Adsorption and hydrolysis reactions of poly(hydroxybutyric acid) depolymerases secreted from Ralstonia pickettii T1 and Penicillium funiculosum onto poly[(R)-3-hydroxybutyric acid]
Adsorption and hydrolysis reactions of poly(hydroxybutyric acid) depolymerases secreted from Ralstonia pickettii T1 and Penicillium funiculosum onto poly[(R)-3-hydroxybutyric acid]
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DOI：
10.1021/bm070231z
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发表时间：
2007-07-01
影响因子：
6.2
通讯作者：
Abe, Hideki
中科院分区：
文献类型：
--
作者：
Numata, Keiji;Yamashita, Koichi;Abe, Hideki
采用原子力显微镜（AFM）和石英晶体微天平（QCM）表征了聚[(R)-3-羟基丁酸]（P(3HB)）与两种聚（羟基丁酸）解聚合酶（PHB）的反应过程。聚(R)-3-羟基丁酸]（P(3HB)）分别来自匹克Ralstonia pickkettii T1和真菌青霉菌。pickettii T1的PHB解聚合酶由催化、连接和底物结合结构域组成，而P. funiculosum的PHB解聚合酶则缺乏底物结合结构域。利用实时原子力显微镜（AFM）观察了皮氏菌T1中PHB解聚合酶单分子在P（3HB）单晶上的吸附以及P（3HB）单晶在磷酸盐缓冲液中37℃下的降解过程。相反，尽管酶降解过程确实在进行，但在P（3HB）单晶表面，用实时原子力显微镜几乎观察不到来自真菌的酶分子。通过对P（3HB）单晶在空气中的原子力显微镜观察，结果表明，在P（3HB）单晶表面吸附的酶不仅有来自皮氏菌T1的PHB解聚合酶，还有来自P. funiculosum的PHB解聚合酶，而且两种酶的浓度几乎相同。这意味着两种酶都吸附在P（3HB）单晶的表面上。此外，QCM测量定量地阐明了两种类型的PHB解聚合酶之间的分离行为差异，即在水条件下，来自匹克氏菌T1的酶几乎不分离，而来自真菌的酶很容易从P（3HB）晶体表面释放。
Reaction processes of poly[(R)-3-hydroxybutyric acid] (P(3HB)) with two types of poly(hydroxybutyric acid) (PHB) depolymerases secreted from Ralstonia pickettii T1 and Penicillium funiculosum were characterized by means of atomic force microscopy (AFM) and quartz crystal microbalance (QCM). The PHB depolymerase from R. pickettii T1 consists of catalytic, linker, and substrate-binding domains, whereas the one from P. funiculosum lacks a substrate-binding domain. We succeeded in observing the adsorption of single molecules of the PHB depolymerase from R. pickettii T1 onto P(3HB) single crystals and the degradation of the single crystals in a phosphate buffer solution at 37 degrees C by real-time AFM. On the contrary, the enzyme molecule from P. funiculosum was hardly observed at the surface of P(3HB) single crystals by real-time AFM, even though the enzymatic degradation of the single crystals was surely progressed. On the basis of the AFM observations in air of the P(3HB) single crystals after the enzymatic treatments, however, not only the PHB depolymerase from R. pickettii T1 but also that from P. funiculosum adsorbed onto the surface of P(3HB) crystals, and both concentrations of the enzymes on the surface were nearly identical. This means both enzymes were adsorbed onto the surface of P(3HB) single crystals. Moreover, QCM measurements clarified quantitatively the differences in detachment behavior between two types of PHB depolymerases, namely the enzyme from R. pickettii T1 was hardly detached but the enzyme from P. funiculosum was released easily from the surface of P(3HB) crystals under an aqueous condition.