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1.
Streptomyces sp. strain K30 was isolated from soil next to a city high way in Münster (Germany) according to its ability to degrade natural and synthetic poly(cis-1,4-isoprene) rubber and to form clear zones on natural rubber latex agar plates. The clear zone forming phenotype was used to clone the responsible gene by phenotypic complementation of a clear zone negative mutant. An open reading frame (lcp) of 1,191 bp was identified, which was preceded by a putative signal sequence and restored the capability to form clear zones on natural rubber latex in the mutant. The putative translation product exhibited strong homologies (50% aa identity) to a putative secreted protein from Streptomyces coelicolor strain A3(2), another clear zone forming strain. Heterologous expression of lcp of Streptomyces sp. strain K30 in Streptomyces lividans strain TK23 enabled the latter to form clear zones on latex-overlay agar plates and to accumulate a degradation product of about 12 kDa containing aldehyde groups. Two ORFs putatively encoding a heterodimeric molybdenum hydroxylase (oxiAB) were identified downstream of lcp in Streptomyces sp. strain K30 strain which exerted a positive effect on clear zone formation and enabled the strain to oxidize the resulting aldehydes. Heterologous expression of a fragment harboring lcp plus oxiAB in S. lividans TK23 resulted in accumulation of aldehydes only in the presence of 10 mM tungstate. Determination of protein content during cultivation on poly(cis-1,4-isoprene) revealed an increase of the cellular protein, and gel permeation chromatography analysis indicated a shift of the molecular weight distribution of the rubber to lower values in the transgenic S. lividans strains and in the wild type, thus confirming utilization and degradation of rubber. Therefore, for the first time, genes responsible for clear zone formation on natural rubber latex and synthetic cis-1,4-polyisoprene degradation in Gram-positive bacteria were identified and characterized.  相似文献   

2.
Since polymeric materials do not decompose easily, disposal of waste polymers is a serious environmental concern. Widespread studies on the biodegradation of rubbers have been carried out in order to overcome the environmental problems associated with rubber waste. This report provides an overview on the microbial degradation of natural and synthetic rubbers. Rubber degrading microbes, bacteria and fungi, are ubiquitous in the environment especially soil. The qualitative data like plate assay, scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) and Sturm test indicated that both natural and synthetic rubbers can be degraded by microorganisms. It has confirmed that the enzymes latex clearing protein (Lcp) and rubber oxygenase A (RoxA) are responsible for the degradation of natural and synthetic rubbers. Lcp was obtained from Gram-positive bacterium Streptomyces sp. strain K30 and RoxA from Gram-negative bacterium Xanthomonas sp. strain 35Y. Analysis of degradation products of natural and synthetic rubbers indicated the oxidative cleavage of double bonds in polymer backbone. Aldehydes, ketones and other carbonyl groups were detected as degradation products from cultures of various rubber degrading strains. This review emphasizes the importance of biodegradation in environmental biotechnology for waste rubber disposal.  相似文献   

3.
A comparative study on biodegradation of di-cumyl peroxide (DCP) crosslinked and uncrosslinked natural rubber by Pseudomonas sp. was carried out. Decrease in organic carbon content along with the changes in tensile strength of the treated rubber, both DCP crosslinked and uncrosslinked natural rubber, indicated rubber hydrocarbon utilization by the Pseudomonas sp. A decrease in 60.88% MPa and 41.66% MPa was observed after five month's old treated uncrosslinked natural rubber and DCP crosslinked rubber, respectively. Biodegradation was more pronounced in natural uncrosslinked rubber, which was further confirmed by the formation of aldehydic compounds with decrease in CH2 stretching frequencies.  相似文献   

