Abstract
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.
Highlights
Biodegradation of rubber 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
Taxonomic identification of isolate RPK1 Isolate RPK1 had a high rubber-degrading activity compared to other rubber degraders in liquid culture, as revealed by pronounced disintegration of rubber pieces (Fig. 1a)
Identification of the gene coding for the latex clearing protein in R. rhodochrous strain RPK1 basic local alignment search tool (BLAST) analysis revealed that many Actinobacteria and all known rubber-degrading Actinobacteria for which the genome sequences have been determined have at least one gene that codes for a so-called latex clearing protein that is suspected to be responsible for the
Summary
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. 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 (12oxo-4,8-dimethyl-trideca-4,8-diene-1-al, ODTD) as the main product [11,12,13]. The other rubber cleaving enzyme is a protein designated as latex clearing protein (Lcp) [1]. It shares no significant sequence homology with RoxA, with cytochrome c peroxidases or with dihaeme 7,10-diol synthases [14] and is present in Gram-positive rubber degrading bacteria such as 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 [15,16,17]. K30 and of G. polyisoprenivorans VH2 [15, 17, 18], and at present there are currently only two biochemically characterized Lcp proteins
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