ABSTRACT

An extracellular protein with strong absorption at 406 nm was purified from cell-free culture fluid of latex-grown Xanthomonas sp. strain 35Y. This protein was identical to the gene product of a recently characterized gene cloned from Xanthomonas sp., as revealed by determination of m/z values and sequencing of selected isolated peptides obtained after trypsin fingerprint analysis. The purified protein degraded both natural rubber latex and chemosynthetic poly(cis-1,4-isoprene) in vitro by oxidative cleavage of the double bonds of poly(cis-1,4-isoprene). 12-Oxo-4,8-dimethyltrideca-4,8-diene-1-al (m/z 236) was identified and unequivocally characterized as the major cleavage product, and there was a homologous series of minor metabolites that differed from the major degradation product only in the number of repetitive isoprene units between terminal functions, CHO-CH₂ and CH₂-COCH₃. An in vitro enzyme assay for oxidative rubber degradation was developed based on high-performance liquid chromatography analysis and spectroscopic detection of product carbonyl functions after derivatization with dinitrophenylhydrazone. Enzymatic cleavage of rubber by the purified protein was strictly dependent on the presence of oxygen; it did not require addition of any soluble cofactors or metal ions and was optimal around pH 7.0 at 40°C. Carbon monoxide and cyanide inhibited the reaction; addition of catalase had no effect, and peroxidase activity could not be detected. The purified protein was specific for natural rubber latex and chemosynthetic poly(cis-1,4-isoprene). Analysis of the amino acid sequence deduced from the cloned gene (roxA [rubber oxygenase]) revealed the presence of two heme-binding motifs (CXXCH) for covalent attachment of heme to the protein. Spectroscopic analysis confirmed the presence of heme, and approximately 2 mol of heme per mol of RoxA was found.

INTRODUCTION

Natural rubber (NR) is a biopolymer that is synthesized by many plants and some fungi. This polymer has been commercially exploited for more than 100 years by cultivating and tapping the rubber tree (Hevea brasiliensis). NR is a polymer of many isoprene units [poly(cis-1,4-isoprene)] that, after cross-linking of the linear polymer chains by sulfur bridges (vulcanization), has superior physical properties. Despite the development of chemosynthetic rubbers, NR is still a necessary raw material for products such as tires, latex gloves, condoms, and seals.

NR does not accumulate in the environment. Many reports on the biodegradability of rubbers were published during the last century (for recent studies see references   and references therein). Even chemically cross-linked (vulcanized) rubbers have been shown to be biodegradable . Two biological strategies for microbial NR degradation have been described so far. (i) A large number of bacteria, most of which belong to the actinomycetes, are able to grow and to produce clearing zones on agar media containing NR latex in the form of a milky opaque emulsion as a carbon source . So far, Xanthomonas sp. strain 35Y is the only known gram-negative NR-degrading bacterium belonging to this group . (ii) The members of the other group of NR-utilizing bacteria do not produce clearing zones on NR latex agar; rather, they are able to solubilize solid pieces of NR and to use the resulting emulsion as a carbon source. Gordonia polyisoprenivorans and Gordonia westfalica belong to this class of bacteria.

The basic molecular mechanism by which rubber is degraded is not known. Tsuchi and coworkers were the first researchers to isolate and identify low-molecular-mass oligo(cis-1,4-isoprene) derivatives with aldehyde and keto end groups from rubber-grown cultures of Xanthomonas and Nocardia species . It is assumed that degradation of the polymer backbone is initiated by statistical oxidative cleavage of one double bond in the polymer chain. The resulting low-molecular-mass oligo(cis-1,4-isoprene) derivatives then are further degraded, presumably by ß-oxidation. Analysis of NR degradation products produced by Streptomyces coelicolor 1A and Streptomyces griseus 1D after 70 days of growth on latex gloves revealed an oligomer pattern similar to that observed for Xanthomonas sp. However, products with different end groups were detected . Since all these studies were performed with undefined culture broth, it is not known whether the products identified were formed in one or more enzymatic steps. To our knowledge, no enzyme involved in rubber degradation has been isolated in an active form or described. Recently, a gene of Xanthomonas sp. whose gene product could be involved in rubber degradation was cloned , but a particular function could not be assigned to the gene. In this study we succeeded in purifying an extracellular protein with polyisoprene oxygenase activity and in characterizing the cleavage reaction.