Enzymatic Surface Hydrolysis of PET: Effect of Structural Diversity on Kinetic Properties of Cutinases from Thermobifida

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• 作者：Enrique Herrero Acero ; Doris Ribitsch ; Georg Steinkellner ; Karl Gruber ; Katrin Greimel ; Inge Eiteljoerg ; Eva Trotscha ; Ren Wei ; Wolfgang Zimmermann ; Manfred Zinn ; Artur Cavaco-Paulo ; Giuliano Freddi ; Helmut Schwab ; Georg Guebitz
• 刊名：Macromolecules
• 出版年：2011
• 出版时间：June 28, 2011
• 年：2011
• 卷：44
• 期：12
• 页码：4632-4640
• 全文大小：999K
• 年卷期：v.44,no.12(June 28, 2011)
• ISSN：1520-5835

文摘

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鈥揳cetate were in the ranges 2.4鈥?11.9 s^鈥? and 127鈥?00 渭M while on pNP鈥揵utyrate they showed k_cat and K_m values between 5.3 and 195.1 s^鈥? and between 1483 and 2133 渭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 A₁₃₄₀/A₁₄₁₀ 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 O 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.