Hiessl Sebastian, Böse Dietrich, Oetermann Sylvia, Eggers Jessica, Pietruszka Jörg, Steinbüchel Alexander
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.
Institut für Bioorganische Chemie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich, Jülich, Germany.
Appl Environ Microbiol. 2014 Sep;80(17):5231-40. doi: 10.1128/AEM.01502-14. Epub 2014 Jun 13.
Gordonia polyisoprenivorans strain VH2, a potent rubber-degrading actinomycete, harbors two latex clearing proteins (Lcps), which are known to be essential for the microbial degradation of rubber. However, biochemical information on the exact role of this protein in the degradation of polyisoprene was lacking. In this study, the gene encoding Lcp1VH2 was heterologously expressed in strains of Escherichia coli, the corresponding protein was purified, and its role in rubber degradation was examined by measurement of oxygen consumption as well as by chromatographic and spectroscopic methods. It turned out that active Lcp1VH2 is a monomer and is responsible for the oxidative cleavage of poly(cis-1,4-isoprene) in synthetic as well as in natural rubber by the addition of oxygen (O2) to the cis double bonds. The resulting oligomers possess repetitive isoprene units with aldehyde (CHO-CH2-) and ketone (-CH2-CO-CH3) functional groups at the termini. Two fractions with average isoprene contents of 18 and 10, respectively, were isolated, thus indicating an endocleavage mechanism. The activity of Lcp1VH2 was determined by applying a polarographic assay. Alkenes, acyclic terpenes, or other rubber-like polymers, such as poly(cis-1,4-butadiene) or poly(trans-1,4-isoprene), are not oxidatively cleaved by Lcp1VH2. The pH and temperature optima of the enzyme are at pH 7 and 30°C, respectively. Furthermore, it was demonstrated that active Lcp1VH2 is a Cu(II)-containing oxygenase that exhibits a conserved domain of unknown function which cannot be detected in any other hitherto-characterized enzyme. The results presented here indicate that this domain might represent a new protein family of oxygenases.
戈登氏聚异戊二烯ivorans菌株VH2是一种高效的橡胶降解放线菌，含有两种乳胶清除蛋白（Lcps），已知这两种蛋白对橡胶的微生物降解至关重要。然而，关于该蛋白在聚异戊二烯降解中的确切作用的生化信息尚缺乏。在本研究中，编码Lcp1VH2的基因在大肠杆菌菌株中进行了异源表达，纯化了相应的蛋白，并通过测量氧气消耗以及色谱和光谱方法研究了其在橡胶降解中的作用。结果表明，活性Lcp1VH2是一种单体，通过向顺式双键添加氧气（O2），负责合成橡胶和天然橡胶中聚（顺式-1,4-异戊二烯）的氧化裂解。生成的低聚物在末端具有带有醛（CHO-CH2-）和酮（-CH2-CO-CH3）官能团的重复异戊二烯单元。分离出了平均异戊二烯含量分别为18和10的两个馏分，从而表明存在一种内切裂解机制。通过极谱法测定了Lcp1VH2的活性。烯烃、无环萜烯或其他类似橡胶的聚合物，如聚（顺式-1,4-丁二烯）或聚（反式-1,4-异戊二烯），不会被Lcp1VH2氧化裂解。该酶的最适pH和温度分别为pH 7和30°C。此外，还证明活性Lcp1VH2是一种含铜（II）的加氧酶，它具有一个功能未知的保守结构域，在迄今已表征的任何其他酶中都无法检测到。此处给出的结果表明，该结构域可能代表一个新的加氧酶蛋白家族。
