Degradation of polyisoprene rubber by newly isolated bacillus sp. AF-666 from soil

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.     

16S rRNA gene sequences analysis of Ficus elastica rubber latex degrading thermophilic Bacillus strain ASU7 isolated from Egypt

A thermophilic Bacillus strain ASU7 was isolated from soil sample collected from Assiut governorate in Upper Egypt on latex rubber-containing medium at 45?°C. Genetically, the 16S bacterial ribosomal RNA gene of the strain ASU7 was amplified by the polymerase chain reaction (PCR) and sequenced. The sequence of the PCR product was compared with known 16S rRNA gene sequences in the GenBank database. Based on phylogenetic analyses, strain ASU7 was identified as Bacillus amyloliquefaciens. The strain was able to utilize Ficus elastica rubber latex as a sole source for carbon and energy. The ability for degradation was determined by measuring the increase in protein content of bacterium (mg/g dry wt), reduction in molecular weight (g/mol), and inherent viscosity (dl/g) of the latex. Moreover, the degradation was also confirmed by observing the growth of bacterium and formation of aldehyde or keto group using scanning electron microscopy (SEM) and shiff's reagent, respectively.     

Identification and characterization of genes from Streptomyces sp. strain K30 responsible for clear zone formation on natural rubber latex and poly(cis-1,4-isoprene) rubber degradation

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.     

Degradation of Ficus elastica rubber latex by Aspergillus terreus,Aspergillus flavus and Myceliophthora thermophila

Since isolates recovered on medium containing-Ficus elastica latex showed good growth on the respective natural rubber than those recovered on Euphorbia pulcherrima or Ficus nitida, 16 of these isolates were selected for further growth experiments on natural rubber to determine their protein content as well as rubber viscosity. Of these, the mesophilic strains Aspergillus terreus AUMC 4682, Aspergillus flavus AUMC 4795 and the thermophilic strain Myceliophthora thermophila AUMC 4653 showed low rubber viscosity and high mycelia protein content indicating high biodegradation ability of rubber. The strains were subjected for further analysis. They showed high ability to degrade poly (cis-1, 4-isoprene) rubber fig. The ability was also determined by measuring the increase in protein content of each fungus (mg g⁻¹ dry wt), reduction in molecular weight (g mol⁻¹) and inherent viscosity (dl g⁻¹). Moreover the degradation was characterized by determining aldehyde or keto group by Schiff reagent and observing the growth using scanning electron microscopy (SEM).     

Effect of pretreatment of rubber material on its biodegradability by various rubber degrading bacteria

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.     

Biodegradation of natural and synthetic rubbers: A review

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.     

Extraction and characterization of a natural rubber from Euphorbia characias latex

A natural rubber was identified and characterized for the first time in the latex of the perennial Mediterranean shrub Euphorbia characias. Four different methods, i.e., acetone, acetic acid, trichloroacetic acid, and Triton® X‐100, followed by successive treatments with cyclohexane/ethanol, were employed to extract the natural rubber. The rubber content was shown to be 14% (w/v) of the E. characias latex, a low content compared with that of Hevea brasiliensis (30–35%) but a similar content to other rubber producing plants. E. characias rubber showed a molecular weight of 93,000 with a M_w/M_n of 2.9. ¹H NMR, ¹³C NMR, and FTIR analysis revealed the characteristic of the cis‐1,4‐polyisoprene typical of natural rubber. These results provided novel insight into latex components and will ultimately benefit the broader understanding of E. characias latex composition. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 589–594, 2012.     

Degradation of latex and of natural rubber by Streptomyces strain La 7

Streptomyces strain La 7 was isolated from the banquete of a city high way in Karlsruhe. According to partial 16S rRNA gene sequencing it was identical with Streptomyces albogriseolus and Streptomyces viridodiastaticus. DNA-DNA-similarity studies revealed 80.3-82.4% similarity between each of two of the three strains. Although phylogenetically closely related, Streptomyces strain La 7 differed from the two reference strains by morphological as well as physiological features and might represent a new species aside of S. albogriseolus and S. viridodiastaticus. The new Streptomyces strain La 7 was grown in a medium containing a latex emulsion or squares of natural rubber gloves as the only carbon source. On agar plates with a latex overlay agar, translucent halo formation around the colonies was observed. The unvulcanized latex was metabolized and the carbon from the isoprene units was apparently used for cell growth. In shake cultures with unlimited oxygen supply, during 60 days of incubation, 140 mg of the 175 mg totally emulgated latex were degraded exponentially. In sterile control flasks about 3% of the initial amount of latex could not be recovered after incubation on a shaker, presumably due to photochemical transformation. During static incubation of sterile medium, the latex formed a sticky layer at the surface of the medium and on the glass walls and recovery of the material was more difficult. Estimation of the protein content of cells from total nitrogen resulted in about 50% of the degraded latex being incorporated into cells, if a standard cell composition was assumed. Direct protein analysis according to Bradford (1976) gave much lower estimates, presumably due to a low content of aromatic amino acids. Stripes of natural rubber were degraded by Streptomyces strain La 7 during 70 days to an extent of about 30%. Scanning electron microscopy demonstrated, that hyphes of Streptomyces strain La 7 colonized and penetrated the latex surface with a concomitant deterioration of the latex material.     

