Identification of new rubber-degrading bacterial strains from aged latex

Chia, Kim-Hou; Nanthini, Jayaram; Thottathil, Gincy P.; Najimudin, Nazalan; Haris, Mas Rosemal Hakim Mas; Sudesh, Kumar

doi:10.1016/j.polymdegradstab.2014.07.027

Cited by 24 publications

(8 citation statements)

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“…Most of the rubber degrading actinomycetes such as Streptomyces sp. K30 [ 1 ], 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.…”

Section: Resultsmentioning

confidence: 99%

Biochemical and spectroscopic characterization of purified Latex Clearing Protein (Lcp) from newly isolated rubber degrading Rhodococcus rhodochrous strain RPK1 reveals novel properties of Lcp

et al. 2016

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BackgroundBiodegradation 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.ResultsA 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 (lcpRr) 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.ConclusionsOur data point to substantial differences in the active sites of Lcp proteins obtained from different rubber degrading bacteria.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0703-x) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Biochemical and spectroscopic characterization of purified Latex Clearing Protein (Lcp) from newly isolated rubber degrading Rhodococcus rhodochrous strain RPK1 reveals novel properties of Lcp

et al. 2016

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“…Most of these materials are released to the environment in the form of waste or by abrasion in the case of tires. As a natural compound, rubber is a fully biodegradable material, and many rubber-degrading microorganisms have been isolated from various ecosystems in the past (1)(2)(3)(4)(5)(6)(7)(8). The initial step of rubber biodegradation is the enzymatic cleavage of the polymeric carbon backbone to smaller products.…”

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confidence: 99%

Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30 Is a b -Type Cytochrome and Differs from Rubber Oxygenase A (RoxA) in Its Biophysical Properties

Birke

Röther

Jendrossek

2015

Appl Environ Microbiol

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Specific polyisoprene-cleaving activities of 1.5 U/mg and 4.6 U/mg were determined for purified Strep-tagged latex clearing protein (Lcp) of Streptomyces sp. strain K30 at 23°C and 37°C, respectively. Metal analysis revealed the presence of approximately one atom of iron per Lcp molecule. Copper, which had been identified in Lcp1 VH2 of Gordonia polyisoprenivorans previously, was below the detection limit in Lcp K30 . Heme was identified as a cofactor in purified Lcp K30 by (i) detection of characteristic ␣-, ␤-, and ␥ (Soret)-bands at 562 nm, 532 nm, and 430 nm in the visible spectrum after chemical reduction, (ii) detection of an acetoneextractable porphyrin molecule, (iii) determination of a heme b-type-specific absorption maximum (556 nm) after chemical conversion of the heme group to a bipyridyl-heme complex, and (iv) detection of a b-heme-specific m/z value of 616.2 via mass spectrometry. Spectroscopic analysis showed that purified Lcp as isolated contains an oxidized heme-Fe 3؉ that is free of bound dioxygen. This is in contrast to the rubber oxygenase RoxA, a c-type heme-containing polyisoprene-cleaving enzyme present in Gram-negative rubber degraders, in which the covalently bound heme firmly binds a dioxygen molecule. Lcp K30 also differed from RoxA in the lengths of the rubber degradation cleavage products and in having a higher melting point of 61.5°C (RoxA, 54.3°C). In summary, RoxA and Lcp both are equipped with a heme cofactor and catalyze an oxidative C-C cleavage reaction but differ in the heme subgroup type and in several biochemical and biophysical properties. These findings suggest differences in the catalytic reaction mechanisms.T he hydrocarbon natural rubber [poly(cis-1,4-isoprene)] is an important biopolymer that is produced by many plants and some fungi. Natural rubber, as well as chemically synthesized poly(cis-1,4-isoprene) (synthetic rubber), has been in use by mankind for more than 100 years. Huge amounts of rubber derived from the rubber tree Hevea brasiliensis are the basis for the manufacturing of tires, sealers, latex gloves, condoms, and many other items. Most of these materials are released to the environment in the form of waste or by abrasion in the case of tires. As a natural compound, rubber is a fully biodegradable material, and many rubber-degrading microorganisms have been isolated from various ecosystems in the past (1-8). The initial step of rubber biodegradation is the enzymatic cleavage of the polymeric carbon backbone to smaller products. Two types of rubber-cleaving enzymes have been characterized so far. One is rubber oxygenase A, RoxA, which is found in Gram-negative clearing zone formers. The other is latex clearing protein, Lcp, which has been identified only in Gram-positive organisms so far. RoxA first was isolated and biochemically characterized from Xanthomonas sp. strain 35Y (9). Biochemical and biophysical investigation revealed that RoxA is an extracellular dioxygenase with two covalently attached heme groups (10, 11) and is structurally but not functio...