4.
The effect of pretreatment of several cis-1,4-polyisoprene containing rubbers on their biodegradability was examined. Tests were carried out with six recently isolated and characterized rubber degrading bacteria belonging to the genera Gordonia (strains Kb2, Kd2 and VH2), Mycobacterium, Micromonospora and Pseudomonas. All strains were able to use natural rubber (NR) as well as NR latex gloves as sole carbon source. Extraction of NR latex gloves by organic solvents resulted in an enhancement of growth for three of the selected strains. On the other hand, growth of Gordonia sp. (strain Kb2 and Kd2), Mycobacterium fortuitum NF4 and Micromonospora aurantiaca W2b on synthetic cis-1,4-polyisoprene did only occur after removal of the antioxidants, that are usually added during manufacture to prevent aging of the materials. Detailed degradation studies performed with Gordonia sp. Kb2 revealed an enhanced mineralization of pretreated NR latex gloves and mineralization of purified natural rubber (NR), indicating the actual mineralization of cis-1,4-polyisoprene rubber constituent even after removal of non-rubber constituent that may act as co-metabolic substrate and support microbial growth. Further analysis by scanning electron microscopy (SEM) clearly demonstrated the enhanced colonization efficiency of these bacteria towards pretreated NR latex gloves. Colonization was additionally visualized by staining of overgrown NR latex gloves with Schiff's reagent, and the purple color produced in the area of degradation was an evidence for the accumulation of aldehydes containing oligomers. Further enhancement of latex gloves degradation could be achieved after successive replacement of mineral salts medium during cultivation. Thereby, a rapid disintegration of untreated NR latex gloves material was accomplished by Gordonia sp. strain VH2.  相似文献   

5.
Various microorganisms were screened for their ability to degrade polyisoprene rubber (natural rubber latex gloves). Strain AF-666, newly isolated from a soil sample, was selected as the best strain having the ability to grow on polyisoprene containing plates. The strain identified as Bacillus sp. AF-666, was found to degrade polyisoprene rubber, both on basal agar plates (latex overlay) as well as in liquid medium. Qualitative analysis of degradation was done through scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy SEM showed changes in surface morphology, like appearance of pits and cracks, and marked difference in transmittance spectra of test and control due to changes in the functional groups, was detected through FTIR. CO2 evolution as a result of rubber degradation, was calculated gravimetrically by Sturm Test. About 4.43 g/1 of CO2 was produced in case of test, whereas, 1.57 g/1 in case of control. The viable number of cells (CFU/ml) was also higher in test than in control. Present study may provide an opportunity for further studies on the applications of biotechnological processes as a tool for rubber waste management.  相似文献   

6.
Various microorganisms were screened for their ability to degrade polyisoprene rubber (natural rubber latex gloves). Strain AF-666, newly isolated from a soil sample, was selected as the best strain having the ability to grow on polyisoprene containing plates. The strain identified as Bacillus sp. AF-666, was found to degrade polyisoprene rubber, both on basal agar plates (latex overlay) as well as in liquid medium. Qualitative analysis of degradation was done through scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. SEM showed changes in surface morphology, like appearance of pits and cracks, and marked difference in transmittance spectra of test and control due to changes in the functional groups, was detected through FTIR. CO2 evolution as a result of rubber degradation, was calculated gravimetrically by Sturm Test. About 4.43 g/1 of CO2 was produced in case of test, whereas, 1.57 g/1 in case of control. The viable number of cells (CFU/ml) was also higher in test than in control. Present study may provide an opportunity for further studies on the applications of biotechnological processes as a tool for rubber waste management.  相似文献   

7.
An extracellular protein with strong absorption at 406 nm was purified from cell-free culture fluid of latex-grown Xanthomonas sp. strain 35Y. This protein was identical to the gene product of a recently characterized gene cloned from Xanthomonas sp., as revealed by determination of m/z values and sequencing of selected isolated peptides obtained after trypsin fingerprint analysis. The purified protein degraded both natural rubber latex and chemosynthetic poly(cis-1,4-isoprene) in vitro by oxidative cleavage of the double bonds of poly(cis-1,4-isoprene). 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (m/z 236) was identified and unequivocally characterized as the major cleavage product, and there was a homologous series of minor metabolites that differed from the major degradation product only in the number of repetitive isoprene units between terminal functions, CHO-CH2--and--H2-COCH3. An in vitro enzyme assay for oxidative rubber degradation was developed based on high-performance liquid chromatography analysis and spectroscopic detection of product carbonyl functions after derivatization with dinitrophenylhydrazone. Enzymatic cleavage of rubber by the purified protein was strictly dependent on the presence of oxygen; it did not require addition of any soluble cofactors or metal ions and was optimal around pH 7.0 at 40 degrees C. Carbon monoxide and cyanide inhibited the reaction; addition of catalase had no effect, and peroxidase activity could not be detected. The purified protein was specific for natural rubber latex and chemosynthetic poly(cis-1,4-isoprene). Analysis of the amino acid sequence deduced from the cloned gene (roxA [rubber oxygenase]) revealed the presence of two heme-binding motifs (CXXCH) for covalent attachment of heme to the protein. Spectroscopic analysis confirmed the presence of heme, and approximately 2 mol of heme per mol of RoxA was found.  相似文献   

