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. BMC Microbiology (2016) 16:92 DOI 10.1186/s12866-016-0703-x 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 Sirimaporn Watcharakul1,2, Wolf Röther1, Jakob Birke1, Kamontam Umsakul2, Brian Hodgson2 and Dieter Jendrossek1* Abstract Background: 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. Results: 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 (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. Conclusions: Our data point to substantial differences in the active sites of Lcp proteins obtained from different rubber degrading bacteria. Keywords: Latex clearing protein (Lcp), Rubber oxygenase, Dioxygenase, Rhodococcus, Biodegradation Background 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 * Correspondence: Sirimaporn Watcharakul and Wolf Röther, shared first authorship 1 Institute of Microbiology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany Full list of author information is available at the end of the article component of rubber latex is the hydrocarbon poly(cis1,4-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 © 2016 Watcharakul et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Watcharakul et al. BMC Microbiology (2016) 16:92 Page 2 of 13 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 [10]. 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 (12oxo-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) [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 [1] 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 cofactor 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 rubberprocessing 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 [19]. 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. while known clear zone formers such as Xanthomonas sp. 35Y [8] or Streptomyces coelicolor strain 1A [3] formed large clearing zones. Isolate RPK1 developed colonies with an intense red colour upon growth and prolonged incubation on NB agar (Fig. 1b). Microscopic examination revealed non-motile cells. Depending on the growth phase the cells were coccoid (cells from late stationary phase), rod-shaped (cells from early and late log phase) or long rods (up to 1 ×5 μm), partially branched and star- (...truncated)