APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2005, p. 2803–2812
`0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.6.2803–2812.2005
`Copyright © 2005, American Society for Microbiology. All Rights Reserved.
`
`Vol. 71, No. 6
`
`MINIREVIEWS
`
`Biodegradation of Natural Rubber and Related Compounds:
`Recent Insights into a Hardly Understood
`Catabolic Capability of Microorganisms
`Karsten Rose and Alexander Steinbu¨chel*
`Institut fu¨r Molekulare Mikrobiologie und Biotechnologie, Westfa¨lische Wilhelms-Universita¨t Mu¨nster,
`Corrensstrasse 3, D-48149 Mu¨nster, Germany
`
`Natural rubber latex is produced by over 2,000 plant species,
`and its main constituent is poly(cis-1,4-isoprene), a highly un-
`saturated hydrocarbon. Since 1914 there have been efforts to
`investigate microbial rubber degradation; however, only re-
`cently have the first proteins involved in this process been
`identified and characterized and have the corresponding genes
`been cloned. Analyses of the degradation products of natural
`and synthetic rubbers isolated from various bacterial cultures
`indicated without exception that there was oxidative cleavage
`of the double bond in the polymer backbone. A similar deg-
`radation mechanism was postulated for the cleavage of
`squalene, which is a triterpene intermediate and precursor of
`steroids and triterpenoids. Aldehyde and/or carbonyl groups
`were detected in most of the analyzed degradation products
`isolated from cultures of various rubber-degrading strains. The
`transient formation of intermediate degradation products with
`molecular masses of about 104 Da from poly(cis-1,4 isoprene)
`having a molecular mass of about 106 Da by nearly all rubber-
`degrading bacteria investigated without detection of other in-
`termediates requires an explanation. Knowledge of rubber deg-
`radation at the protein and gene levels and detailed analyses of
`detectable degradation products should result in a detailed un-
`derstanding of these obviously new enzymatic reactions.
`
`OCCURRENCE AND CHEMICAL STRUCTURE OF
`NATURAL RUBBER
`The term natural rubber or caoutchouc (from Indian: caa ⫽
`tears; ochu ⫽ tree; cahuchu ⫽ weeping tree) refers to a coag-
`ulated or precipitated product obtained from latex of rubber
`plants (Hevea brasiliensis), which forms nonlinked but partially
`vulcanizable polymer chains having molecular masses of about
`106 Da with elastic properties; at higher temperatures natural
`rubber is plastically ductile and useful for production of elas-
`tomers. Latex serves as a clogging material during healing of
`wounds caused by mechanical injury of plants.
`Natural rubber consists of C5H8 units (isoprene), each con-
`taining one double bond in the cis configuration (Fig. 1). How-
`ever, polyisoprene of H. brasiliensis contains in addition two
`
`* Corresponding author. Mailing address: Institut fu¨r Molekulare
`Mikrobiologie und Biotechnologie, Westfa¨lische Wilhelms-Universita¨t
`Mu¨nster, Corrensstrasse 3, D-48149 Mu¨nster, Germany. Phone: 49-
`251-8339821. Fax: 49-251-8338388. E-mail:steinbu@uni-muenster.de.
`
`trans-isoprene units in the terminal region (52). Although ap-
`proximately 2,000 plants synthesize poly(cis-1,4-isoprene), only
`natural rubber of H. brasiliensis (99% of the world market) and
`guayule rubber of Parthenium argentatum (1% of the world
`market) are produced commercially (52). Latex of Hevea
`plants contains about 30% poly(cis-1,4-isoprene) and is har-
`vested by a “tapping” procedure after the bark of the plants is
`notched diagonally, which yields 100 to 200 ml latex resin
`within 3 h. Such “tapping” is usually carried out every 2 to 3
`days, yielding up to 2,500 kg of natural rubber per year per ha.
`In 1998, the world production of natural rubber was about 6.6
`million tons; more than 70% of this rubber was produced in
`only three countries (Thailand, Indonesia, and Malaysia), and
`about 40% was purchased by only three countries (United
`States, China, and Japan). Most of the natural rubber (75%) is
`used for production of automobile tires (33).