8.
Several actinomycetes isolated from nature were able to use both natural rubber (NR) and synthetic cis-1,4-polyisoprene rubber (IR) as a sole source of carbon. According to their degradation behavior, they were divided into two groups. Representatives of the first group grew only in direct contact to the rubber substrate and led to considerable disintegration of the material during cultivation. The second group consisted of weaker rubber decomposers that did not grow adhesively, as indicated by the formation of clear zones (translucent halos) around bacterial colonies after cultivation on NR dispersed in mineral agar. Taxonomic analysis of four selected strains based on 16S rRNA similarity examinations revealed two Gordonia sp. strains, VH2 and Kb2, and one Mycobacterium fortuitum strain, NF4, belonging to the first group as well as one Micromonospora aurantiaca strain, W2b, belonging to the second group. Schiff's reagent staining tests performed for each of the strains indicated colonization of the rubber surface, formation of a bacterial biofilm, and occurrence of compounds containing aldehyde groups during cultivation with NR latex gloves. Detailed analysis by means of scanning electron microscopy yielded further evidence for the two different microbial strategies and clarified the colonization efficiency. Thereby, strains VH2, Kb2, and NF4 directly adhered to and merged into the rubber material, while strain W2b produced mycelial corridors, especially on the surface of IR. Fourier transform infrared spectroscopy comprising the attenuated total reflectance technique was applied on NR latex gloves overgrown by cells of the Gordonia strains, which were the strongest rubber decomposers. Spectra demonstrated the decrease in number of cis-1,4 double bonds, the formation of carbonyl groups, and the change of the overall chemical environment, indicating that an oxidative attack at the double bond is the first metabolic step of the biodegradation process.  相似文献   

9.
PHA-rubber blends: synthesis, characterization and biodegradation   总被引:1,自引:0,他引:1  
Medium chain length polyhydroxyalkanoates (mcl-PHA) and different rubbers; namely natural rubber, nitrile rubber and butadiene rubber were blended at room temperature using solution blending technique. Blends constituted 5%, 10% and 15% of mcl-PHA in different rubbers. Thermogravimetric analysis of mcl-PHA showed the melting temperature of the polymer around 50 degrees C. Thermal properties of the synthesized blend were studied by Differential Scanning Calorimetry which confirmed effective blending between the polymers. Blending of mcl-PHA with natural rubber led to the synthesis of a different polymer having the melting point of 90 degrees C. Degradation studies of the blends were carried out using a soil isolate, Pseudomonas sp. 202 for 30 days. Extracellular protein concentration as well as OD660 due to the growth of Pseudomonas sp. 202 was studied. The degradation of blended plastic material, as evidenced by % weight loss after degradation and increase in the growth of organism correlated with the amount of mcl-PHA present in the sample. Growth of Pseudomonas sp. 202 resulted in 14.63%, 16.12% and 3.84% weight loss of PHA:rubber blends (natural, nitrile and butadiene rubber). Scanning electron microscopic studies after 30 days of incubation further confirmed biodegradation of the films.  相似文献   