Degradation of vulcanized and nonvulcanized polyisoprene rubbers by lipid peroxidation catalyzed by oxidative enzymes and transition metals

Despite numerous reports concerning the biodegradation of rubber materials, there has been no report of rubber degradation by fully characterized enzymes. In the present paper, we presented a new method to decompose nonvulcanized and vulcanized polyisoprene rubbers by controlling the free radical chain reactions of lipids using oxidative enzymes, manganese peroxidase (MnP), laccase (Lac), and horseradish peroxidase (HRP). Nonvulcanized synthetic polyisoprene (IR) was degraded by the free radicals from unsaturated fatty acids produced by MnP, HRP, and a combination of Lac/1-hydroxybenzotriazole. In contrast, lipoxygenase caused no apparent degradation. Degradation of IR was also observed in lipid peroxidation initiated by the Fenton reaction (FR) and Mn(III), an oxidation product produced by MnP. Vulcanized polyisoprene rubber sheets were degraded by the lipid peroxidation initiated by HRP, MnP, Mn(III), and FR. Pyrolysis GC-MS analysis demonstrated that the lipid peroxidation liberated isoprenoid fragments from the vulcanized rubbers.     

Rhizobacter gummiphilus NS21 has two rubber oxygenases (RoxA and RoxB) acting synergistically in rubber utilisation

Biodegradation of poly(cis-1,4-isoprene) (rubber) by Gram-negative bacteria has been investigated on the enzymatic level only in Steroidobacter cummioxidans 35Y (previously Xanthomonas sp. 35Y). This species produces two kinds of rubber oxygenases, RoxA_35Y and RoxB_35Y, one of which (RoxB_35Y) cleaves polyisoprene to a mixture of C₂₀- and higher oligoisoprenoids while the other (RoxA_35Y) cleaves polyisoprene and RoxB_35Y-derived oligoisoprenoids to the C₁₅-oligoisoprenoid 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). ODTD can be taken up by S. cummioxidans and used as a carbon source. Gram-positive rubber-degrading bacteria employ another type of rubber oxygenase, latex clearing protein (Lcp), for the initial oxidative attack of the polyisoprene molecule. In this contribution, we examined which type of rubber oxygenase is present in the only other well-documented Gram-negative rubber-degrading species, Rhizobacter gummiphilus NS21. No homologue for an Lcp protein but homologues for a putative RoxA and a RoxB protein (the latter identical to a previously postulated LatA-denominated rubber cleaving enzyme) were identified in the genome of strain NS21. The roxA_NS21 and roxB_NS21 genes were separately expressed in a ∆roxA_35Y/∆roxB_35Y background of S. cummioxidans 35Y and restored the ability of the mutant to produce oligoisoprenoids. The RoxA_NS21 and RoxB_NS21 proteins were each purified and biochemically characterised. The results—in combination with in silico analysis of databases—indicate that Gram-negative rubber-degrading bacteria generally utilise two synergistically acting rubber oxygenases (RoxA/RoxB) for efficient cleavage of polyisoprene to ODTD.

     

Construction and intergeneric conjugative transfer of a pSG5-based cosmid vector from Escherichia coli to the polyisoprene rubber degrading strain Micromonospora aurantiaca W2b

A non-rubber degrading mutant of the polyisoprene rubber degrading bacterium Micromonospora aurantiaca W2b lacking the capability to form halos on latex overlay agar plates was isolated after N-methyl-N-nitro-N-nitrosoguanidine mutagenesis. A 10.3-kb shuttle cosmid vector pGM446 was constructed from the Streptomyces cloning vectors pGM160 and pOJ446. This vector was transferred by conjugation from Escherichia coli to M. aurantiaca W2b. The frequency of formation of exconjugants with pGM446 was 3.6 x 10(-3). This vector could be useful for shotgun cloning of genes into the non-rubber degrading mutant L1 from M. aurantiaca W2b.     

