Cleavage of poly(cis-1,4-isoprene) by the latex clearing protein (Lcp) and involvement of a molybdenum-dependent oxidoreductase (OxiBA) in early steps of rubber degradation

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This project investigates initial cleavage of poly(cis-1,4-isoprene) by Lcp (Latex clearing protein) from Streptomyces sp. K30. Lcp homologues occur in all other Gram-positive rubber and gutta percha degrading bacteria; this protein seems to be generally responsible for cleavage of polyisoprenoides by Actinobacteria. The aims are as follows: (i) Secretion of Lcp via the Tat pathway will be investigated. (ii) Random mutagenesis employing a mutator strain and an E.coli-Streptomyces shuttle vector will unravel essential amino acid residues of Lcp, (iii) which will be verified by site specific mutagenesis. (iv) An enzyme activity assay for Lcp will be developed to characterize wild type and mutated Lcp proteins and to investigate regulation of Lcp. (v) During cleavage of poly(cis-1,4-isoprene), oxygen is incorporated into the degradation products. An enzyme activity assay and knowledge of amino acids involved in cleavage will enable to determine the molecular mechanism of rubber cleavage by Lcp. In this context, putative co-factors and the 3D structure of Lcp will be identified. (vi) A putative aldehyde dehydrogenase encoded by oxiA and oxiB located downstream of lcp obviously oxidizes the cleavage products produced by Lcp. Function and regulation of oxiAB will be investigated. (vii) 2D gel electrophoresis employing Nocardia farcinica IFM10152, the only rubber degrading bacterium with known genome sequence, will unravel further proteins involved in poly(cis-1,4-isoprene) degradation.