title: Biochemical and spectroscopic characterization of purified Latex Clearing Protein (Lcp) from newly isolated rubber degrading Rhodococcus rhodochrous strain RPK1 reveals novel properties of Lcp.
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 (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.
  
  
  CONCLUSIONS
  Our data point to substantial differences in the active sites of Lcp proteins obtained from different rubber degrading bacteria.
authors.affiliation: Institute of Microbiology, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.|Prince of Songkla University, Songkla, Thailand.
authors.forename: Sirimaporn
authors.initials: S
authors.lastname: Watcharakul
authors.affiliation: Institute of Microbiology, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
authors.forename: Wolf
authors.initials: W
authors.lastname: Röther
authors.affiliation: Institute of Microbiology, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
authors.forename: Jakob
authors.initials: J
authors.lastname: Birke
authors.affiliation: Prince of Songkla University, Songkla, Thailand.
authors.forename: Kamontam
authors.initials: K
authors.lastname: Umsakul
authors.affiliation: Prince of Songkla University, Songkla, Thailand.
authors.forename: Brian
authors.initials: B
authors.lastname: Hodgson
authors.affiliation: Institute of Microbiology, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany. dieter.jendrossek@imb.uni-stuttgart.de.
authors.forename: Dieter
authors.initials: D
authors.lastname: Jendrossek
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001673:Biodegradation, Environmental; D020134:Catalytic Domain; D003001:Cloning, Molecular; D007218:Industrial Microbiology; D007220:Industrial Waste; D013058:Mass Spectrometry; D012240:Rhodococcus / Q000145:classification / Q000254:growth & development* / Q000302:isolation & purification / Q000378:metabolism*; D012408:Rubber / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007220:Industrial Waste; D012408:Rubber
keywords: Biodegradation; Dioxygenase; Latex clearing protein (Lcp); Rhodococcus; Rubber oxygenase