一株溴氰菊酯降解菌的分离鉴定及其降解条件优化

【背景】拟除虫菊酯类农药的降解已成为食品安全和环境卫生领域的研究热点,而生物降解被认为是一种绿色高效的解决方法。【目的】从长期受拟除虫菊酯类农药污染的草莓根系土壤分离一株溴氰菊酯(deltamethrin,DM)降解菌,并优化其培养基及降解条件,从而提高DM降解菌的降解效率。【方法】采用富集驯化、分离纯化法筛选DM降解菌,通过形态学和生理生化特征,以及16S rRNA基因序列分析进行鉴定。通过Plackett-Burman因素筛选试验、最陡爬坡试验和Box-Behnken试验优化菌株降解条件。【结果】筛选获得一株DM降解菌LH-1-1,96h对DM(100mg/L)的降解率为53.43%,经鉴定为琼氏不动杆菌(Acinetobacter junii);通过优化后,在DM浓度75mg/L、胰蛋白胨3 g/L、pH值6.8、硫酸铵1.5 g/L、氯化铁0.01 g/L、接种量为5%(体积比)、菌龄12 h、培养温度30℃条件下,菌株LH-1-1对DM降解率达82.36%,较未优化前提高了28.93%。【结论】A. junii LH-1-1具有较高的DM降解能力,该菌可为生物修复受DM或拟除虫菊酯类农药污染的环境提供优良的微生物资源。     

Isolation and characterization of Streptomyces, Actinoplanes, and Methylibium strains that are involved in degradation of natural rubber and synthetic poly(cis-1,4-isoprene)

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.     

A gram-negative bacterium, identified as Pseudomonas aeruginosa AL98, is a potent degrader of natural rubber and synthetic cis-1, 4-polyisoprene

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.     

Taxonomic characterization of two rubber degrading bacteria belonging to the species Gordonia polyisoprenivorans and analysis of hyper variable regions of 16S rDNA sequences

Two cis-1,4-polyisoprene (isoprene rubber) degrading bacteria, strains VH2 and Y2K, were identified as strains of the species Gordonia polyisoprenivorans belonging to the Corynebacterineae, a suborder of the order Actinomycetales. Both showed characteristic growth and degradation of isoprene rubber as described previously for the type strain of G. polyisoprenivorans Kd2 (DSM 44302(T)). For strain VH2 the chemotaxonomic properties were investigated, and DNA-DNA hybridization experiments with the type strain revealed the affiliation to the species G. polyisoprenivorans. The comparison of the 16S rDNA sequences, and especially hyper variable regions of these, led to the classification of strain Y2K to the same species. At present, the species G. polyisoprenivorans comprises three different isolates which share the ability to degrade isoprene rubber potently but which were obtained from different geographic regions.     

Oligo(cis-1,4-isoprene) aldehyde-oxidizing dehydrogenases of the rubber-degrading bacterium Gordonia polyisoprenivorans VH2

The actinomycete Gordonia polyisoprenivorans strain VH2 is well-known for its ability to efficiently degrade and catabolize natural rubber [poly(cis-1,4-isoprene)]. Recently, a pathway for the catabolism of rubber by strain VH2 was postulated based on genomic data and the analysis of mutants (Hiessl et al. in Appl Environ Microbiol 78:2874–2887, 2012). To further elucidate the degradation pathway of poly(cis-1,4-isoprene), 2-dimensional-polyacrylamide gel electrophoresis was performed. The analysis of the identified protein spots by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry confirmed the postulated intracellular pathway suggesting a degradation of rubber via β-oxidation. In addition, other valuable information on rubber catabolism of G. polyisoprenivorans strain VH2 (e.g. oxidative stress response) was provided. Identified proteins, which were more abundant in cells grown with rubber than in cells grown with propionate, implied a putative long-chain acyl-CoA-dehydrogenase, a 3-ketoacyl-CoA-thiolase, and an aldehyde dehydrogenase. The amino acid sequence of the latter showed a high similarity towards geranial dehydrogenases. The expression of the corresponding gene was upregulated > 10-fold under poly(cis-1,4-isoprene)-degrading conditions. The putative geranial dehydrogenase and a homolog were purified and used for enzyme assays. Deletion mutants for five aldehyde dehydrogenases were generated, and growth with poly(cis-1,4-isoprene) was investigated. While none of the mutants had an altered phenotype regarding growth with poly(cis-1,4-isoprene) as sole carbon and energy source, purified aldehyde dehydrogenases were able to catalyze the oxidation of oligoisoprene aldehydes indicating an involvement in rubber degradation.

