinase.     

Evidence for a constitutive cytoplasmic cutinase in ungerminated conidia of Botrytis cinerea Pers.: Fr.

Cytoplasmic soluble proteins from ungerminated conidia of Botrytis cinerea exhibited cutinase activity, while cell wall binding proteins lacked this activity. Cutinase activity in proteins extracted from cell walls and cytoplasm of ungerminated conidia of Botrytis cinerea was determined using p-nitrophenyl butyrate (PNB) and TLC analysis of products derived from hydrolysis of [³H]cutin. Treatment of conidia with indoxyl acetate, a substrate indicative of non-specific esterase and cutinase activity, also gave a positive reaction in the cytoplasm of ungerminated conidia. The possible role of a putative constitutive cutinase in the cytoplasm of conidia in the early stages of infection of plants by B. cinerea is discussed.     

Synthesis and characterization of a new cutinase substrate, 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate

Phytopathogenic fungi penetrate plants by breaking down the cuticular barrier with cutinase. Cutinases are extracellular hydrolytic enzymes that degrade cutin, a polyester composed of hydroxy and epoxy fatty acids. Until now, cutinase has been recognized by its ability to release labeled cutin monomers or by a non-specific esterase assay based on the hydrolysis of p-nitrophenyl esters of short fatty acids. In this work, an insoluble p-nitrophenyl derivative was synthesized and purified, and its structure was determined to be 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate (pNMSEH) by nuclear magnetic resonance (H+ NMR) analysis. pNMSEH was tested as a new cutinase substrate with Pseudomonas mandocino cutinase and porcine liver esterase. While a linear release over time of p-nitrophenol (pNP) was recorded in the presence of cutinase, no response was obtained with the esterase. The calculated kinetic parameters of pNMSEH hydrolysis by cutinase revealed a high specificity (Km=1.8mM), albeit a low catalytic rate (Vmax=10.5 micromol min(-l)l(-1)). This new synthetic substrate may be helpful for detecting and assaying cutinase activity in mixed solutions, such as crude fungal extracellular extracts.     

Metabolism of butyl benzyl phthalate by Gordonia sp. strain MTCC 4818

The microbial degradative characteristics of butyl benzyl phthalate (BBP) were investigated by the Gordonia sp. strain MTCC 4818 isolated from creosote-contaminated soil. The test organism can utilize a number of phthalate esters as sole sources of carbon and energy, where BBP was totally degraded within 4 days under shake culture conditions. High performance liquid chromatography profile of the metabolites isolated from spent culture indicated the accumulation of two major products apart from phthalic acid (PA), which were characterized by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy as mono-n-butyl phthalate (MBuP) and monobenzyl phthalate (MBzP). Neither of the metabolites, MBuP, MBzP or PA, supported growth of the test organism, while in resting cell transformation, the monoesters were hydrolyzed to PA to a very minor extent, which was found to be a dead-end product in the degradation process. On the other hand, the test organism grew well on benzyl alcohol and butanol, the hydrolyzed products of BBP. The esterase(s) was found to be inducible in nature and can hydrolyze in vitro the seven different phthalate diesters tested to their corresponding monoesters irrespective of their support to the growth of the test organism.     

The effect of additives and mechanical agitation in surface modification of acrylic fibres by cutinase and esterase

The surface of an acrylic fibre containing about 7% of vinyl acetate was modified using Fusarium solani pisi cutinase and a commercial esterase, Texazym PES. The effect of acrylic solvents and stabilising polyols on cutinase operational stability was studied. The half-life time of cutinase increased by 3.5-fold with the addition of 15% N,N-dimethylacetamide (DMA) and by 3-fold with 1M glycerol. The impact of additives and mechanical agitation in the protein adsorption and in the hydrolysis of vinyl acetate from acrylic fabric was investigated. The hydroxyl groups produced on the surface of the fibre were able to react specifically with Remazol Brilliant Blue R (cotton reactive dye) and to increase the colour of the acrylic-treated fabric. The best staining level was obtained with a high level of mechanical agitation and with the addition of 1% DMA. Under these conditions, the raise in the acrylic fabric colour depth was 30% for cutinase and 25% for Texazym. The crystallinity degree, determined by X-ray diffraction, was not significantly changed between control samples and samples treated with cutinase. The results showed that the outcome of the application of these enzymes depends closely on the reaction media conditions.     

