Yoshie Naoko, Oike Yoshihiro, Kasuya Ken-ichi, Doi Yoshiharu, Inoue Yoshio
Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Biomacromolecules. 2002 Nov-Dec;3(6):1320-6. doi: 10.1021/bm020077a.
The change in the surface structure of poly[(R)-3-hydroxybutyrate] [PHB] films upon the enzymatic hydrolysis was analyzed by attenuated total reflection infrared [ATR/IR] spectrometry. As enzymes, PHB depolymerases isolated from Ralstonia pickettii T1 and Pseudomonas stutzeri were used. By curve decomposition of the carbonyl stretching band of ATR/IR spectra, the change in the surface crystallinity of PHB films by exposure to buffer containing 0, 1, and 4 microg of PHB depolymerases was estimated. It has been widely believed that the enzymatic hydrolysis first occurs in the amorphous phase, followed by the degradation in the crystalline phase, and extracellular PHB depolymerase can degrade only polymer chains in the surface layer of the film. Therefore, the surface crystallinity had been expected to increase upon the enzymatic degradation. However, the results were contrary to this expectation. The surface crystallinity was decreased by the enzymatic attack. Because ATR/IR spectrometry is sensitive to a small change in molecular structure of the sample surface, the decrease in the crystallinity shown by ATR/IR experiments probably does not indicate the complete loss of regularity of the crystalline phase. Because the chains at crystalline surface are more mobile than those inside the crystals, the C=O band for crystalline surface may appear at a position similar to those of the amorphous or interfacial phase in ATR/IR spectra of PHB. Only the chains inside the crystals may contribute to the C=O band of the crystalline phase. Thus, we rather suppose that the decrease in the crystalline peak of the ATR/IR spectra reflects the change in chain mobility or the increase of crystalline surface area by cracking of lamellas at the surface layers of PHB films or both.
采用衰减全反射红外光谱法（ATR/IR）分析了聚（R）-3-羟基丁酸酯（PHB）薄膜在酶促水解后表面结构的变化。使用从皮氏罗尔斯通氏菌T1和施氏假单胞菌中分离出的PHB解聚酶作为酶。通过对ATR/IR光谱中羰基伸缩带进行曲线分解，估算了PHB薄膜在暴露于含有0、1和4微克PHB解聚酶的缓冲液后表面结晶度的变化。人们普遍认为酶促水解首先发生在非晶相中，随后在晶相中降解，并且细胞外PHB解聚酶只能降解薄膜表层的聚合物链。因此，预计酶促降解后表面结晶度会增加。然而，结果与这一预期相反。酶促作用使表面结晶度降低。由于ATR/IR光谱法对样品表面分子结构的微小变化敏感，ATR/IR实验显示的结晶度降低可能并不表明晶相规则性的完全丧失。因为晶体表面的链比晶体内的链更具流动性，在PHB的ATR/IR光谱中，晶体表面的C=O带可能出现在与非晶相或界面相类似的位置。只有晶体内的链可能对晶相的C=O带有所贡献。因此，我们宁愿认为ATR/IR光谱中结晶峰的降低反映了链流动性的变化，或者是由于PHB薄膜表层片晶的破裂导致晶体表面积的增加，或者两者兼而有之。
