- Research article
- Open Access
Biochemical and spectroscopic characterization of purified Latex Clearing Protein (Lcp) from newly isolated rubber degrading Rhodococcus rhodochrous strain RPK1 reveals novel properties of Lcp
© Watcharakul et al. 2016
- Received: 19 January 2016
- Accepted: 10 May 2016
- Published: 23 May 2016
Biodegradation of rubber (polyisoprene) is initiated by oxidative cleavage of the polyisoprene backbone and is performed either by an extracellular rubber oxygenase (RoxA) from Gram-negative rubber degrading bacteria or by a latex clearing protein (Lcp) secreted by Gram-positive rubber degrading bacteria. Only little is known on the biochemistry of polyisoprene cleavage by Lcp and on the types and functions of the involved cofactors.
A rubber-degrading bacterium was isolated from the effluent of a rubber-processing factory and was taxonomically identified as a Rhodococcus rhodochrous species. A gene of R. rhodochrous RPK1 that coded for a polyisoprene-cleaving latex clearing protein (lcp Rr ) was identified, cloned, expressed in Escherichia coli and purified. Purified LcpRr had a specific activity of 3.1 U/mg at 30 °C and degraded poly(1,4-cis-isoprene) to a mixture of oligoisoprene molecules with terminal keto and aldehyde groups. The pH optimum of LcpRr was higher (pH 8) than for other rubber-cleaving enzymes (≈ pH 7). UVvis spectroscopic analysis of LcpRr revealed a cytochrome-specific absorption spectrum with an additional feature at long wavelengths that has not been observed for any other rubber-cleaving enzyme. The presence of one b-type haem in LcpRr as a co-factor was confirmed by (i) metal analysis, (ii) solvent extraction, (iii) bipyridyl assay and (iv) detection of haem-b specific m/z values via mass-spectrometry.
Our data point to substantial differences in the active sites of Lcp proteins obtained from different rubber degrading bacteria.
- Latex clearing protein (Lcp)
- Rubber oxygenase
Natural rubber is an important biopolymer that has been produced for more than a century by cultivating the rubber tree (Hevea brasiliensis). Natural rubber obtained by tapping of the rubber trees is used for countless applications, for example for the production of tires, sealings, latex gloves and many, many other items. The main component of rubber latex is the hydrocarbon poly(cis-1,4-isoprene). Chemosynthetic rubber is also produced at a scale that is almost comparable to that of the natural compound.
Despite the economic importance of rubber and the enormous amounts of rubber waste materials that are permanently released into the environment, complete degradation in nature is rarely detected and wastes continue to accumulate. Knowledge of the reasons for this is limited. In fact, application is made of this extremely slow natural degradation for example in the use of rubber tyres to provide attachment sites for creating artificial coral reefs. However, microorganisms that can attack rubber have been detected in many ecosystems in which the physical parameters (temperature, pH, salinity) are moderate [1–7]. It is also well known that the initial microbial attack on rubber depends on the ability to produce and secrete rubber-cleaving enzymes into the environment. Only two types of rubber-cleaving enzymes are known. One is the rubber oxygenase RoxA that was first isolated from Xanthomonas sp. 35Y [8, 9] and so far has been found only in Gram-negative bacteria . RoxA of Xanthomonas sp. 35Y is a c-type dihaem dioxygenase and cleaves poly(cis-1,4-isoprene) into a C15 compound with a terminal keto and aldehyde group (12-oxo-4,8-dimethyl-trideca-4,8-diene-1-al, ODTD) as the main product [11–13]. The other rubber cleaving enzyme is a protein designated as latex clearing protein (Lcp) . It shares no significant sequence homology with RoxA, with cytochrome c peroxidases or with dihaeme 7,10-diol synthases  and is present in Gram-positive rubber degrading bacteria such as Streptomyces sp. K30  and other Actinobacteria. G. polyisoprenivorans VH2 and Streptomyces sp. K30, two well-studied Gram-positive rubber degraders, oxidatively cleave poly(cis-1,4-isoprene) to products of different sizes but with the same keto and aldehyde end groups as in RoxA-generated ODTD [15–17]. There have been different reports published for the co-factor and metal-contents of the Lcps from Streptomyces sp. K30 and of G. polyisoprenivorans VH2 [15, 17, 18], and at present there are currently only two biochemically characterized Lcp proteins.