Cutinase gene disruption in Fusarium solani f sp pisi decreases its virulence on pea.

Fusarium solani f sp pisi (Nectria haematococca) isolate 77-2-3 with one cutinase gene produced 10 to 20% of the cutinase produced by isolate T-8 that has multiple cutinase genes, whereas cutinase gene-disrupted mutant 77-102 of isolate 77-2-3 did not produce cutinase. On the surface of pea stem segments, lesion formation was most frequent and most severe with T-8, less frequent and less severe with 77-2-3, and much less frequent and much milder with the gene-disrupted mutant. Microscopic examination of the lesions caused by the mutant strongly suggest that it penetrated the host mostly via the stomata. In seedling assays, 77-2-3 caused severe lesions on every seedling and stunted growth, whereas the mutant showed very mild lesions on one-third of the seedlings with no stunting. Thus, cutinase gene disruption resulted in a significant decrease in the pathogenicity of F. s. pisi on pea.     

Hydrolysis of plant cuticle by plant pathogens. Properties of cutinase I, cutinase II, and a nonspecific esterase isolated from Fusarium solani pisi.

The properties of the homogeneous cutinase I, cutinase II, and the nonspecific esterase isolated from the extracellular fluid of cutin-grown Fusarium solani F. pisi (R.E. Purdy and P.E. Kolattukudy (1975), Biochemistry, preceding paper in this issue) were investigated. Using tritiated apple cutin as substrate, the two cutinases showed similar substrate concentration dependence, protein concentration dependence, time course profiles, and pH dependence profiles with optimum near 10.0. Using unlabeled cutin, the rate of dihydroxyhexadecanoic acid release from apple fruit cutin by cutinase I was determined to be 4.4 mumol per min per mg. The cutinases hydrolyzed methyl hexadecanoate, cyclohexyl hexadecanoate, and to a much lesser extent hexadecyl hexadecanoate but not 9-hexadecanoyloxyheptadecane, cholesteryl hexadecanoate, or hexadecyl cinnamate. The extent of hydrolysis of these model substrates by cutinase I was at least three times that by cutinase II. The nonspecific esterase hydrolyzed all of the above esters except hexadecyl cinnamate, and did so to a much greater extent than did the cutinases. None of the enzymes hydrolyzed alpha- or beta-glucosides of p-nitrophenol. p-Nitrophenyl esters of fatty acids from C2 through C18 were used as substrates and V's and Kms were determined...     

Cutinolytic esterase activity of bacteria isolated from mixed-plant compost and characterization of a cutinase gene from Pseudomonas pseudoalcaligenes

The objective of the current study was to examine cutinolytic esterase (i.e., cutinase) activity by pseudomonads and bacteria isolated from mixed-plant compost. Approximately 400 isolates representing 52 taxa recovered from mixed-plant compost using cuticle baits, along with 117 pseudomonad isolates obtained from a culture collection (i.e., non-compost habitats), were evaluated. The ability of isolates to degrade the synthetic cutin polycaprolactone (PCL) was initially measured. Isolates from 23 taxa recovered from the compost degraded PCL. As well, isolates from 13 taxa of pseudomonads cleared PCL. Secondary screening measured esterase activity induced by the presence of apple cuticle using the chromogenic substrate p-nitrophenyl butyrate. Eighteen isolates representing four taxa (Alcaligenes faecalis , Bacillus licheniformis , Bacillus pumilus , and Pseudomonas pseudoalcaligenes) recovered from compost exhibited substantial esterase activity when grown with cuticle. In contrast, none of the pseudomonad isolates from the culture collection produced appreciable esterase activity. Although degradation of PCL was not correlated with esterase activity, isolates that were unable to degrade PCL failed to produce measureable esterase activities. Zymogram analysis indicated that the esterases produced by bacteria from compost ranged in size from 29 to 47 kDa. A gene from P. pseudoalcaligenes (cutA) was found to code for a cutin-induced esterase consisting of 302 amino acids and a theoretical protein size of 32 kDa. The enzyme was unique and was most closely related to other bacterial lipases (≤48% similarity).     