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“…This washing step was repeated. The top layer or NR latex concentrate was removed and used to prepare the NR latex agar [29].…”

Section: Screening For Clear Zone Forming Strains On Natural Rubber (Nr) Latex Agarmentioning

confidence: 99%

Rubber Degrading Strains: Microtetraspora and Dactylosporangium

et al. 2021

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Rubber composed of highly unsaturated hydrocarbons, modified through addition of chemicals and vulcanization are widely used to date. However, the usage of rubber, faces many obstacles. These elastomeric materials are difficult to be re-used and recovered, leading to high post-consumer waste and vast environmental problems. Tyres, the major rubber waste source can take up to 80 years to naturally degrade. Experiments show that the latex clearing proteins (Lcp) found in Actinobacteria were reportedly critical for the initial oxidative cleavage of poly(cis-1,4-isoprene), the major polymeric unit of rubber. Although, more than 100 rubber degrading strains have been reported, only 8 Lcp proteins isolated from Nocardia (3), Gordonia (2), Streptomyces (1), Rhodococcus (1), and Solimonas (1) have been purified and biochemically characterized. Previous studies on rubber degrading strains and Lcp enzymes, implied that they are distinct. Following this, we aim to discover additional rubber degrading strains by randomly screening 940 Actinobacterial strains isolated from various locations in Sarawak on natural rubber (NR) latex agar. A total of 18 strains from 5 genera produced clearing zones on NR latex agar, and genes encoding Lcp were identified. We report here lcp genes from Microtetraspora sp. AC03309 (lcp1 and lcp2) and Dactylosporangium sp. AC04546 (lcp1, lcp2, lcp3), together with the predicted genes related to rubber degradation. In silico analysis suggested that Microtetraspora sp. AC03309 is a distinct species closely related to Microtetraspora glauca while Dactylosporangium sp. AC04546 is a species closely related to Dactylosporangium sucinum. Genome-based characterization allowed the establishment of the strains taxonomic position and provided insights into their metabolic potential especially in biodegradation of rubber. Morphological changes and the spectrophotometric detection of aldehyde and keto groups indicated the degradation of the original material in rubber samples incubated with the strains. This confirms the strains’ ability to utilize different rubber materials (fresh latex, NR product and vulcanized rubber) as the sole carbon source. Both strains exhibited different levels of biodegradation ability. Findings on tyre utilization capability by Dactylosporangium sp. AC04546 is of interest. The final aim is to find sustainable rubber treatment methods to treat rubber wastes.

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Identification of new rubber-degrading bacterial strains from aged latex

Cited by 24 publications

References 25 publications

Biochemical and spectroscopic characterization of purified Latex Clearing Protein (Lcp) from newly isolated rubber degrading Rhodococcus rhodochrous strain RPK1 reveals novel properties of Lcp

Biochemical and spectroscopic characterization of purified Latex Clearing Protein (Lcp) from newly isolated rubber degrading Rhodococcus rhodochrous strain RPK1 reveals novel properties of Lcp

Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30 Is a b -Type Cytochrome and Differs from Rubber Oxygenase A (RoxA) in Its Biophysical Properties

Rubber Degrading Strains: Microtetraspora and Dactylosporangium

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