Abstract

Nocardia nova SH22a is an actinobacterium capable of degrading the polyisoprenes poly(cis -1,4-isoprene) and poly(trans-1,4-isoprene). Seq uencing and annotating the genome of this strain led to the identification of a single gene coding for the key enzyme for the degradation of rubber: the latex clearing protein (Lcp). In this study, we showed that Lcp(SH22a)-contrary to other already characterized rubber cleaving enzymes-is responsible for the initial cleavage of both polyisoprene isomers. For this purpose, lcp(sH22). was heterologously expressed in an Escherichia coli strain and purified with a functional His(6)- or Strep-tag. Applying liquid chromatography electrospray ionization time-of-flight mass spectrometry (LC/ESI-ToF-MS ) and a spectrophotometric pyridine hemochrome assay, heme b was identified as a cofactor. Furthermore, hemeassociated iron was identified using total reflection X-ray fluorescence (TXRF) analysis and inhibition tests. The enzyme's temperature and pH optima at 30 degrees C and 7, respectively, were determined using an oxygen consumption assay. Cleavage of poly(cis-1,4-isoprene) and poly(trans-1,4-isoprene) by the oxygenase was confirmed via detection of carbonyl functional groups containing cleavage products, using Schiff's reagent and electrospray ionization mass spectrometry (ESI-MS).