Vivod Robin, Andler Rodrigo, Oetermann Sylvia, Altenhoff Anna-Lena, Seipel Nele, Holtkamp Michael, Hogeback Jens, Karst Uwe, Steinbüchel Alexander
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität.
Facultad de Ciencias Agrarias y Forestales, Universidad Católica del Maule.
J Gen Appl Microbiol. 2020 Jan 31;65(6):293-300. doi: 10.2323/jgam.2019.01.003. Epub 2019 Jul 16.
Nocardia nova SH22a is an actinobacterium capable of degrading the polyisoprenes poly(cis-1,4-isoprene) and poly(trans-1,4-isoprene). Sequencing 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-contrary to other already characterized rubber cleaving enzymes-is responsible for the initial cleavage of both polyisoprene isomers. For this purpose, lcp was heterologously expressed in an Escherichia coli strain and purified with a functional His- 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, heme-associated iron was identified using total reflection X-ray fluorescence (TXRF) analysis and inhibition tests. The enzyme's temperature and pH optima at 30°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).
新星诺卡氏菌SH22a是一种能够降解聚异戊二烯——聚（顺式-1,4-异戊二烯）和聚（反式-1,4-异戊二烯）的放线菌。对该菌株的基因组进行测序和注释后，鉴定出了一个编码橡胶降解关键酶的单一基因：乳胶清除蛋白（Lcp）。在本研究中，我们发现，与其他已表征的橡胶裂解酶不同，Lcp负责两种聚异戊二烯异构体的初始裂解。为此，lcp在大肠杆菌菌株中进行了异源表达，并通过功能性His标签或链霉亲和素标签进行了纯化。应用液相色谱电喷雾电离飞行时间质谱（LC/ESI-ToF-MS）和分光光度吡啶血色原测定法，血红素b被鉴定为一种辅因子。此外，使用全反射X射线荧光（TXRF）分析和抑制试验鉴定了与血红素相关的铁。使用耗氧测定法确定了该酶的最适温度和pH分别为30°C和7。通过使用席夫试剂和电喷雾电离质谱（ESI-MS）检测含羰基官能团的裂解产物，证实了加氧酶对聚（顺式-1,4-异戊二烯）和聚（反式-1,4-异戊二烯）的裂解。