`Dehydrated natural rubber of H. brasiliensis contains ap-
`proximately 6% nonpolyisoprene constituents. Depending on
`the clone, seasonal effects, and the state of the soil, the average
`composition of latex is as follows: 25 to 35% (wt/wt) polyiso-
`prene; 1 to 1.8% (wt/wt) protein; 1 to 2% (wt/wt) carbohy-
`drates; 0.4 to 1.1% (wt/wt) neutral lipids; 0.5 to 0.6% (wt/wt)
`polar lipids; 0.4 to 0.6% (wt/wt) inorganic components; 0.4%
`(wt/wt) amino acids, amides, etc.; and 50 to 70% (wt/wt) water
`(51). The polymer is present in 3- to 5-␮m so-called rubber
`particles, which are covered by a layer of proteins and lipids
`(20), which separate the hydrophobic rubber molecules from
`the hydrophilic environment. Because some Hevea proteins
`have allergenic potential, methods were developed to remove
`these proteins. An efficient method involves cleaning the latex by
`centrifugation and employing enzymatic digestion with alkaline
`proteases or papain or treatment with sodium or potassium hy-
`droxide. This allows production of condoms and latex gloves with
`low protein contents (less than 20 ␮g/g of natural rubber).
`Only a few plant species synthesize polyisoprenes in the trans
`configuration (Fig. 1). Chicle (Manikara zapota), gutta-percha
`(Pallaquium gutta), and balata (Manikara bidentata) are typical
`representatives of trans-polyisoprene-synthesizing plants. Gut-
`ta-percha and balata produce trans-polyisoprenes with high
`molecular weights (1.4 ⫻ 105 to 1.7 ⫻ 105). The chicle tree is
`unique, because it produces latex with about equal amounts of
`cis- and trans-polyisoprenes.
`The discovery of the classical vulcanization process by
`Goodyear in 1839 allowed production of materials with im-
`
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`2804
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`R
`
`R
`
`R
`
`Poly(cis-1,4-isoprene) = natural rubber
`
`R
`
`Poly(tran s-1,4-isoprene) = G utta Percha
`
`Squalene
`
`Squalane
`
`beta-Carotene
`
`OH
`
`Citronellol
`FIG. 1. Structural formulas of polyisoprenoids and putative low-
`molecular-weight model substances.
`
`proved properties from natural rubber. The polyisoprene mol-
`ecules are covalently linked by bridges of elemental sulfur at
`the double bonds (13). Alternatively, vulcanization is also
`achieved by employing organic peroxides (32) or radiation
`(51); such vulcanized materials have lower long-term stability
`since the polymer chains are cross-linked solely by carbon
`bonds. Although the first synthetic rubbers were produced at
`the beginning of the last century, only after 1950, after the
`development of stereospecific catalysts, could polyisoprene be
`synthesized in the cis and trans configurations (52). Today it is
`possible to produce synthetic polyisoprene that has physical
`properties similar to those of natural rubber with a purity of 98
`to 99%. However, the stress stability, processability, and other
`parameters of synthetic polyisoprene are still less satisfying
`than those of natural rubber (52).
`
`OCCURRENCE AND CHEMICAL STRUCTURE
`OF SQUALENE
`
`Squalene (2,6,10,15,19,23-hexamethyltetracosa-2,6E,10E,
`14E,18E,22E-hexaene) (Fig. 1) was discovered first in the liver
`of “dogfish” (Squalus acanthias), an organism belonging to the
`
`class Squalidae that was the origin of the name squalene (55).
`Squalene is a natural triterpene which plays an important role
`as a precursor in the biosynthesis of steroids and triterpenoids.
`Biosynthesis of squalene results from a “tail-to-tail” conden-
`sation of two molecules of the sequiterpene farnesylpyrophos-
`phate (16). It occurs, for example, in human sebum and in olive
`oil. In the latter, the squalene content decreases significantly
`only after 6 to 8 months, indicating that the molecule has
`considerable stability (35). Squalene was also identified as an
`essential molecule in anal gland secretions of beavers that keep
`their pelts water repellent (41). Squalene also occurs in many
`microorganisms; e.g., 0.4% (wt/wt) of the cell dry mass of
`Nannocystis exedens is composed of squalene (25).
`
`PROBLEMS AND DIFFICULTIES HAMPERING STUDIES
`OF THE MICROBIAL DEGRADATION OF RUBBER
`
`Several serious difficulties hamper investigation of microbial
`rubber degradation. Rubber biodegradation is a slow process,
`and the growth of bacteria utilizing rubber as a sole carbon
`source is also slow. Therefore, incubation periods extending
`over weeks or even months are required to obtain enough cell
`mass or degradation products of the polymers for further anal-
`ysis. This is particularly true for members of the clear-zone-
`forming group (see below). Periods of 10 to 12 weeks have to
`be considered for Streptomyces coelicolor 1A (8), Thermomono-
`spora curvata E5 (22), or Streptomyces sp. strain K30 (40); the
`only exception is Xanthomonas sp. strain 35Y (54). Although
`members of the non-clear-zone-forming group exhibit slightly
`faster growth, cultivation periods of at least 6 weeks are also
`required for Gordonia westfalica (11), e.g.,
`to determine
`whether a putative mutant is able to grow on the polymer.
