Identification of Poly( cis -1,4-Isoprene) Degradation Intermediates during Growth of Moderately Thermophilic Actinomycetes on Rubber and Cloning of a Functional lcp Homologue from Nocardia farcinica Strain E1

Abstract

ABSTRACT The enrichment and isolation of thermophilic bacteria capable of rubber [poly( cis -1,4-isoprene)] degradation revealed eight different strains exhibiting both currently known strategies used by rubber-degrading mesophilic bacteria. Taxonomic characterization of these isolates by 16S rRNA gene sequence analysis demonstrated closest relationships to Actinomadura nitritigenes , Nocardia farcinica , and Thermomonospora curvata . While strains related to N. farcinica exhibited adhesive growth as described for mycolic acid-containing actinomycetes belonging to the genus Gordonia , strains related to A. nitritigenes and T. curvata formed translucent halos on natural rubber latex agar as described for several mycelium-forming actinomycetes. For all strains, optimum growth rates were observed at 50°C. The capability of rubber degradation was confirmed by mineralization experiments and by gel permeation chromatography (GPC). Intermediates resulting from early degradation steps were purified by preparative GPC, and their analysis by infrared spectroscopy revealed the occurrence of carbonyl carbon atoms. Staining with Schiff's reagent also revealed the presence of aldehyde groups in the intermediates. Bifunctional isoprenoid species terminated with a keto and aldehyde function were found by matrix-assisted laser desorption ionization-time-of-flight and electrospray ionization mass spectrometry analyses. Evidence was obtained that biodegradation of poly( cis -1,4-isoprene) is initiated by endocleavage, rather than by exocleavage. A gene ( lcp ) coding for a protein with high homology to Lcp ( l atex- c learing p rotein) from Streptomyces sp. strain K30 was identified in Nocardia farcinica E1. Streptomyces lividans TK23 expressing this Lcp homologue was able to cleave synthetic poly( cis -1,4-isoprene), confirming its involvement in initial polymer cleavage.