Degradation of di-ethylhexyl phthalate (DEHP) in bioslurry phase reactor and identification of metabolites by HPLC and MS

Di-ethylhexyl phthalate (DEHP) belongs to the class of phthalate esters and is used as an additive in many products including plastics, paints and inks or as a solvent in industrial formulations. However, it is used mostly for its plasticizing ability in polyvinyl chloride (PVC) products, in which it is often added in concentrations exceeding 40% by mass. DEHP is one of the more recalcitrant phthalate esters, which has xeno-estrogenic, carcinogenic and mutagenic effects. Five different bioslurry reactors were operated under different conditions to study the degradation of DEHP (1 mg g⁻¹ soil) in soil. The process performance was assessed by monitoring DEHP concentration periodically using high performance liquid chromatography (HPLC). The ongoing biological process was monitored by analyzing pH, oxidation–reduction potential (ORP), dissolved oxygen (DO), oxygen uptake rate (OUR) and colony forming units (CFU) for every 24 h. More than 90% degradation was observed within 12 days of the cycle period in the augmented reactors. Metabolites formed during the degradation of DEHP in the slurry phase reactor were identified and the pathway was also established. The degradation process was found to follow zero-order kinetic model.     

Biodegradation of dimethyl phthalate by an entomopathogenic nematode symbiont Xenorhabdus indica strain KB-3

Dimethyl phthalate (DMP) a common environmental pollutant is well known for its endocrine disrupting activities. Wide spread use of the plastics and pesticides in agriculture have resulted in DMP pollution in soils. An endosymbiotic bacteria Xenorhabdus indica isolated from entomopathogenic nematode Steinernema thermophilum was investigated for biodegradation of DMP. Biodegradation experiment was conducted for 12 days in minimal salt medium supplemented with beef extract. Quantification of residual DMP by High performance liquid chromatography (HPLC) revealed that maximum degradation (98.75%) occurred at 9th day of incubation along with higher esterase activity (46.94 IU/ml) and growth of bacteria (263.75 μg/ml). The efficacy of purified esterase for degradation of DMP was also investigated. It was observed that enzyme alone transform more than 25.6% of DMP into mono-methyl phthalate (MMP) and phthalic acid (PA) within 24 h, which confirms its role in degradation of DMP. Activity of carboxyl esterase enzyme was also positively correlated (r = 0.88) with biodegradation of DMP. Xenorhabdus completely mineralized the DMP as the two intermediates mono-methyl phthalate (MMP) and phthalic acid were not detected after the incubation period of 12 days. The results suggest that the X. indica was highly efficient in degrading DMP and can be employed for bioremediation of contaminated sites.     

Cutinase is not required for fungal pathogenicity on pea.

Cutinase, a fungal extracellular esterase, has been proposed to be crucial in the early events of plant infection by many pathogenic fungi. To test the long-standing hypothesis that cutinase of Nectria haematococca (Fusarium solani f sp pisi) is essential to pathogenicity, we constructed cutinase-deficient mutants by transformation-mediated gene disruption of the single cutinase gene of a highly virulent N. haematococca strain. Four independent mutants were obtained lacking a functional cutinase gene, as confirmed by gel blot analyses and enzyme assays. Bioassays of the cutinase-deficient strains showed no difference in pathogenicity and virulence on pea compared to the wild type and a control transformant. We conclude that the cutinase of N. haematococca is not essential for the infection of pea.     