`Frequently, newly isolated strains must be used to study
`rubber biodegradation. These isolates are often members of
`poorly characterized taxa, and established genetic tools are not
`applicable. Therefore, for a newly isolated strain of the clear-
`zone-forming bacterium Micromonospora aurantiaca W2b and
`for some representatives of the genus Gordonia, efficient trans-
`formation systems based on conjugation and electroporation
`were established (3, 39). For example, it was shown that the
`origin of replication (oriV) of the native Rhodococcus rhodo-
`chrous plasmid pNC903 permitted replication of this plasmid
`in some Gordonia species. In addition, oriV of the megaplas-
`mid pKB1 from the rubber-degrading bacterium G. westfalica
`Kb1 was used for construction of Escherichia-Gordonia shuttle
`vectors, which were also applicable to other Gordonia species
`and other bacteria (11). In addition, the genome sequence of
`no rubber-degrading bacterium has been determined.
`Additional problems arise from the presence of other natu-
`ral biodegradable compounds in natural rubber and latex (see
`above) or from additives which are required for vulcanization
`or to influence the material properties. To avoid allocation of
`growth or CO2 release to degradation of, e.g., proteins and
`lipids present in the material, growth and mineralization ex-
`periments must be performed carefully. Additives can promote
`(e.g., fillers and stoppers) or inhibit (accelerators, antioxidants,
`and preservation material) biodegradation of rubber material
`(20, 31). The inhibitory effect of antioxidants extracted from
`synthetic polyisoprene, which was prepared for tire production,
`on the growth of G. westfalica was demonstrated by Berekaa et
`
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`VOL. 71, 2005
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`MINIREVIEWS
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`2805
`
`curred in species of the genus Corynebacterium if the cells were
`cultivated in squalene medium supplemented with yeast ex-
`tract, and the metabolites resisted further degradation and
`were excreted into the culture broth.
`The third pathway involves oxygenase-catalyzed cleavage of
`the internal double bonds and leads to geranylacetone and
`5,9,13-trimethyltetradec-4E,8E,12-trienic acid (Fig. 3) (58).
`This pathway is of particular interest with regard to microbial
`rubber cleavage, because all internal double bonds in squalene
`involve carbon atoms that carry a methyl group like that in
`polyisoprene. The hypothetical degradation pathway shown in
`Fig. 3 was postulated for Arthrobacter sp. and for Marinobacter
`squalenivorans (36). Investigations of the latter organism led to
`detection of several metabolites that occur during growth on
`squalene. With regard to these metabolites, oxygenase-cata-
`lyzed cleavage of internal double bonds, oxidation of keto-
`terminal methyl groups, decarboxylation of the resulting keto
`acid, and esterase activity were proposed for squalene degra-
`dation by M. squalenivorans, although no enzymes or genes
`were identified. Microbial epoxidation of alkenes, proposed for
`squalene cleavage by M. squalenivorans, was first demonstrated
`for cells of Pseudomonas aeruginosa when the formation of
`1,2-epoxyoctane from 1-octene was observed (56).
`In contrast to aerobic degradation of squalene, information
`about the anaerobic catabolism of squalene is scarce. Incom-
`plete conversion of squalene by a methanogenic enrichment
`culture was studied by Sawada et al. (44). Several denitrifying
`and squalene-degrading bacteria were recently isolated and
`characterized (9). In a denitrifying Marinobacter species hydra-
`tion of double bonds to tertiary alcohols occurred as the first
`step (Fig. 4), and methyl ketones were formed as products of
`carbon chain cleavage (37). The methyl ketones may be car-
`boxylated, yielding acids, which are then probably metabolized
`via ␤-oxidation and ␤-decarboxylation reactions; asymmetric
`diols have not been detected.
`So far, no enzymes or genes involved in microbial squalene
`degradation have been identified. Only squalene epoxidase has
`been characterized in detail. However, this epoxidase is an
`anabolic enzyme that catalyzes the conversion of squalene to
`(3S)-2,3-oxidosqualene. Together with the cyclization of (3S)-
`2,3-oxidosqualene to sterols, it catalyzes a key step in the con-
`version of acyclic lipids into sterols in plants, fungi, and verte-
`brates (1, 27, 59). Inhibition of squalene epoxidase is an
`important target in the design of therapeutically important
`antifungal agents like terbinafin (1, 12, 43).