10.
Rubber-degrading bacteria were screened for the production of clearing zones around their colonies on latex overlay agar plates. Novel three bacteria, Streptomyces sp. strain LCIC4, Actinoplanes sp. strain OR16, and Methylibium sp. strain NS21, were isolated. To the best of our knowledge, this is the first report on the isolation of a Gram-negative rubber-degrading bacterium other than γ-proteobacteria. Gel permeation chromatography analysis revealed that these strains degraded poly(cis-1,4-isoprene) to low-molecular-weight products. The occurrence of aldehyde groups in the degradation products by NS21 was suggested by staining with Schiff's reagent and 1H-nuclear magnetic resonance spectroscopy. The lcp gene of LCIC4, which showed 99% amino acid sequence identity with that of Streptomyces sp. strain K30, was cloned, and contained a putative twin-arginine motif at its N terminus. It is located next to oxiB, which is estimated to be responsible for oxidation of degradation intermediate of rubber in K30. Southern hybridization analysis using LCIC4 lcp probe revealed the presence of a lcp-homolog in OR16. These results suggest that the lcp-homologs are involved in rubber degradation in LCIC4 and OR16.  相似文献   

11.
Several actinomycetes isolated from nature were able to use both natural rubber (NR) and synthetic cis-1,4-polyisoprene rubber (IR) as a sole source of carbon. According to their degradation behavior, they were divided into two groups. Representatives of the first group grew only in direct contact to the rubber substrate and led to considerable disintegration of the material during cultivation. The second group consisted of weaker rubber decomposers that did not grow adhesively, as indicated by the formation of clear zones (translucent halos) around bacterial colonies after cultivation on NR dispersed in mineral agar. Taxonomic analysis of four selected strains based on 16S rRNA similarity examinations revealed two Gordonia sp. strains, VH2 and Kb2, and one Mycobacterium fortuitum strain, NF4, belonging to the first group as well as one Micromonospora aurantiaca strain, W2b, belonging to the second group. Schiff's reagent staining tests performed for each of the strains indicated colonization of the rubber surface, formation of a bacterial biofilm, and occurrence of compounds containing aldehyde groups during cultivation with NR latex gloves. Detailed analysis by means of scanning electron microscopy yielded further evidence for the two different microbial strategies and clarified the colonization efficiency. Thereby, strains VH2, Kb2, and NF4 directly adhered to and merged into the rubber material, while strain W2b produced mycelial corridors, especially on the surface of IR. Fourier transform infrared spectroscopy comprising the attenuated total reflectance technique was applied on NR latex gloves overgrown by cells of the Gordonia strains, which were the strongest rubber decomposers. Spectra demonstrated the decrease in number of cis-1,4 double bonds, the formation of carbonyl groups, and the change of the overall chemical environment, indicating that an oxidative attack at the double bond is the first metabolic step of the biodegradation process.  相似文献   

12.
An extracellular protein with strong absorption at 406 nm was purified from cell-free culture fluid of latex-grown Xanthomonas sp. strain 35Y. This protein was identical to the gene product of a recently characterized gene cloned from Xanthomonas sp., as revealed by determination of m/z values and sequencing of selected isolated peptides obtained after trypsin fingerprint analysis. The purified protein degraded both natural rubber latex and chemosynthetic poly(cis-1,4-isoprene) in vitro by oxidative cleavage of the double bonds of poly(cis-1,4-isoprene). 12-Oxo-4,8-dimethyltrideca-4,8-diene-1-al (m/z 236) was identified and unequivocally characterized as the major cleavage product, and there was a homologous series of minor metabolites that differed from the major degradation product only in the number of repetitive isoprene units between terminal functions, CHO-CH2— and —CH2-COCH3. An in vitro enzyme assay for oxidative rubber degradation was developed based on high-performance liquid chromatography analysis and spectroscopic detection of product carbonyl functions after derivatization with dinitrophenylhydrazone. Enzymatic cleavage of rubber by the purified protein was strictly dependent on the presence of oxygen; it did not require addition of any soluble cofactors or metal ions and was optimal around pH 7.0 at 40°C. Carbon monoxide and cyanide inhibited the reaction; addition of catalase had no effect, and peroxidase activity could not be detected. The purified protein was specific for natural rubber latex and chemosynthetic poly(cis-1,4-isoprene). Analysis of the amino acid sequence deduced from the cloned gene (roxA [rubber oxygenase]) revealed the presence of two heme-binding motifs (CXXCH) for covalent attachment of heme to the protein. Spectroscopic analysis confirmed the presence of heme, and approximately 2 mol of heme per mol of RoxA was found.  相似文献   

