Fusion of Binding Domains to Thermobifida cellulosilytica Cutinase to Tune Sorption Characteristics and Enhancing PET Hydrolysis

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• 作者：Doris Ribitsch ; Antonio Orcal Yebra ; Sabine Zitzenbacher ; Jing Wu ; Susanne Nowitsch ; Georg Steinkellner ; Katrin Greimel ; Ales Doliska ; Gustav Oberdorfer ; Christian C. Gruber ; Karl Gruber ; Helmut Schwab ; Karin Stana-Kleinschek ; Enrique Herrero Acero ; Georg M. Guebitz
• 刊名：Biomacromolecules
• 出版年：2013
• 出版时间：June 10, 2013
• 年：2013
• 卷：14
• 期：6
• 页码：1769-1776
• 全文大小：395K
• 年卷期：v.14,no.6(June 10, 2013)
• ISSN：1526-4602

文摘

A cutinase from Thermomyces cellullosylitica (Thc_Cut1), hydrolyzing the synthetic polymer polyethylene terephthalate (PET), was fused with two different binding modules to improve sorption and thereby hydrolysis. The binding modules were from cellobiohydrolase I from Hypocrea jecorina (CBM) and from a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM). Although both binding modules have a hydrophobic nature, it was possible to express the proteins in E. coli. Both fusion enzymes and the native one had comparable k_cat values in the range of 311 to 342 s^鈥? on pNP-butyrate, while the catalytic efficiencies k_cat/K_m decreased from 0.41 s^鈥?/ 渭M (native enzyme) to 0.21 and 0.33 s^鈥?/渭M for Thc_Cut1+PBM and Thc_Cut1+CBM, respectively. The fusion enzymes were active both on the insoluble PET model substrate bis(benzoyloxyethyl) terephthalate (3PET) and on PET although the hydrolysis pattern was differed when compared to Thc_Cut1. Enhanced adsorption of the fusion enzymes was visible by chemiluminescence after incubation with a 6xHisTag specific horseradish peroxidase (HRP) labeled probe. Increased adsorption to PET by the fusion enzymes was confirmed with Quarz Crystal Microbalance (QCM-D) analysis and indeed resulted in enhanced hydrolysis activity (3.8脳 for Thc_Cut1+CBM) on PET, as quantified, based on released mono/oligomers.