`For squalane degradation by Mycobacterium spp., a pathway
`based on carboxylation and deacetylation was proposed (5), as
`such a pathway was also found for the degradation of
`citronellol (17). However, for both molecules cleavage at the
`double-bond positions did not occur. In contrast, ␤-carotene
`cleavage of the double bond by a ␤-carotene 15,15⬘-monoox-
`ygenase occurred at the C-15 position (57); however, this dou-
`ble bond does not involve a carbon atom carrying a methyl
`group like all double bonds in polyisoprene and squalene.
`
`MICROBIAL DEGRADATION OF NATURAL AND
`SYNTHETIC RUBBER
`
`Microbial degradation of natural rubber has been investi-
`gated for 100 years (48) (Table 1). It became obvious that
`
`the terminal methyl groups of
`FIG. 2. Proposed oxidation of
`squalene to squalenedioic acid (pathway A) and hydration of squalene
`to mono- and dihydrated squalene (pathway B). Evidence for these
`pathways was obtained by using Corynebacterium sp. strain SY-79 (47)
`and Corynebacterium sp. strain S-401 (46).
`
`al. (6). It was also shown that extraction of latex gloves with
`organic solvents before incubation enhanced the growth of
`some rubber-degrading strains (6).
`Various difficulties in the study of microbial rubber degra-
`dation could be overcome by the use of low-molecular-weight
`model substances. Molecules like squalene, squalane, ␤-caro-
`tene, or citronellol may be suitable for this purpose (Fig. 1),
`although the chemical structures of all these compounds differ
`from that of natural rubber with regard to the configuration of
`the methyl groups or the existence of double bonds. Oligomers
`exactly matching the chemical structure of natural rubber are
`not available.
`
`MICROBIAL DEGRADATION OF SQUALENE
`AND SQUALANE
`
`Squalene can be regarded with some restrictions as a low-
`molecular-weight model substance to study microbial polyiso-
`prene degradation, although the configuration of the methyl
`groups is trans. Interestingly, all rubber-degrading bacteria
`which do not form clear zones on latex agar (see below) are
`able to metabolize squalene, whereas all clear-zone-forming
`rubber-degrading strains (see below) are unable to use
`squalene as a sole carbon source (unpublished data).
`Examination of the aerobic degradation of squalene re-
`vealed three different metabolic pathways, including (i) oxida-
`tion of the terminal methyl groups that leads to squalenedioic
`acid (Fig. 2) (47) and (ii) hydratation of the double bond that
`leads to tertiary alcohols (Fig. 2) (46). These pathways oc-
`
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`2806
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`MINIREVIEWS
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`APPL. ENVIRON. MICROBIOL.
`
`O
`
`H 2O
`
`O H
`
`O H
`
`*
`
`O
`
`*
`
`Com pound #1
`
`Com pound #2
`
`COH
`
`Dehydrogenase
`
`O
`
`CO OH
`
`*
`
`- CO 2
`
`C O O H
`
`Com pound #4
`
`beta-O xidation
`
`beta-Decarboxylation
`
`C2 units
`
`Cellular m aterial + CO 2
`
`O
`
`O
`
`*
`
`CH 2OH
`
`Com pound #3
`
`C O O H
`
`Com pound #5
`
`Esterase
`
`Com pound #6
`
`FIG. 3. Proposed oxygenase-catalyzed cleavage of squalene and pathways for aerobic metabolism. Evidence for this pathway was obtained by
`using Marinobacter strain 2Asq64 (36). Compound 1, geranylacetone; compound 2, 5,9,13-trimethyltetradeca-4E,8E,12-trienal; compound 3,
`5,9,13-trimethyltetradeca-4E,8E,12-trien-1-ol; compound 4, 5,9-dimethyldeca-4E,8-dienoic acid; compound 5, 5,9,12-trimethyltetradeca-4E,8E,12-
`trienoic acid; compound 6, 5,9,13-trimethyltetradecyl-5,9,13-trimethyltetradecanoate. Detected metabolites are indicated by asterisks.
