Enrique Herrero Acero - Enzymatic Surface Hydrolysis of PET: Effect of Structural Diversity on Kinetic Properties of Cutinases from Thremobifida

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  Publication Details (including relevant citation   information):

  Macromolecules, 2011, 44 (12), pp 4632–4640

  DOI: 10.1021/ma200949p

  Publication Date (Web): May 20, 2011


  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 kcat and Km values on pNP–acetate were in the   ranges 2.4–211.9 s–1 and   127–200 μM while on pNP–butyrate they showed   kcat and   Km values between   5.3 and 195.1 s–1 and   between 1483 and 2133 μM. 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.

  Address (URL): http://pubs.acs.org/doi/abs/10.1021/ma200949p