Kasuya K, Ohura T, Masuda K, Doi Y
Polymer Chemistry Laboratory, The Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
Int J Biol Macromol. 1999 May;24(4):329-36. doi: 10.1016/s0141-8130(99)00046-x.
The substrate specificities of three extracellular polyhydroxybutyrate (PHB) depolymerases from Alcaligenes faecalis (PhaZ Afa), Pseudomonas stutzeri (PhaZ Pst), and Comamonas acidovorans (PhaZ Cac), which are grouped into types A and B based on the position of a lipase box sequence in the catalytic domain, were examined for films of 12 different aliphatic polyesters. Each of these PHB depolymerases used was capable of hydrolyzing poly(3-hydroxybutyrate) (P(3HB)), poly(3-hydroxypropionate) (P(3HP)), poly(4-hydroxybutyrate) (P(4HB)), poly(ethylene succinate) (PESU), and poly(ethylene adipate) (PEA) but could not hydrolyze another seven polyesters. In addition, the binding characteristics of substrate binding domains from PhaZ Afa, PhaZ Cac, and PHB depolymerase from Comamonas testosteroni (PhaZ Cte) were studied by using fusions with glutathione S-transferase (GST). All of fusion proteins adsorbed strongly on the surfaces of polyester granules of P(3HB), P(3HP), and poly(2-hydroxypropionate) (P(2HP)) which was not hydrolyzed by the PHB depolymerases used in this study, while they did not bind on Avicel and chitin granules. The adsorption kinetics of the fusion proteins to the surface of P(3HB) and P(2HP) granules were found to obey the Langmuir isotherm. The cross-area per molecule of fusion protein bound to P(3HB) granules was estimated to be 12+/-4 nm2/molecule. It has been suggested that the active sites in catalytic domains of PHB depolymerases have a similar conformational structure, and that several amino acids in substrate-binding domains of PHB depolymerases interact specifically with the surface of polyesters.
对粪产碱杆菌（PhaZ Afa）、施氏假单胞菌（PhaZ Pst）和食酸丛毛单胞菌（PhaZ Cac）的三种细胞外聚羟基丁酸酯（PHB）解聚酶的底物特异性进行了研究，这三种酶根据催化结构域中脂肪酶盒序列的位置分为A和B型，研究对象为12种不同脂肪族聚酯薄膜。所使用的每种PHB解聚酶都能够水解聚（3-羟基丁酸酯）（P(3HB)）、聚（3-羟基丙酸酯）（P(3HP)）、聚（4-羟基丁酸酯）（P(4HB)）、聚琥珀酸乙二酯（PESU）和聚己二酸乙二酯（PEA），但不能水解其他七种聚酯。此外，通过与谷胱甘肽S-转移酶（GST）融合，研究了PhaZ Afa、PhaZ Cac和睾丸酮丛毛单胞菌的PHB解聚酶（PhaZ Cte）的底物结合结构域的结合特性。所有融合蛋白都强烈吸附在P(3HB)、P(3HP)和聚（2-羟基丙酸酯）（P(2HP)）的聚酯颗粒表面，而本研究中使用的PHB解聚酶不能水解P(2HP)，同时它们不与微晶纤维素和几丁质颗粒结合。发现融合蛋白与P(3HB)和P(2HP)颗粒表面的吸附动力学符合朗缪尔等温线。与P(3HB)颗粒结合的融合蛋白每分子的交叉面积估计为12±4 nm2/分子。有人提出，PHB解聚酶催化结构域中的活性位点具有相似的构象结构，并且PHB解聚酶底物结合结构域中的几个氨基酸与聚酯表面特异性相互作用。