`
`bacteria, as well as fungi, are capable of degrading rubber and
`that rubber biodegradation is a slow process (14, 19, 21, 23, 34,
`50). The introduction of latex overlay agar plates, which con-
`sisted of a bottom agar layer of mineral salt medium and a
`layer of latex or latex agar on top, for isolation and cultivation
`of
`rubber-degrading microorganisms was an important
`achievement (50). Microorganisms growing on such plates
`formed clear zones around their colonies. When 1,220 different
`bacteria were investigated for the ability to degrade rubber
`employing the latex overlay agar plate technique, 50 clear-
`zone-forming, rubber-degrading strains all belonging to the
`mycelium-forming actinomycetes (Table 1) were identified
`(23). Formation of clear zones was inhibited by addition of
`glucose, indicating that there was regulation of the expression
`of rubber-degrading enzymes. Growth of some of the strains
`on natural rubber led to significant weight loss (10 to 30%,
`
`wt/wt) of the material used and to a decrease in the average
`molecular weight of the polymer from 640,000 to about 25,000.
`One disadvantage of latex overlay agar plates is that not all
`rubber-degrading bacteria can be cultivated in this way, be-
`cause many do not form halos on such plates and because too
`little polyisoprene is locally available to allow formation of
`visible colonies by these organisms. Rubber-degrading bacteria
`were therefore divided into two groups according to the growth
`type and other characteristics (29). With one exception, rep-
`resentatives of the first group belong to the clear-zone-forming
`actinomycetes mentioned above and metabolize the polyiso-
`prene by secretion of one or several enzymes. Most represen-
`tatives of this group show relatively weak growth on natural or
`synthetic rubber. Members of the second group do not form
`halos and do not grow on latex plates; they require direct
`contact with the polymer, and growth on rubber is adhesive in
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`MINIREVIEWS
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`2807
`
`FIG. 4. Proposed pathway for the anaerobic degradation of squalene. Evidence for this pathway was obtained by using Marinobacter sp. strain
`2sq31 (37). Compound 1 (2,6,10,15,19,23-hexamethyltetracosa-2,6E,18E,22E-tetraen-10,15-diol) and compound 2 (7,11,15-trimethylhexadeca-
`6E,10E,14-trien-2-one) were detected in the cultivation broth.
`
`an obligatory sense. Members of this group show relatively
`strong growth on polyisoprene and belong to the Corynebacte-
`rium-Nocardia-Mycobacterium group. Some new rubber-de-
`grading strains belonging to the Corynebacterium-Nocardia-
`
`TABLE 1. Rubber-degrading bacteria mentioned
`
`Bacterium
`
`Type of rubber
`degradationa
`
`Reference
`
`Actinomadura sp.
`Actinomyces candidus
`Actinomyces elastica
`Actinomyces elasticus
`Actinomyces fuscus
`Actinoplanes (three species)
`Dactylosporangium sp.
`Gordonia polyisoprenivorans VH2
`Gordonia polyisoprenivorans Y2K
`Gordonia westfalica Kb1
`Micromonospora aurantiaca W2b
`Micromonospora (five strains)
`Mycobacterium fortuitum NF4
`Nocardia sp.
`Nocardia sp. strain 835A
`Nocardia farcinica S3
`Proactinomyces ruber
`Streptomyces (31 strains)
`Streptomyces sp.
`Streptomyces sp.
`Streptomyces sp. strain La7
`Streptomyces sp. strain K30
`Thermomonospora sp. strain E5
`Xanthomonas sp. strain 35Y
`
`B
`?
`?
`?
`?
`B
`B
`A
`A
`A
`B
`B
`A
`B
`?
`A
`?
`B
`B
`B
`B
`B
`B
`B
`
`23
`34
`48
`34
`48
`23
`23
`29
`2
`30
`29
`23
`29
`23
`53
`22
`34
`23
`28
`38
`19
`40
`22
`54
`
`Mycobacterium group, such as Gordonia polyisoprenivorans
`strains VH2 and Y2K, G. westfalica strain Kb1, and Mycobac-
`terium fortuitum strain NF4, were isolated recently (2, 30) (Ta-
`ble 1). Species of the genus Gordonia very frequently are rub-
`ber degraders (4).
`Biodegradation of vulcanized rubber material is also possi-
`ble, although it is even more difficult due to the interlinkages of
`the poly(cis-1,4-isoprene) chains, which result in reduced water
`absorption and gas permeability of the material (45). Two
`Streptomyces strains were isolated from vulcanized gaskets of
`cement water tubes, which were the cause of 1.5-mm-diameter
`holes in the material after 12 months of incubation (38). Con-
`tinuation of these studies led to development of the so-called
`Leeflang test bath, in which rubber material is examined in a
`steady aquatic stream with regard to its stability against micro-
`bial degradation (28).