13.
Rubber-degrading activity was found in the extracellular culture medium of Xanthomonas sp. strain 35Y which was grown on natural rubber latex. Natural rubber in the latex state was degraded by the crude enzyme, and two fractions were separately observed by gel permeation chromatography of the reaction products. One fraction was of higher molecular weight (HMW) with a very wide MW distribution from 10 to 10, and the other fraction was of lower molecular weight (LMW) with a MW of a few hundred. H-nuclear magnetic resonance spectra of the partially purified fractions were those expected of cis-1,4-polyisoprene mixtures with the structure OHC-CH(2)-(-CH(2)-C(-CH(3)) = CH-CH(2)-)(n)-CH(2)-C(=O)-CH(3), with average values of n of about 113 and 2 for HMW and LMW fractions, respectively. The LMW fraction consisted mostly of one component in gas-liquid chromatography as well as in gel permeation chromatography, and the main component was identified as 12-oxo-4,8-dimethyl trideca-4,8-diene-1-al (acetonyl diprenyl acetoaldehyde, A(l)P(2)A(t)) by C-nuclear magnetic resonance and gas chromatography-mass spectra. Not only the latices of natural and synthetic isoprene rubber, but also some kinds of low-MW polyisoprene compounds of cis-1,4 type, were degraded by the crude enzyme. The rubber-degrading reaction was found to be at least partly oxygenase catalyzed from the incorporation of O into A(l)P(2)A(t) under an O(2) atmosphere.  相似文献   

14.
Rubber-degrading bacteria were screened for the production of clearing zones around their colonies on latex overlay agar plates. Novel three bacteria, Streptomyces sp. strain LCIC4, Actinoplanes sp. strain OR16, and Methylibium sp. strain NS21, were isolated. To the best of our knowledge, this is the first report on the isolation of a Gram-negative rubber-degrading bacterium other than γ-proteobacteria. Gel permeation chromatography analysis revealed that these strains degraded poly(cis-1,4-isoprene) to low-molecular-weight products. The occurrence of aldehyde groups in the degradation products by NS21 was suggested by staining with Schiff's reagent and 1H-nuclear magnetic resonance spectroscopy. The lcp gene of LCIC4, which showed 99% amino acid sequence identity with that of Streptomyces sp. strain K30, was cloned, and contained a putative twin-arginine motif at its N terminus. It is located next to oxiB, which is estimated to be responsible for oxidation of degradation intermediate of rubber in K30. Southern hybridization analysis using LCIC4 lcp probe revealed the presence of a lcp-homolog in OR16. These results suggest that the lcp-homologs are involved in rubber degradation in LCIC4 and OR16.  相似文献   

15.
16.
Colonization and degradation of rubber pieces by Nocardia sp.   总被引:1,自引:0,他引:1  
The growth of a Nocardia sp. occurs essentially on the insoluble rubber substrate and the cells are tightly bound to the rubber in the initial stage of the growth in spite of vigorous stirring of the cultures. The colonization of rubber pieces was followed by staining with Schiff reagent, and it was revealed that not only the thickness of rubber pieces, but also their length and width greatly influenced microbial colonization and degradation of natural rubber products. Among rubber pieces of various shapes, long strips were most rapidly covered by many microbial colonies and experienced the highest rate of rubber degradation. The rate of degradation (expressed by % weight loss) of the long strips of rubber was a linear function of surface area per unit weight of rubber. Thin and wide films of rubber were also rapidly colonized and degraded, while the colonization and degradation of short and narrow pieces were substantially slower and less extensive.  相似文献   

