Numata Keiji, Yamashita Koichi, Fujita Masahiro, Tsuge Takeharu, Kasuya Ken-Ichi, Iwata Tadahisa, Doi Yoshiharu, Abe Hideki
Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama, Japan.
Biomacromolecules. 2007 Jul;8(7):2276-81. doi: 10.1021/bm070231z. Epub 2007 Jun 5.
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.
利用原子力显微镜（AFM）和石英晶体微天平（QCM）对聚（R）-3-羟基丁酸酯（P(3HB)）与皮氏罗尔斯通氏菌T1和绳状青霉分泌的两种聚羟基丁酸酯（PHB）解聚酶的反应过程进行了表征。皮氏罗尔斯通氏菌T1的PHB解聚酶由催化结构域、连接结构域和底物结合结构域组成，而绳状青霉的PHB解聚酶缺乏底物结合结构域。我们通过实时AFM成功观察到皮氏罗尔斯通氏菌T1的PHB解聚酶单分子在37℃的磷酸盐缓冲溶液中吸附到P(3HB)单晶上以及单晶的降解过程。相反，尽管单晶的酶促降解确实在进行，但通过实时AFM在P(3HB)单晶表面几乎观察不到绳状青霉的酶分子。然而，根据酶处理后在空气中对P(3HB)单晶的AFM观察，不仅皮氏罗尔斯通氏菌T1的PHB解聚酶，而且绳状青霉的PHB解聚酶都吸附到了P(3HB)晶体表面上，并且两种酶在表面的浓度几乎相同。这意味着两种酶都吸附到了P(3HB)单晶表面。此外，QCM测量定量地阐明了两种PHB解聚酶在脱离行为上的差异，即在水性条件下，皮氏罗尔斯通氏菌T1的酶几乎不脱离，但绳状青霉的酶很容易从P(3HB)晶体表面释放。