`So far, there have been no reports which have definitely dem-
`onstrated biodegradation of poly(trans-1,4-isoprene), the main
`constituent of gutta-percha and balata. Although isolation of sev-
`eral microorganisms capable of destroying cast films of gutta
`extracted from Eucommia was reported by Kupletskaya et al.
`(26), no further details were determined. Intensive attempts in
`our laboratory to enrich and isolate poly(trans-1,4-isoprene)-de-
`grading bacteria or to demonstrate poly(trans-1,4-isoprene) deg-
`radation by known rubber degraders failed.
`
`BIOCHEMICAL ANALYSIS OF RUBBER
`BIODEGRADATION
`
`a A, rubber-degrading bacteria which are unable to grow or form clear zones
`on latex overlay plates B, rubber-degrading bacteria which form clear zones on
`latex overlay agar plates. For the type of rubber degradation see reference 29.
`
`Enzymes involved in rubber biodegradation, particularly en-
`zymes catalyzing cleavage of the rubber backbone, were one of
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`TABLE 2. Degradation products obtained from natural rubber or synthetic polyisoprene after incubation with different bacteria
`
`Strain
`
`Poly(cis-1,4-isoprene)a
`
`Mol wt of degradation
`products
`
`No. of isoprene
`units
`
`Method of
`identificationb
`
`Functional
`groupsc
`
`Reference(s)
`
`Nocardia sp. strain 835A
`
`Xanthomonas sp. strain 35Y
`
`S. coelicolor 1A
`
`S. lividans TK23 pIJ702::lcp
`Nocardia farcinica S3
`Thermomonospora sp. strain E5
`
`NR
`
`NR
`
`NR
`
`IR
`IR
`IR
`
`⬃7,800
`⬃1,300
`⬃7,700
`236
`226
`196
`264
`⬃12,000
`⬃13,000
`⬃13,000
`⬃570
`
`114
`19
`113
`2
`2
`2
`3
`⬃180
`⬃190
`⬃190
`⬃8
`
`NMR, GPC
`NMR, GPC
`NMR, GPC
`NMR, GPC
`NMR, EI
`NMR, EI
`NMR, EI
`GPC
`GPC
`GPC
`GPC
`
`A, K
`A, K
`A, K
`A, K
`K, Ac
`K
`K
`A
`A, C
`A
`K?
`
`53
`
`54
`10, 54
`7
`
`40
`22
`22
`
`a NR, natural rubber; IR, poly(cis-1,4-isoprene).
`b EI, electron ionization mass spectrometry.
`c Functional groups were detected by NMR, infrared spectrometry, or staining with Schiff’s reagent. A, aldehyde group; K, keto group; Ac, acid group; C, carbonyl
`group.
`
`the last obstacles to biopolymer degradation and were un-
`known until recently. Chemical analysis of degradation prod-
`ucts which were transiently formed due to incomplete biodeg-
`radation, analysis of mutants not capable of using natural
`rubber as a carbon source for growth, and finally identification
`of the first genes coding for enzymes catalyzing cleavage of
`polyisoprene revealed some information about the biochemis-
`try of rubber biodegradation.
`Rubber biodegradation by Gordonia sp. The occurrence of
`isoprene oligomers containing aldehyde and ketone groups
`after incubation of latex gloves with G. polyisoprenivorans and
`other bacteria and a decrease in the number of double bonds
`in the polyisoprene chain were demonstrated by staining with
`Schiff’s reagent and using Fourier transform infrared spectros-
`copy with attenuated total reflectance (29). This was consistent
`with oxidative cleavage of the polyisoprene molecules.
`Analyses of plasmid-free mutants of G. westfalica strain Kb1,
`which had lost the ability to grow on natural rubber as a sole
`carbon source, suggested that genes located on a 101-kbp
`megaplasmid comprising 105 open reading frames play an es-
`sential role in rubber degradation (11). In addition, transposon
`mutagenesis of Gordonia species using a transposon based on
`the IS493 element (49) from Streptomyces lividans TK66 re-
`vealed mutants defective in pigmentation, anabolic pathways,
`and also mutants with defects in rubber utilization that are
`currently being investigated in our laboratory (4).
