Cleavage of poly(cis-1,4-isoprene) by the latex clearing protein (Lcp) and involvement of a molybdenum-dependent oxidoreductase (OxiBA) in early steps of rubber degradation

本项目研究了链霉菌（Streptomyces sp. K30）的Lcp （Latex clearing protein）对聚顺式-1,4-异戊二烯的初始裂解作用。Lcp同源物存在于所有其他革兰氏阳性橡胶和胶过胶降解细菌中；这种蛋白质似乎通常负责放线菌对多异戊二烯类的裂解。目的如下：(i)通过Tat途径研究Lcp的分泌。（ii）采用突变菌株和大肠杆菌-链霉菌穿梭载体进行随机诱变，将解开Lcp的必需氨基酸残基；（iii）将通过位点特异性诱变进行验证。（iv）将开发Lcp的酶活性测定，以表征野生型和突变的Lcp蛋白，并研究Lcp的调控。(v)在聚（顺-1,4-异戊二烯）裂解过程中，氧被纳入降解产物。酶活性测定和氨基酸参与切割的知识将有助于确定Lcp切割橡胶的分子机制。在这种情况下，假定的辅助因素和Lcp的三维结构将被确定。（vi）位于lcp下游的一种被推测为由氧和氧b编码的醛脱氢酶明显地氧化lcp产生的裂解产物。我们将研究oxiAB的功能和调控。（vii）利用farcinica诺卡菌IFM10152（唯一已知基因组序列的橡胶降解细菌）进行二维凝胶电泳，将揭示更多参与聚顺式-1,4-异戊二烯降解的蛋白质。

This project investigates initial cleavage of poly(cis-1,4-isoprene) by Lcp (Latex clearing protein) from Streptomyces sp. K30. Lcp homologues occur in all other Gram-positive rubber and gutta percha degrading bacteria; this protein seems to be generally responsible for cleavage of polyisoprenoides by Actinobacteria. The aims are as follows: (i) Secretion of Lcp via the Tat pathway will be investigated. (ii) Random mutagenesis employing a mutator strain and an E.coli-Streptomyces shuttle vector will unravel essential amino acid residues of Lcp, (iii) which will be verified by site specific mutagenesis. (iv) An enzyme activity assay for Lcp will be developed to characterize wild type and mutated Lcp proteins and to investigate regulation of Lcp. (v) During cleavage of poly(cis-1,4-isoprene), oxygen is incorporated into the degradation products. An enzyme activity assay and knowledge of amino acids involved in cleavage will enable to determine the molecular mechanism of rubber cleavage by Lcp. In this context, putative co-factors and the 3D structure of Lcp will be identified. (vi) A putative aldehyde dehydrogenase encoded by oxiA and oxiB located downstream of lcp obviously oxidizes the cleavage products produced by Lcp. Function and regulation of oxiAB will be investigated. (vii) 2D gel electrophoresis employing Nocardia farcinica IFM10152, the only rubber degrading bacterium with known genome sequence, will unravel further proteins involved in poly(cis-1,4-isoprene) degradation.