     

Biodegradation of diazinon by Serratia marcescens DI101 and its use in bioremediation of contaminated environment

Four diazinon-degrading bacteria were isolated from agricultural soil by using an enrichment technique. The biochemical analysis and molecular method including RFLP indicated that these isolates were identical, and one strain designated DI101 was selected for further study. Phylogenetic analysis based on 16S rDNA sequencing indicated that the strain DI101 clearly belongs to the Serratia marcescens group. The ability of the strain to utilize diazinon as a source of carbon and phosphorus was investigated under different culture conditions. The DI101 strain was able to completely degrade 50 mg/l diazinon in MSM within 11 days with a degradation rate of 0.226 day-1. The inoculation of sterilized soil treated with 100 mg/kg of diazinon with 10(6) CFU/g DI101 resulted in a faster degradation rate than was recorded in non-sterilized soil. The diazinon degradation rate by DI101 was efficient at temperatures from 25 to 30degrees C and at pHs from 7.0 to 8.0. The degradation rate of diazinon was not affected by the absence of a phosphorus supplement, and addition of other carbon sources (glucose or succinate) resulted in the slowing down of the degradation rate. The maximum degradation rate (Vmax) of diazinon was 0.292 day-1 and its saturation constant (Ks) was 11 mg/l, as determined by a Michaelis-Menten curve. The strain was able to degrade diethylthiophosphate-containing organophosphates such as chlorpyrifos, coumaphos, parathion, and isazofos when provided as a source of carbon and phosphorus, but not ethoprophos, cadusafos, and fenamiphos. These results propose useful information for the potential application of the DI101 strain in bioremediation of pesticide-contaminated environments.     

Role of lutoid membrane transport and protein synthesis in the regeneration mechanism of latex in different <Emphasis Type="Italic">Hevea</Emphasis> clones

Natural rubber (cis-1,4 polyisoprene) is synthesised in the milky cytoplasm, the latex, of specialized cells called laticifers in the bark tissues of the rubber tree (Hevea brasiliensis). Regeneration mechanism of latex after each tapping (controlled wounding of the bark) was studied in relation to lutoid membrane enzymes and protein synthesis in twelve rubber clones with varying yield potentials during the peak rubber yielding season. High activity of membrane enzymes and better availability of biochemical energy [ATP] were observed in clones viz; RRII 105, RRIM 600, PB 260, RRII 422 and RRII 430. The highest protein biosynthetic capacity was noticed in clone PB 260 and RRIM 600. However, high ATP content, increased invertase activity and protein biosynthesis were observed in the medium yielding clone GT1 compared to clones with low rubber yield potential. Very low sugar content and increased invertase activity in the latex of clone PB 260 indicated intense latex metabolism with high protein turnover that implies fast recouping of the cellular metabolites lost during latex harvesting. Clone PB 217 was characterized by very high sucrose and low ATP concentration and ATPase activity in latex indicating slow metabolism and hence be suitable for inducing latex metabolism using ethylene stimulant. Low rubber yielding clones such as RRII 33 and RRII 38 were consistently recorded a high sucrose content but very low activity of membrane enzymes, reduced ATP concentration and low protein biosynthesis in latex. Among the recently released modern clones (RRII 400 series), latex regeneration capacity was higher in RRII 422 and RRII 430. The significance of lutoid membrane transport and protein synthesis is discussed in relation to general latex metabolism of these rubber clones. The outcome of this study would be helpful to design suitable latex harvesting systems and yield stimulation methods for optimizing latex production in each clone based on metabolic profiling.     

Purification of a prenyltransferase that elongates cis-polyisoprene rubber from the latex of Hevea brasiliensis

We have purified "rubber transferase" from latex of the commercial rubber tree Hevea brasiliensis and find that it is a dimer with a monomeric molecular mass of 38,000 Da, requires Mg2+, and is stabilized by thiols in agreement with studies of a partially purified preparation previously described (Archer, B. L., and Cockbain, E. G. (1969) Methods Enzymol. 15, 476-480). Greater than 90% of the [1-14C]isopentenyl pyrophosphate which is incorporated into deproteinated rubber particles by the purified prenyltransferase is added to high molecular mass polyisoprene (greater than 20,000 Da). Purified prenyltransferase and deproteinated rubber particles reconstitute 40-60% of the biosynthetic activity of whole latex in samples matched for rubber content. Incorporation is linear with added rubber particles up to at least 10 mg/ml rubber or 20 microM rubber molecules (based on a number average molecular mass of 500,000 Da). Prenyltransferase concentrations estimated in whole latex (0.37% or 160 nM) are sufficient to saturate all elongation sites in whole latex, and addition of purified prenyltransferase does not increase [1-14C]isopentenyl pyrophosphate incorporation. Deproteinated rubber particles can be titrated with the pure enzyme (Kd = 9 nM) demonstrating that the fraction of rubber molecules available for addition is low (approximately 0.01%). An estimated 7,000 isoprene units are added per complex at a rate of 1/s in a typical assay. Hevea prenyltransferase catalyzes the formation of cis-isoprene in the presence of rubber particles. However, in the absence of rubber particles and in the presence of dimethylallyl pyrophosphate, the purified prenyltransferase catalyzes the formation of geranyl pyrophosphate and all trans-farnesyl pyrophosphate as demonstrated by thin layer chromatography, gas chromatography, and molecular exclusion chromatography.     

Rubber-degrading enzyme from a bacterial culture

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.