`Rubber biodegradation by Nocardia sp. strain 835A. Nocar-
`dia sp. strain 835A, which exhibited reasonable growth on
`natural and synthetic rubber, was one of the first strains that
`was investigated in detail with regard to rubber biodegrada-
`tion, and it was postulated that there was oxidative cleavage of
`poly(cis-1,4-isoprene) at
`the double-bond position (53).
`Weight losses of the rubber material used of 75 and 100%
`(wt/wt) after 2 and 8 weeks of incubation, respectively, and of
`the latex glove material used of 90% (wt/wt) after 8 weeks were
`obtained. Gel permeation chromatography (GPC) of the chlo-
`roform-soluble fraction of degraded glove material revealed
`two fractions of fragments with molecular masses of 1 ⫻ 104
`and 1.6 ⫻ 103 Da, comprising 114 and 19 isoprene molecules,
`respectively. Both fractions exhibited infrared spectra identical
`to those of aldehyde derivatives of dolichol, and based on these
`
`results together with the results of 1H nuclear magnetic reso-
`nance (1H-NMR) and 13C-NMR studies molecules with alde-
`hyde and keto groups having the following formula were pos-
`tulated: OHC-CH2-[CH2-C(-CH3)ACH-CH2]n-CH2-C(AO)-
`CH3
`(Table 2). Unfortunately,
`investigations of
`rubber
`degradation by this strain were not continued at a biochemical
`or molecular level.
`Rubber biodegradation by Streptomyces sp. Species of the
`genus Streptomyces have frequently been investigated with re-
`gard to rubber biodegradation. The protein content of cultures
`of the clear-zone-forming organism S. coelicolor strain 1A in-
`creased from 240 ␮g/ml to 620 ␮g/ml during incubation of the
`cells with natural rubber latex after 10 weeks of incubation (7).
`GPC analysis of the rubber material remaining after cultiva-
`tion of this strain for 6 weeks with synthetic poly(cis-1,4-iso-
`prene) showed a shift in the molecular mass distribution from
`about 800 kDa to about 2 ⫻ 104 Da. Analysis of the degrada-
`tion products of disintegrated latex gloves revealed several
`compounds, which could be separated by high-performance
`thin-layer chromatography. Three of the compounds isolated
`were identified by one- and two-dimensional 1H-NMR spec-
`troscopy as 2,6-dimethyl-10-oxo-undec-6-enoic acid, 5,6-meth-
`yl-undec-5-ene-2,9-dione, and 5,9-6,10-dimethyl-pentadec-5,9-
`diene-2,13-dione. From this analysis and the occurrence of
`acetonyldiprenylacetoaldehyde (Ap2A), which was first identi-
`fied as a rubber degradation product in a Nocardia sp. strain
`835A culture (see above), the hypothetical pathway for degra-
`dation of poly(cis-1,4-isoprene) shown in Fig. 5 was suggested.
`However, the authors pointed out that the compounds identi-
`fied (compounds 2 to 4) were not necessarily intermediates of
`rubber degradation, so that these metabolites may have been
`dead end products. Unfortunately, UV-induced mutants of
`Streptomyces griseus 1D and S. coelicolor 1A which were not
`able to form clear zones on latex overlay agar plates, which
`showed no increase in protein content in liquid culture con-
`taining latex as a sole carbon source, and which did not pro-
`duce a weight loss in glove material or changes in the molec-
`ular weight of the polymer were not analyzed further (8).
`Streptomyces sp. strain K30 is another strain from which
`UV-induced mutants defective in rubber degradation were
`obtained (40). About 1% of the mutants analyzed exhibited a
`
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`

`VOL. 71, 2005
`
`MINIREVIEWS
`
`2809
`
`R
`
`R
`
`Lcp
`
`RoxA
`
`O
`
`O
`
`O
`
`n
`
`*
`
`O
`
`OxiAB
`
`O
`
`tungstate
`
`n
`
`HSCoA
`
`n
`
`O
`
`S-CoA
`
`AcCoA
`
`O
`HSCoA
`
`n
`
`H
`
`H2O
`
`beta-oxidation
`
`O
`
`OH
`
`O
`
`S-CoA
`
`oxidation
`
`HSCoA
`
`O
`
`O
`
`n
`
`S-CoA
`
`O
`
`O
`
`CO2
`
`HSCoA
`
`omega-oxidation
`
`ProCoA
`
`O
`
`HSCoA
`
`O
`
`O
`
`n
`
`S-CoA
`
`O
`
`HSCoA
`
`OH
`
`n
`
`#
`
`O
`n=1
`
`O
`
`n
`
`# n=1; n=2
`
`subterminal
`oxidation
`
`O
`
`n
`
`O
`
`AcCoA
`
`EtOH
`
`O
`
`beta-oxidation
`
`S-CoA
`
`O
`
`n
`
`FIG. 5. Hypothetical pathway for rubber degradation. Evidence for this pathway was obtained by using S. coelicolor 1A (7) and Streptomyces
`sp. strain K30 (40). Metabolites detected by Tsuchii and Takeda (54) are indicated by asterisks (n ⫽ 1), and metabolites detected by Bode et al.
