Handrick René, Technow Ulrike, Reichart Thomas, Reinhardt Simone, Sander Till, Jendrossek Dieter
Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70550, Stuttgart, Germany.
FEMS Microbiol Lett. 2004 Jan 30;230(2):265-74. doi: 10.1016/S0378-1097(03)00919-4.
Hydrolysis of native (amorphous) polyhydroxybutyrate (nPHB) granules isolated from different sources by soluble PHB depolymerase of Rhodospirillum rubrum in vitro requires the presence of a heat-stable compound (activator). The activator was purified and was resistant against various physical and chemical stresses such as heat (up to 130 degrees C), pH 1-12, dryness, oxidation by H2O2, reducing and denaturing compounds (2-mercaptoethanol, 5 M guanidinium-HCl) and many solvents including phenol/chloroform. The activator coding gene was identified by N-terminal sequencing of the purified protein, and the deduced protein showed significant homology to magnetosome-associated protein (Mms16) of magnetotactic bacteria. Analysis of the activation process in vitro showed that the activator acts on nPHB granules but not on the depolymerase. The effect of the activator could be mimicked by pretreatment of nPHB granules with trypsin or other proteases but protease activity of the purified activator was not detected. Evidence is shown that different mechanisms were responsible for activation of nPHB by trypsin and activator, respectively. PHB granule-associated protein (PhaP) of Ralstonia eutropha nPHB granules were cleaved by trypsin but no cleavage occurred after activator treatment. Hydrolysis of artificial protein-free PHB granules coated with negatively charged detergents (sodium dodecyl sulfate (SDS), cholate but not cetyltrimethyl-ammonium bromide (CTAB)) did not require activation and confirmed that surface layer proteins of nPHB granules are the targets of the activator rather than lipids. All experimental data are in agreement with the assumption that trypsin and the activator enable the PHB depolymerase to find and to bind to the polymer surface: trypsin by removing a portion of proteins from the polymer surface, the activator by modifying the surface structure in a not yet understood manner presumably by interaction with phasins of the proteinous surface layer of nPHB.
体外利用红螺菌的可溶性聚羟基丁酸酯解聚酶水解从不同来源分离得到的天然（无定形）聚羟基丁酸酯（nPHB）颗粒，需要一种热稳定化合物（激活剂）的存在。该激活剂被纯化出来，并且能抵抗各种物理和化学应激，如高温（高达130摄氏度）、pH值1至12、干燥、过氧化氢氧化、还原和变性化合物（2-巯基乙醇、5M盐酸胍）以及包括苯酚/氯仿在内的多种溶剂。通过对纯化蛋白进行N端测序鉴定出激活剂编码基因，推导的蛋白与趋磁细菌的磁小体相关蛋白（Mms16）具有显著同源性。体外激活过程分析表明，激活剂作用于nPHB颗粒而非解聚酶。用胰蛋白酶或其他蛋白酶预处理nPHB颗粒可模拟激活剂的作用，但未检测到纯化激活剂的蛋白酶活性。有证据表明，胰蛋白酶和激活剂分别通过不同机制激活nPHB。真养产碱菌nPHB颗粒的聚羟基丁酸酯颗粒相关蛋白（PhaP）被胰蛋白酶切割，但激活剂处理后未发生切割。用带负电荷的去污剂（十二烷基硫酸钠（SDS）、胆酸盐，但不是十六烷基三甲基溴化铵（CTAB））包被的人工无蛋白PHB颗粒的水解不需要激活，这证实了nPHB颗粒的表面层蛋白是激活剂的作用靶点而非脂质。所有实验数据都与以下假设一致：胰蛋白酶和激活剂使聚羟基丁酸酯解聚酶能够找到并结合到聚合物表面，胰蛋白酶通过从聚合物表面去除一部分蛋白质，激活剂则通过以一种尚未明确的方式修饰表面结构，可能是通过与nPHB蛋白质表面层的相蛋白相互作用。
