Enzymatic Surface Hydrolysis of PET: Effect of Structural Diversity on Kinetic Properties of Cutinases from Thermobifida
Enzymatic Surface Hydrolysis of PET: Effect of Structural Diversity on Kinetic Properties of Cutinases from Thermobifida
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DOI：
10.1021/ma200949p
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发表时间：
2011-06-28
期刊：
影响因子：
5.5
通讯作者：
Guebitz, Georg
中科院分区：
文献类型：
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作者：
Acero, Enrique Herrero;Ribitsch, Doris;Guebitz, Georg
在本研究中，来自 Thermobifida cellulosilytica DSM44535（Thc_Cut1 和 Thc_Cut2）和 Thermobifida fusca DSM44342（Thf42_Cut1）水解聚对苯二甲酸乙二醇酯（PET）的角质酶在大肠杆菌 BL21-Gold（DE3）中成功克隆和表达。将它们水解 PET 的能力与其他水解天然聚酯的酶进行了比较，包括来自荧光假单胞菌的 PHA 解聚酶 (ePhaZmcl) 和来自 T. fusca KW3 的两种角质酶。三种分离的 Thermobifida 角质酶非常相似（最多只有 18 个氨基酸差异），但对可溶性底物的动力学参数不同。它们在 pNP-乙酸盐上的 k(cat) 和 K-M 值在 2.4-211.9 s(-1) 和 127-200 AIM 范围内，而在 pNP-丁酸盐上，它们的 k(cat) 和 K-m 值在 5.3 和 195.1 s(-1) 之间以及 1483 和 2133 mu M 之间。Thc_Cut1 释放最高量的 MHET 和PET 和对苯二甲酸双(苯甲酰氧基乙基)酯 (3PET) 中的对苯二甲酸伴随着 PET 亲水性的最高增加，如水接触角 (WCA) 降低所示。 FTIR-ATR 分析显示酶处理后结晶度指数 A(1340)/A(1410) 增加，并且使用 2-(溴甲基)萘衍生化测量了羧基和羟基含量的增加。对由角质酶和 3PET 组成的共价结合四面体中间体进行建模表明，活性位点 His-209 位于底物的 0 附近，因此允许水解。另一方面，模型表明，静电和疏水表面特性不同的 Thc_Cut1 和 Thc_Cut2 区域能够与 PET 接触/相互作用，这可以解释它们不同的水解效率。
In this study cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) and Thermobifida fusca DSM44342 (Thf42_Cut1) hydrolyzing poly(ethylene terephthalate) (PET) were successfully cloned and expressed in E.coli BL21-Gold(DE3). Their ability to hydrolyze PET was compared with other enzymes hydrolyzing natural polyesters, including the PHA depolymerase (ePhaZmcl) from Pseudomonas fluorescens and two cutinases from T. fusca KW3. The three isolated Thermobifida cutinases are very similar (only a maximum of 18 amino acid differences) but yet had different kinetic parameters on soluble substrates. Their k(cat) and K-M values on pNP-acetate were in the ranges 2.4-211.9 s(-1) and 127-200 AIM while on pNP-butyrate they showed k(cat) and K-m values between 5.3 and 195.1 s(-1) and between 1483 and 2133 mu M. Thc_Cut1 released highest amounts of MHET and terephthalic acid from PET and bis(benzoyloxyethyl) terephthalate (3PET) with the highest concomitant increase in PET hydrophilicity as indicated by water contact angle (WCA) decreases. FTIR-ATR analysis revealed an increase in the crystallinity index A(1340)/A(1410) upon enzyme treatment and an increase of the amount of carboxylic and hydroxylic was measured using derivatization with 2-(bromomethyl)naphthalene. Modeling the covalently bound tetrahedral intermediate consisting of cutinase and 3PET indicated that the active site His-209 is in the proximity of the 0 of the substrate thus allowing hydrolysis. On the other hand, the models indicated that regions of Thc_Cut1 and Thc_Cut2 which differed in electrostatic and in hydrophobic surface properties were able to reach/interact with PET which may explain their different hydrolysis efficiencies.