`(7) are indicated by number signs. The position of inhibition of this pathway by tungstate is indicated (40). Lcp, latex clearing protein from
`Streptomyces sp. strain K30; OxiAB, oxidoreductase from Streptomyces sp. strain K30; RoxA, rubber oxygenase from Xanthomonas sp. (10); CoA,
`coenzyme A.
`
`clear-zone-negative phenotype on latex overlay plates. How-
`ever, only a few of these latex-negative mutants retained the
`ability to form clear zones on xylan like the wild type, thus
`indicating a correlation between rubber and xylan degradation
`possibly due to defects in protein secretion. One of these
`rubber-negative mutants was used to identify three genes en-
`coding the rubber-degrading capability of Streptomyces sp.
`strain K30 by phenotypic complementation (40). The cloned
`lcp (latex clearing protein) gene restored clear zone formation
`in the rubber-negative mutants described above and also en-
`abled a recombinant strain of S. lividans TK23 to grow and to
`form clear zones on latex overlay agar plates. Furthermore,
`genes for a heterodimeric molybdenum hydroxylase homo-
`logue (oxiAB) were located downstream of lcp in Streptomyces
`
`sp. strain K30 (40). Whereas heterologous expression of lcp in
`S. lividans TK23 resulted in the accumulation of 12-kDa deg-
`radation products containing aldehyde groups, heterologous
`expression of lcp plus oxiAB yielded aldehydes only if 10 mM
`tungstate was present. Since tungstate is known to be a specific
`inhibitor of molybdenum hydroxylases, OxiAB probably oxi-
`dized the aldehydes formed by Lcp to the corresponding acids,
`which could then be further metabolized via the ␤-oxidation
`pathway (Fig. 5). This is consistent with the observation that
`the presence of 0.1% acrylic acid in the medium prevented
`growth of Streptomyces species on latex (8; Rose, unpublished
`data).
`Rubber biodegradation by Xanthomonas sp. strain 35Y. In-
`cubation with the gram-negative, clear-zone-forming organism
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`2810
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`MINIREVIEWS
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`APPL. ENVIRON. MICROBIOL.
`
`Xanthomonas sp. strain 35Y resulted in a weight loss of 60% in
`natural rubber after only 7 days (54). GPC analysis of the
`degradation products obtained after incubation of natural rub-
`ber with a crude enzyme extract revealed compounds with
`apparent molecular weights of less than 104 and 103 (Table 2),
`comprising about 113 and only 2 isoprene units, respectively.
`1H-NMR and 13C-NMR analyses revealed the same molecular
`structure for the degradation products as that obtained with
`Nocardia sp. strain 835A (see above). Gas chromatography
`(GC)-mass spectrometry (MS) analysis identified the com-
`pound in the low-molecular-weight fraction as acetonyldipre-
`nylacetoaldehyde. A crude enzyme extract prepared from the
`supernatant of a culture of this Xanthomonas strain incubated
`with natural rubber latex for 5 days revealed activity with
`natural rubber, poly(cis-1,4-isoprene), dolichol, and ficaprenol
`but not with the trans oligoisoprenoid squalene. Degradation
`studies with crude enzyme and latex in the presence of 18O
`revealed incorporation of 18O into AP2A. After incubation for
`1 h, the incorporation of one 18O atom into AP2A was 77%
`and the incorporation of two atoms of 18O was 4%. Under a
`nitrogen atmosphere, no detectable AP2A was produced.
`Therefore, it was concluded that molecular oxygen is necessary
`for rubber cleavage at the double-bond position of the poly-
`mer.
`This Xanthomonas strain secretes a protein having an ap-
`parent molecular mass of 65 kDa during growth on latex (10,
`24), which was referred to as rubber oxygenase (RoxA). Anal-
`ysis of the sequence of the cloned gene resulted in identifica-
`tion of a signal peptide sequence in the nonmature protein and
`two heme-binding motifs and a 20-amino-acid region con-
`served in diheme cytrochrome c peroxidases. RoxA o