角质酶在生物可降解聚酯聚己二酸/对苯二甲酸丁二醇酯降解中的应用

随着废弃塑料带来的环境污染越来越严重,生物可降解聚酯已成为大众关注的焦点。聚己二酸/对苯二甲酸丁二醇酯[poly(butylene adipate-co-terephthalate),PBAT]是脂肪族和芳香族共聚形成的生物可降解聚酯,兼具两者的优异性能。针对PBAT在自然条件下对降解环境要求严格且降解周期长的不足之处,本研究探究了角质酶在PBAT降解中的应用和对苯二甲酸-丁二醇酯(butylene terephthalate,BT)含量对PBAT生物降解性的影响,以实现对PBAT降解速率的提升。选取5种不同来源的聚酯降解酶对PBAT进行降解应用并比较出降解效果最优的酶,并测定了含有不同BT含量的PBAT聚酯的降解效率。结果表明,角质酶ICCG为降解效果最好的酶,且BT含量越高PBAT的降解率越低。此外,还确定了角质酶ICCG对高BT含量的PBAT(H)降解的最适温度、最适缓冲液类型、最适pH、最适E/S(enzyme to substrate)和最适底物浓度比分别为75℃、Tris-HCl、9.0、0.4%和1.0%。本研究结果可为角质酶在PBAT降解中的应用提供一定的理论依据和实验...     

Metabolism and toxicity of synthetic analogues of macrocyclic diester pyrrolizidine alkaloids

Seven macrocyclic diesters analogous to hepatotoxic pyrrolizidine alkaloids have been tested in male weanling Wistar rats. The compounds were the succinate (VII), 2,3-dimethylsuccinate (VIII), phthalate (IX), glutarate (X), 2,4-dimethylglutarate (XI), 3,3-dimethylglutarate (XII) and 3,3-pentamethyleneglutarate (XIII) of the synthetic amino dialcohol, synthanecine A. Single doses of these compounds were given i.p. to rats, and liver levels of pyrrolic metabolites were measured 2 h later. For these experiments both normal rats and rats pretreated with the esterase inhibitor tri-orthocresylphosphate (TOCP) were used. In normal rats, low levels of pyrrolic metabolites were formed from compounds VII, IX, X and XI, but these levels were greatly enhanced in rats with inhibited esterase activity. Much higher pyrrole levels were formed from compounds VIII, XII and XIII in normal rats, and esterase inhibition had relatively little effect on their metabolic conversion to pyrroles. This indicated that the last mentioned compounds were relatively resistant to enzymic hydrolysis, whereas VII, IX, X and XI were easily hydrolysed in normal rats, providing an alternative metabolic path which limited their conversion to pyrrolic metabolites. Comparison of results obtained using the 2,4-dimethylglutarate (XI), the 3,3-dimethylglutarate (XII) and the 3,3,-pentamethyleneglutarate (XIII) showed that 3,3-disubstitution but not 2,4-disubstitution in the glutaric acid moiety conferred high resistance to esterase attack. Toxicity tests using four of the compounds confirmed that acute hepatotoxicity was dose related, and associated with the formation of pyrrolic metabolites in the liver. The 3,3-dimethylglutarate (XII) was highly toxic both in normal and in TOCP treated rats, doses of 25-30 mg/kg causing moderate to severe centrilobular necrosis of the liver. In contrast the toxicity of the unsubstituted succinate (VII), glutarate (X) and 2,4-dimethylglutarate (XI) was very low in normal rats but high in rats with inhibited esterase activity. Thus, the glutarate (X) was non-toxic at 200 mg/kg in normal rats, but in TOCP treated rats, in which pyrrolic metabolite formation was enhanced by a factor of 17.5, a 50 mg/kg dose of this compound was severely hepatotoxic. Kidney damage, which was generally limited to the presence of isolated necrotic cells, sometimes accompanied the liver damage caused by these compounds, but acute toxic effects were not observed in any other tissues.(ABSTRACT TRUNCATED AT 400 WORDS)