In this study, we used a waste pond at a rubber-processing factory in Thailand as a natural enrichment environment for rubber-degrading microorganisms and a source for the isolation of new rubber degrading strains. Taxonomic analysis revealed that one isolated strain was a member of the genus Rhodococcus, a taxon that had not been previously identified as having the ability to utilise rubber as a sole source of carbon and energy but that is well known for its members to have a high potential for the biodegradation of recalcitrant compounds . Biochemical and biophysical characterization of the purified recombinant Lcp protein of Rhodococcus rhodochrous strain RPK1 revealed some unexpected properties not previously described for any other rubber-degrading enzyme in addition to properties shared with the two other characterized Lcp proteins.
Taxonomic identification of isolate RPK1
Identification of the gene coding for the latex clearing protein in R. rhodochrous strain RPK1
Properties of biochemically characterized rubber oxygenases
Gene length [bp]
Residues (pre-/mature enzyme)
Molecular mass (apo-/mature protein) [kDa]
strep tagged [kDa]
Molar extinction coefficient [104 M−1 cm−1]
9.5 (407 nm)
8.0 (412 nm)
20.6 (406 nm)
Metal atoms per protein molecule
not known/no haeme
Oxidation state of haeme iron
UVvis effect upon addition of CO
Specific activity [U/mg] (23/30/37 °C)
Conserved DUF2236 residuesc
Expression and purification of LcpRr
Biochemical properties of LcpRr
LcpRr is a b–type cytochrome and revealed remarkable differences to LcpK30
To confirm that LcpRr is a haem-containing protein and to determine its haem type, a metal analysis and a spectral analysis by the haem-bipyridyl assay were performed. 6.5 μg Fe/mL LcpRr protein solution (5.3 mg protein/mL) were determined. This corresponded almost perfectly with a stoichiometry of one atom Fe per one LcpRr molecule. It was of interest that low amounts of copper (2.8 μg/mL) were also identified and corresponded to 0.36 atoms Cu per one LcpRr molecule. Zinc was detected at the detection limit (0.1 μg/mL) and Nickel was below the detection limit (<0.1 μg/mL); other metals (vanadium to zinc tested) were also not detected in significant amounts. Divalent cations such as magnesium or calcium were not present (below the detection limit of 0.1 μg/mL) (Table 1). The presence of approximately one third of an atom% Cu per LcpRr molecule was unexpected because only traces of copper had been previously detected in LcpK30 . The determined amount of copper in LcpRr, however, was too high to be explained by an error in the determination of the metal or protein concentration. One possibility could be that the amount of copper was due to a contamination of the protein by traces of copper present in either the growth medium or in the buffer ingredients. For example, the used batch of NaCl that was present in some purification buffers was only of 98 % purity and could contain traces of heavy metals. However, sub-stoichiometric amounts of copper (precise concentration not known) had been also detected in Lcp1VH2 of G. polyisoprenivorans . Addition of an equimolar concentration or of a 10-fold molar excess of copper ions [Cu(II)Cl2] to the assay mixture with purified LcpRr had no detectable effect on the UVvis spectrum or on the activity of LcpRr. The addition of 50 μM CuSO4 to the LcpRr-expression culture produced no increased activity or yield of LcpRr. At present, there is no convincing explanation for the finding of variable sub-stoichiometric amounts of copper in the purified Lcp proteins from R. rhodochrous RPK1.
An absorption maximum of 556 nm was determined using the bipyridyl assay for LcpRr and for haemoglobin that was used as a b-type cytochrome control protein (Additional file 3). This result indicated the presence of a b-type haem in LcpRr. In contrast to the covalently linked c-type cytochromes, the haem groups of the b-type cytochromes are not covalently linked to the peptide chain and can be therefore extracted by an acid solvent extraction . Acid solvent extraction of the purified LcpRr yielded a coloured supernatant and a non-coloured precipitate. In contrast, solvent extraction of the c-type cytochromes such as RoxAXsp or of other commercially available cytochrome c enzymes yielded a non-coloured supernatant and a red precipitate which is in agreement with the covalent attachment of porphyrin to the polypeptide. MALDI-ToF analysis of the purified LcpRr resulted in the identification of ions with m/z values of 616 (data not shown) which is typical for haem b . Taken together, all these results indicated that LcpRr is a b-type cytochrome similar to LcpK30  Notably, MALDI-ToF analysis of LcpRr also revealed an ion species with m/z values of 619 besides that of 616 which could correspond to a verdo-haem . As the activity of purified LcpRr rapidly and substantially decreased during storage, the haem species with m/z value of 619 could represent a haem degradation product of the inactivated LcpRr.
