Kasuya Ken-ichi, Takano Tsutomu, Tezuka Yoko, Hsieh W-C, Mitomo Hiroshi, Doi Yoshiharu
Material Science Laboratory, Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, 1-5-1 Tenjin, Kiryu-shi, Gunma 376-8515, Japan.
Int J Biol Macromol. 2003 Dec;33(4-5):221-6. doi: 10.1016/j.ijbiomac.2003.08.006.
A DNA fragment carrying the gene encoding poly(3-hydroxybutyrate) (P(3HB)) depolymerase was cloned from the genomic DNA of Marinobacter sp. DNA sequencing analysis revealed that the Marinobacter sp. P(3HB) depolymerase gene is composed of 1734bp and encodes 578 amino acids with a molecular mass of 61,757Da. A sequence homology search showed that the deduced protein contains the signal peptide, catalytic domain (CD), cadherin-type linker domain (LD), and two substrate-binding domain (SBD). The fusion proteins of glutathione S-transferase (GST) with the CD showed the hydrolytic activity for denatured P(3HB) (dP(3HB)), P(3HB) emulsion (eP(3HB)) and p-nitrophenylbutyrate. On the other hand, the fusion proteins lacking the SBD showed much lower hydrolytic activity for dP(3HB) compared to the proteins containing both CD and SBD. In addition, binding tests revealed that the SBDs are specifically bound not to eP(3HB) but dP(3HB). These suggest that the SBDs play a crucial role in the enzymatic hydrolysis of dP(3HB) that is a solid substrate.
从海杆菌属（Marinobacter sp.）的基因组DNA中克隆出一个携带编码聚（3-羟基丁酸酯）（P(3HB)）解聚酶基因的DNA片段。DNA测序分析表明，海杆菌属的P(3HB)解聚酶基因由1734个碱基对组成，编码578个氨基酸，分子量为61757道尔顿。序列同源性搜索显示，推导的蛋白质包含信号肽、催化结构域（CD）、钙黏蛋白型连接结构域（LD）和两个底物结合结构域（SBD）。谷胱甘肽S-转移酶（GST）与CD的融合蛋白对变性的P(3HB)（dP(3HB)）、P(3HB)乳液（eP(3HB)）和对硝基苯基丁酸酯具有水解活性。另一方面，与同时含有CD和SBD的蛋白质相比，缺少SBD的融合蛋白对dP(3HB)的水解活性要低得多。此外，结合试验表明，SBD特异性结合的不是eP(3HB)，而是dP(3HB)。这些结果表明，SBD在作为固体底物的dP(3HB)的酶促水解中起关键作用。
