Isolation of a novel rubber degrading bacterium from a consortium and characterization of its lcp gene products

Abstract

Rubber degrading consortium ST608 was enriched from a rubber processing waste pond using Mineral Salt Medium (MSM) supplemented with sterile rubber glove pieces, representative of a vulcanized rubber, as a sole carbon source. Substantial disintegration of the rubber pieces was visible within 3 weeks of incubation with the consortium, this indicated the presence of active rubber degrading microorganisms. The highest percentage weight loss of the rubber pieces over a 30 day period was 50%, and according to the CO2 released, this accounted for 57% of the total available rubber carbon. The deterioration of rubber pieces was also observed by Scanning Electron Microscope (SEM), numerous microorganisms were associated with the areas of strong disintegration resulted in structural changes, that was detected by Fourier Transform Infrared spectroscopy Attenuated Total Reflectance (FTIR- ATR), which revealed (i) a significant decrease of the numbers of double bonds in the isoprene chains (ii) the formation of aldehyde-ketone groups (iii) the formation of different chemical bondings including fatty acid and polysaccharide of microbial cells. The bacterial consortium ST608 was analyzed by PCR-DGGE, and eight prominent strains were identified based on 16s rRNA genes with most isolates identified belonging to the actinomycetes group. This work clearly showed the potential of one bacterial consortium ST608 to biodegrade rubber while undergoing dynamic changes in the bacterial population. Some strains that played key roles throughout the experiment were identified. Due to little is known about rubber degrading enzymes and their corresponding genes, in particular about the biochemistry of polyisoprene cleavage by Leps and the types and functions of the involved cofactors. In this research study, one of rubber degrading bacteria identified as a Rhodococcus rhodochrous RPK1, was derived from the potential consortium ST608, which has not been previously identified its biochemical properties. Thus, identification and characterization of properties of lcp gene was performed. A lcp gene of R. rhodochrous RPK1 that coded for a polyisoprene-cleaving latex clearing protein (LCPR) 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(cis-1,4-isoprene) to a mixture of oligoisoprene molecules with terminal keto and aldehyde groups. The optimum pH of LCPRr was higher (pH 8) than for other known rubber-cleaving enzymes (≈ pH 7). UVvis spectroscopic analysis of LCPR 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 Leper 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. This study points the potential of bacterial consortium ST608 to degrade vulcanized rubber glove with identification of strains that played important roles in the process, and revealed the substantial differences in the active sites of LCPRr proteins to other rubber degrading proteins.