LcpRr is insensitive to most chelating inhibitors
LcpRr but not LcpK30 is accessible for external ligands
lcp genes are frequently present in the genomes of Actinobacteria  and many rubber degrading species have been described for members from this group [3–5, 23, 31, 32]. Most of the rubber degrading actinomycetes such as Streptomyces sp. K30 , Streptomyces coelicolor 1A, and many others [3, 6, 33] produce clearing zones on opaque polyisoprene latex agar. However several of the most potent rubber degraders do not produce clearing zones and apparently need a close contact to the rubber material they degrade. Two well known rubber degrading Gordonia species (G. polyisoprenivorans and G. westfalica) [20, 22] and also the strain from this study (R. rhodochrous RPK1) belong to this group of non-clearing zone formers. One might speculate that the Lcp proteins of non-clearing zone formers constitute a group that have an open conformation with free access to the active site (no conformational change is needed) and that the other Lcp proteins that have an ability to form a clearing zone have a closed form. The prototype of the first group would be LcpRr and the prototype of the latter would be LcpK30. It will be necessary to biochemically investigate more Lcp proteins and to solve the structure of Lcp proteins to find evidence for or against this hypothesis.
This study extends the list of biochemically characterized rubber-degrading non-clearing zone formers by latex clearing proteins (Lcp) to the genus Rhodococcus (besides Gordonia). The detection of rubber-cleaving activity with purified LcpRr and the absence of clearing zones during growth on polyisoprene latex agar raises the question of whether the designation “latex clearing protein” has been well-chosen. Rubber oxygenase B (RoxB) would be an appropriate alternative. However, the designation Lcp has been used is several previous publications and has also been used for many annotated genes in genome-sequenced Actinobacteria. Re-classification of Lcp as RoxB therefore could lead to the confusion of other research workers.
The isolation and characterization of the Lcp protein of R. rhodochrous RPK1 in this study showed that all the so far studied Lcp proteins can differ in some spectroscopic features and/or in spatial arrangements of their metal ions/cofactors and indicate the presence of two or even more subgroups of Lcp proteins. It will be necessary to study more Lcp proteins to reveal the complete variability of rubber degrading enzymes present in rubber-degrading organisms.
Bacterial strains, plasmids and culture conditions
Bacterial strains, plasmids and oligonucleotides used in this study
Strain or plasmid
E. coli JM109
Plasmid storage and expression of lcp
E. coli XL1-blue
Rhodococcus rhodochrous RPK1
Wild type strain, degrades rubber
pUC9::strep-lcp K30 (SN5339)
Cloning vector for lcp K30 , Apr
pUC9::strep-lcp Rr (SN5759)
Cloning vector for lcp Rr , Apr
Mobilizable broad host range
Expression vector, Kmr
p4782.1::strep-lcp K30 (SN5496)
Coding sequence of strep-lcp K30 under
Rhamnose promoter control, Kmr
p4782.1::strep-lcp Rr (SN5760)
Coding sequence of strep-lcp Rr under
Rhamnose promoter control, Kmr
Enrichment and isolation of rubber-degrading microorganisms
Liquid from a waste pond at a rubber latex processing factory in Thailand (Namom rubber factory at Namom, Songkhla) was used as an inoculum to enrich for rubber-degrading microorganisms in a mineral salts medium (MSM) that had been supplemented with 1×1 cm pieces of rubber gloves as a sole source of carbon and energy. After two weeks of incubation at 30 °C, 0.1 volumes (without pieces of rubber) were transferred to fresh medium and incubated for an additional month. Substantial disintegration of the new rubber pieces became visible and indicated that active rubber-degrading microorganisms were present. Several bacterial strains were isolated from this enrichment culture by repeated purification from streaks onto NB and LB agar plates. Each isolate was subsequently tested for its ability to degrade rubber in liquid MSM with rubber pieces as carbon source. One isolate (designated as isolate RPK1) with strong rubber-degrading activity was selected for this study.
