Characteristic Interactions between Poly(hydroxybutyrate) Depolymerase and Poly[(R)-3-hydroxybutyrate] Film Studied by a Quartz Crystal Microbalance
Characteristic Interactions between Poly(hydroxybutyrate) Depolymerase and Poly[(R)-3-hydroxybutyrate] Film Studied by a Quartz Crystal Microbalance
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通过石英晶体微天平研究聚(羟基丁酸酯)解聚酶和聚[(R)-3-羟基丁酸酯]膜之间的特征相互作用
DOI：
10.1002/mabi.200300004
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发表时间：
2003
影响因子：
4.6
通讯作者：
Y. Doi
中科院分区：
文献类型：
--
作者：
K. Yamashita;T. Funato;Yoichi Suzuki;S. Teramachi;Y. Doi
采用石英晶体微天平（QCM）技术，研究了在0.01 M磷酸盐缓冲溶液（pH7.4）中，37°C条件下，聚（R）-3-羟基丁酸酯（P（3 HB））膜被Ralstonia picketti T1的聚羟基丁酸酶（PHB）解聚酶降解的情况。P（3 HB）膜的酶促降解定量地跟随QCM作为正频移。而吸附在膜上的解聚酶的量可以通过使用在催化位点中没有水解活性的突变酶来评估为负频移。降解速率随酶浓度的增加而增加，在1.0 μg·mL-1时达到最大值，随着酶浓度的增加，降解速率逐渐降低。这种酶的浓度依赖性可以定量地解释在由吸附的酶的膜表面的覆盖率的变化。当QCM池中的野生型酶溶液在反应中间被突变酶溶液替换时，降解速率显著降低，表明吸附在P（3 HB）表面上的野生型酶容易被溶液中的突变酶取代。另一方面，用其他蛋白质或缓冲溶液替换野生型酶溶液根本不影响降解速率，这表明吸附的酶没有从膜表面解吸。因此，吸附的PHB解聚酶仅通过与溶液中相同的解聚酶相互作用而从P（3 HB）表面释放。
Enzymatic degradation of poly[(R)-3-hydroxybutyrate] (P(3HB)) film by the poly(hydroxybutyrase) (PHB) depolymerase from Ralstonia picketti T1 was studied in 0.01 M phosphate buffer solution (pH7.4) at 37°C by using a quartz crystal microbalance (QCM) technique. Enzymatic degradation of P(3HB) film was quantitatively followed by QCM as a positive frequency shift. While, the amount of depolymerases adsorbed on the film could be evaluated as a negative frequency shift by using a mutant enzyme which had no hydrolytic activity in a catalytic site. The degradation rate increased with enzyme concentration to reach a maximum value at 1.0 μg.mL -1 , and then the rate decreased at higher enzyme concentration. This enzyme concentration dependence could be quantitatively explained in terms of a change of coverage of the film surface by the adsorbed enzyme. When the wild-type enzyme solution in a QCM cell was replaced with the mutant enzyme solution in the middle of the reaction, the degradation rate was reduced markedly, indicating that the wild-type enzyme adsorbed on the P(3HB) surface is easily substituted by the nutant enzyme in the solution. On the other hand, replacement of the wild-type enzyme solution with other proteins or buffer solutions did not affect the degradation rate at all, suggesting that the adsorbed enzyme was not desorbed from the film surface. Thus, the adsorbed PHB depolymerase is released from the P(3HB) surface only by interaction with the same depolymerase in solution.