17.
18.
Acinetobacter johnsonii A2 isolated from the natural community of Laguna Azul (Andean Mountains at 4,560 m above sea level), Serratia marcescens MF42, Pseudomonas sp. strain MF8 isolated from the planktonic community, and Cytophaga sp. strain MF7 isolated from the benthic community from Laguna Pozuelos (Andean Puna at 3,600 m above sea level) were subjected to UV-B (3,931 J m-2) irradiation. In addition, a marine Pseudomonas putida strain, 2IDINH, and a second Acinetobacter johnsonii strain, ATCC 17909, were used as external controls. Resistance to UV-B and kinetic rates of light-dependent (UV-A [315 to 400 nm] and cool white light [400 to 700 nm]) and -independent reactivation following exposure were determined by measuring the survival (expressed as CFU) and accumulation of cyclobutane pyrimidine dimers (CPD). Significant differences in survival after UV-B irradiation were observed: Acinetobacter johnsonii A2, 48%; Acinetobacter johnsonii ATCC 17909, 20%; Pseudomonas sp. strain MF8, 40%; marine Pseudomonas putida strain 2IDINH, 12%; Cytophaga sp. strain MF7, 20%; and Serratia marcescens, 21%. Most bacteria exhibited little DNA damage (between 40 and 80 CPD/Mb), except for the benthic isolate Cytophaga sp. strain MF7 (400 CPD/Mb) and Acinetobacter johnsonii ATCC 17909 (160 CPD/Mb). The recovery strategies through dark and light repair were different in all strains. The most efficient in recovering were both Acinetobacter johnsonii A2 and Cytophaga sp. strain MF7; Serratia marcescens MF42 showed intermediate recovery, and in both Pseudomonas strains, recovery was essentially zero. The UV-B responses and recovery abilities of the different bacteria were consistent with the irradiation levels in their native environment.  相似文献   

19.
The enrichment and isolation of thermophilic bacteria capable of rubber [poly(cis-1,4-isoprene)] degradation revealed eight different strains exhibiting both currently known strategies used by rubber-degrading mesophilic bacteria. Taxonomic characterization of these isolates by 16S rRNA gene sequence analysis demonstrated closest relationships to Actinomadura nitritigenes, Nocardia farcinica, and Thermomonospora curvata. While strains related to N. farcinica exhibited adhesive growth as described for mycolic acid-containing actinomycetes belonging to the genus Gordonia, strains related to A. nitritigenes and T. curvata formed translucent halos on natural rubber latex agar as described for several mycelium-forming actinomycetes. For all strains, optimum growth rates were observed at 50 degrees C. The capability of rubber degradation was confirmed by mineralization experiments and by gel permeation chromatography (GPC). Intermediates resulting from early degradation steps were purified by preparative GPC, and their analysis by infrared spectroscopy revealed the occurrence of carbonyl carbon atoms. Staining with Schiff's reagent also revealed the presence of aldehyde groups in the intermediates. Bifunctional isoprenoid species terminated with a keto and aldehyde function were found by matrix-assisted laser desorption ionization-time-of-flight and electrospray ionization mass spectrometry analyses. Evidence was obtained that biodegradation of poly(cis-1,4-isoprene) is initiated by endocleavage, rather than by exocleavage. A gene (lcp) coding for a protein with high homology to Lcp (latex-clearing protein) from Streptomyces sp. strain K30 was identified in Nocardia farcinica E1. Streptomyces lividans TK23 expressing this Lcp homologue was able to cleave synthetic poly(cis-1,4-isoprene), confirming its involvement in initial polymer cleavage.  相似文献   

20.
A Gram-negative bacterium, strain AL98, was isolated from foul water inside of a deteriorated car tire on a farmer's field in Münster, Germany. The strain was able to considerably disintegrate natural rubber (NR), either in the raw state as NR latex concentrate or in the vulcanized state as NR latex glove, as well as raw synthetic cis-1,4-polyisoprene (IR). Determination of carbon dioxide evolution and living cell number during batch cultivation with each of the materials as sole source of carbon, revealed mineralization of the rubber polymer during biomass increase. Surface investigation by scanning electron microscopy gave evidence for an adhesive growth behavior of the strain proceeding by colonizing the rubber surface, merging into the rubber and forming a biofilm prior to disintegration of the material. Schiff's reagent staining performed with NR latex gloves indicated production and accumulation of aldehyde groups during colonization. The solid glove substrate disappeared completely after a prolonged cultivation period as a result of continuous degradation. Taxonomic analyses of the strain, which were also based on similarity examination of the complete 16S rRNA gene, revealed classification of strain AL98 as a strain of Pseudomonas aeruginosa. This is the first report about the isolation of a Gram-negative bacterium exhibiting strong rubber decomposing properties.  相似文献   

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