Cloning and heterologous expression of lcp Rr , and determination of the 16S rRNA gene sequence of isolate RPK1
The lcp Rr gene was amplified using the chromosomal DNA from R. rhodochrous strain RPK1 as template and the oligonucleotides LcpRr-complete_for and LcpRr-complete_rev as PCR primers and Takara Primestar DNA polymerase as the proof-reading polymerizing enzyme. The DNA sequence of the product was determined and is available under the accession no KU140417. Alternatively, the coding sequence of mature LcpRr was amplified from chromosomal DNA using LcpRr-mature-PstI_for and LcpRr-mature-HindIII_rev as primers. The DNA products were purified via agarose gel electrophoresis, cleaved with restriction enzymes PstI and HindIII and ligated into plasmid pUC9::lcp K30 that had been cleaved by the same restriction enzymes. The coding sequence for strep-tagged lcp Rr was cut out using HindIII and NdeI and was subsequently ligated into the expression plasmid p4782.1 and transformed to competent E. coli JM109 cells.
A part of the 16S rRNA gene of the isolate RPK1 was PCR-amplified using the primers 16S-universal_for and 16S-universal_rev. The DNA sequence of the resulting PCR product was determined (1412 bp) and revealed a strong similarity to the 16S rRNA genes of several Rhodococcus sp. strains. The 16S rRNA gene sequence of isolate RPK1 was determined after PCR amplification using the primers (16S-Rr-complete_for and 16S-Rr-complete_rev), that were specific for the ends of the known 16S rRNA gene sequences of R. rhodochrous strains taken form the NCBI data base, and is now available under the accession no KU140418.
Purification of LcpRr, LcpK30 and of RoxAXsp
Purification of the rubber oxygenase of Xanthomonas sp. 35Y (RoxAXsp) and latex clearing protein LcpK30 was performed as described previously [12, 18] LcpRr was purified as follows: eight individual 600 mL LB cultures in 3 litre Erlenmeyer flasks were inoculated each with 0.02 volumes of a seed culture of E. coli JM109 harbouring the plasmid p4782.1::lcp Rr that had been grown with the same medium. It was important that L-rhamnose (0.1 %, wt/vol) was present right from the beginning in the main cultures to maximise the yield of the expressed LcpRr protein. Cells of the main culture were harvested by centrifugation after ≈ 20 h of growth at 22 °C and were immediately used for protein purification. The cell pellet was resuspended in 100 mM potassium phosphate buffer, pH 7.7, containing 150 mM sodium chloride (KPN, 2 mL KPN/g cell wet weight). A soluble cell extract was prepared by two French press steps and subsequent centrifugation at 40,000 g for 40 min. The supernatant (≈ 60 mL) was directly applied to a 10 mL Strep-Tactin HC gravity flow column that had been equilibrated with KPN buffer. The column was washed with at least five volumes of KPN buffer before the LcpRr protein was eluted by ≈ 30 mL of 5 mM desthiobiotin dissolved in KPN. LcpRr-containing fractions were combined, desalted by running through a G25 Sephadex (26/160) Hiprep desalting column (53 mL bed volume) that had been equilibrated with 1 mM potassium phosphate (KP) buffer, pH 7.0 and subsequently concentrated to 1–2 mL via ultrafiltration (10 kDa cut-off). Remaining impurities were removed by chromatography on a Superdex 200 column (16/600, equilibrated with 1 mM KP, pH 7) at a flow rate of 1 mL/min. Combined LcpRr-containing fractions were ultrafiltrated (10 kDa cut-off) and concentrated to ≈ 1.5 mL. Aliquots of the purified LcpRr protein were stored on ice for about 3 days (LcpK30 up to 1 week) or shock-frozen with liquid nitrogen and stored at -70 °C.
Determination of the cytochrome type of LcpRr
The haem type of LcpRr was determined by the bi-pyridyl assay as described elsewhere . Purified RoxAXsp, cytochrome c (horseheart, type III, Sigma, St. Louis, USA) (both c-type cytochromes) and haemoglobin (b-type) (bovine, Sigma, St. Louis, USA) were used as controls for known c-type and b-type cytochromes, respectively. 25 μL of the respective protein stock solution (4–8 mg/mL) was added to 975 μL solution A (100 mM sodium hydroxide, 20 % (v/v) pyridine, 0.3 mM potassium ferricyanide). Subsequently, 2–5 mg sodium dithionite were added and the spectrum of the reduced cytochrome was recorded. The absorption maxima of the resulting α-bands were characteristic for b-type (556 nm) and c-type (550 nm) cytochromes. Bi-pyridyl-haem complexes of α-type cytochromes absorb at 584–588 nm. Additional assays for determination of the haem type were performed via extraction of haem by acidic acetone and by a matrix assisted laser desorption ionization time of flight (MALDI-ToF) analysis as previously described in detail .
