Gamerith Caroline, Vastano Marco, Ghorbanpour Sahar M, Zitzenbacher Sabine, Ribitsch Doris, Zumstein Michael T, Sander Michael, Herrero Acero Enrique, Pellis Alessandro, Guebitz Georg M
Austrian Centre of Industrial BiotechnologyTulln, Austria.
Institute of Environmental Biotechnology, University of Natural Resources and Life Sciences ViennaTulln, Austria.
Front Microbiol. 2017 May 24;8:938. doi: 10.3389/fmicb.2017.00938. eCollection 2017.
To study hydrolysis of aromatic and aliphatic polyesters cutinase 1 from (Thc_Cut1) was expressed in . No significant differences between the expression of native Thc_Cut1 and of two glycosylation site knock out mutants (Thc_Cut1_koAsn and Thc_Cut1_koST) concerning the total extracellular protein concentration and volumetric activity were observed. Hydrolysis of poly(ethylene terephthalate) (PET) was shown for all three enzymes based on quantification of released products by HPLC and similar concentrations of released terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) were detected for all enzymes. Both tested aliphatic polyesters poly(butylene succinate) (PBS) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were hydrolyzed by Thc_Cut1 and Thc_Cut1_koST, although PBS was hydrolyzed to significantly higher extent than PHBV. These findings were also confirmed quartz crystal microbalance (QCM) analysis; for PHBV only a small mass change was observed while the mass of PBS thin films decreased by 93% upon enzymatic hydrolysis with Thc_Cut1. Although both enzymes led to similar concentrations of released products upon hydrolysis of PET and PHBV, Thc_Cut1_koST was found to be significantly more active on PBS than the native Thc_Cut1. Hydrolysis of PBS films by Thc_Cut1 and Thc_Cut1_koST was followed by weight loss and scanning electron microscopy (SEM). Within 96 h of hydrolysis up to 92 and 41% of weight loss were detected with Thc_Cut1_koST and Thc_Cut1, respectively. Furthermore, SEM characterization of PBS films clearly showed that enzyme tretment resulted in morphological changes of the film surface.
为了研究芳香族和脂肪族聚酯的水解情况，来自[具体来源未明确]的角质酶1（Thc_Cut1）在[具体表达宿主未明确]中进行了表达。在总细胞外蛋白浓度和体积活性方面，未观察到天然Thc_Cut1与两个糖基化位点敲除突变体（Thc_Cut1_koAsn和Thc_Cut1_koST）的表达有显著差异。基于通过高效液相色谱法（HPLC）对释放产物的定量分析，所有三种酶均显示出对聚对苯二甲酸乙二酯（PET）的水解作用，并且检测到所有酶释放的对苯二甲酸（TPA）和单（2 - 羟乙基）对苯二甲酸酯（MHET）浓度相似。两种测试的脂肪族聚酯聚丁二酸丁二醇酯（PBS）和聚（3 - 羟基丁酸酯 - 共 - 3 - 羟基戊酸酯）（PHBV）均被Thc_Cut1和Thc_Cut1_koST水解，尽管PBS的水解程度明显高于PHBV。这些发现也通过石英晶体微天平（QCM）分析得到了证实；对于PHBV，仅观察到较小的质量变化，而在用Thc_Cut1进行酶水解后，PBS薄膜的质量下降了93%。尽管两种酶在水解PET和PHBV时导致释放产物的浓度相似，但发现Thc_Cut1_koST对PBS的活性明显高于天然Thc_Cut1。通过重量损失和扫描电子显微镜（SEM）跟踪了Thc_Cut1和Thc_Cut1_koST对PBS薄膜的水解情况。在水解96小时内，Thc_Cut1_koST和Thc_Cut1分别检测到高达92%和41%的重量损失。此外，PBS薄膜的SEM表征清楚地表明，酶处理导致薄膜表面形态发生变化。