Assay of Lcp activity
An HPLC-based assay for LcpRr-derived polyisoprene degradation products was used for most routine assays: poly(cis-1,4-isoprene) latex was diluted with 100 mM KP buffer, pH 7, to 0.2 % (assay volume 0.7 mL) and incubated in the presence of purified Lcp protein for 2 h at a temperature as indicated (for routine assay at room temperature [23 °C]). In the case of inhibition studies, the corresponding compound was added and gently solubilised in the reaction mix before the enzyme was added (final inhibitor concentration 1 mM). The products were extracted with 1 mL ethyl acetate (in a 2 mL Eppendorf tube), dried, and dissolved in 100 μL methanol. Aliquots were applied to an RP8 HPLC column (12 ×4 mm, 5 μm particle size, 0.7 mL/min) with water (A) and methanol (B) as mobile phases. The concentration of B was increased from 50 % (v/v) to 100 % (v/v) within 15 min; products were detected at 210 nm. The C35 product peak (at ≈ 23 min) was used for quantification and compared to a control without inhibitor. Alternatively, activity of LcpRr was assayed by determination of the rate of oxygen consumption in an OXY-4 mini apparatus (PreSens, Regensburg, Germany) as described previously . Triplicates and controls without LcpRr or with heat-inactivated LcpRr were recorded simultaneously. A stability assay was performed by incubation of the purified LcpRr protein in the assay buffer at 37 °C for variable time periods. The remaining activity of the protein was determined as described above.
The concentration of protein solutions was determined by the bicinchoninic acid (BCA) method. The concentrations of purified rubber-cleaving enzymes were also determined from the molar extinction coefficients of LcpRr, LcpK30 and RoxAXsp: LcpRr, ε407 = 9.5 104 M−1 cm−1, LcpK30, ε412 = 8.0 104 M−1 cm−1, and RoxAXsp, ε406 = 2.06 ×105 M−1 cm−1. Separation of proteins was performed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) under reducing (2-mercaptoethanol) conditions. The metal content of the purified Lcp protein was determined using inductively coupled plasma-MS (ICP-MS) by the Spuren-Analytisches Laboratorium Dr. Baumann (Germany). Fuchsin staining of polyisoprene degradation products was performed by addition of a 1 % Fuchsin solution (0.5 g Fuchsin, 12.5 mL acetic acid, 2.5 g Na2S2O3, 0.2 mL HCl (37 %) and 37.5 mL H2O) to the Lcp assay mixture. Staining of the cells for PHB and polyphosphate was performed as described previously .
Consent for publication
Availability of data and materials
The DNA sequence of the R. rhodochrous RPK1 lcp and 16S rRNA gene have been submitted to NCBI and are available under the accession No KU140417 and KU140418. The R. rhodochrous RPK1 strain has been deposited at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ) and can be obtained once the Nagoya protocol regulations have been finished. Until then, strain RPK1 can be obtained from the lab of S.W. and K.U. upon request.
BCA, bicinchoninic acid; BLAST, basic local alignment search tool; DAPI, 4’,6-diamidine-2-phenylindol; HPLC, high pressure liquid chromatography; ICP, inductively coupled plasma; KPN, potassium phosphate-sodium chloride; LB, lysogeny broth; Lcp, latex clearing protein; MALDI-ToF, matrix-assisted laser desorption ionisation time of flight; MS, mass spectrometry; MSM, mineral salts medium; NB, nutrient broth; ODTD, 12-oxo-4,8-dimethyl-trideca-4,8-diene-1-al; PHB, polyhydroxybutyrate; RoxA, rubber oxygenase A; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; UVvis, ultra violet visible.
The help of T. Jurkowski (Institute of Biochemistry, University Stuttgart) in MALDI-MS analysis and the work of Simone Reinhardt and Anna Schweter during some experiments is greatly acknowledged.
This work was funded by grants of the Deutsche Forschungsgemeinschaft to D.J. and of the Strategic Scholarships Fellowships Frontier Research Networks, Thailand to S.W.
SW, and WR. carried out most experiments. JB, KU. and DJ. designed the study; JB and DJ. supervised the experiments; all authors analysed the data, DJ. wrote the manuscript. BH. checked and improved language. All authors read and approved the manuscript.
The authors declare that they have no competing interests.
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