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Functional analysis of chimeric TrCel6A enzymes with different carbohydrate binding modules.
Protein Engineering, Design and Selection ( IF 2.4 ) Pub Date : 2020-02-25 , DOI: 10.1093/protein/gzaa003
Stefan Jarl Christensen 1 , Silke Flindt Badino 1 , Ana Mafalda Cavaleiro 1, 2 , Kim Borch 2 , Peter Westh 3
Affiliation  

The glycoside hydrolase (GH) family 6 is an important group of enzymes that constitute an essential part of industrial enzyme cocktails used to convert lignocellulose into fermentable sugars. In nature, enzymes from this family often have a carbohydrate binding module (CBM) from the CBM family 1. These modules are known to promote adsorption to the cellulose surface and influence enzymatic activity. Here, we have investigated the functional diversity of CBMs found within the GH6 family. This was done by constructing five chimeric enzymes based on the model enzyme, TrCel6A, from the soft-rot fungus Trichoderma reesei. The natural CBM of this enzyme was exchanged with CBMs from other GH6 enzymes originating from different cellulose degrading fungi. The chimeric enzymes were expressed in the same host and investigated in adsorption and quasi-steady-state kinetic experiments. Our results quantified functional differences of these phylogenetically distant binding modules. Thus, the partitioning coefficient for substrate binding varied 4-fold, while the maximal turnover (kcat) showed a 2-fold difference. The wild-type enzyme showed the highest cellulose affinity on all tested substrates and the highest catalytic turnover. The CBM from Serendipita indica strongly promoted the enzyme's ability to form productive complexes with sites on the substrate surface but showed lower turnover of the complex. We conclude that the CBM plays an important role for the functional differences between GH6 wild-type enzymes.

中文翻译:

具有不同碳水化合物结合模块的嵌合TrCel6A酶的功能分析。

糖苷水解酶(GH)家族6是重要的一组酶,它们构成了用于将木质纤维素转化为可发酵糖的工业酶混合物的重要组成部分。实际上,来自该家族的酶通常具有来自CBM家族1的碳水化合物结合模块(CBM)。已知这些模块可促进对纤维素表面的吸附并影响酶活性。在这里,我们调查了GH6家族中发现的CBM的功能多样性。这是通过从软腐真菌里氏木霉(Trichoderma reesei)构建基于模型酶TrCel6A的五种嵌合酶来完成的。该酶的天然CBM与其他GH6酶的CBM交换,这些GH6酶来自不同的纤维素降解真菌。嵌合酶在同一宿主中表达,并在吸附和准稳态动力学实验中进行了研究。我们的结果量化了这些系统发育距离遥远的绑定模块的功能差异。因此,底物结合的分配系数变化了4倍,而最大周转率(kcat)则显示了2倍的差异。野生型酶在所有测试的底物上显示出最高的纤维素亲和力和最高的催化转化率。来自印度Serendipita的CBM大大增强了酶形成具有底物表面位点的生产性复合物的能力,但显示出较低的复合物周转率。我们得出结论,煤层气对GH6野生型酶之间的功能差异起着重要作用。我们的结果量化了这些系统发育距离遥远的绑定模块的功能差异。因此,底物结合的分配系数变化了4倍,而最大周转率(kcat)则显示了2倍的差异。野生型酶在所有测试的底物上显示出最高的纤维素亲和力和最高的催化转化率。来自印度Serendipita的CBM大大增强了酶形成具有底物表面位点的生产性复合物的能力,但显示出较低的复合物周转率。我们得出结论,煤层气对GH6野生型酶之间的功能差异起着重要作用。我们的结果量化了这些系统发育距离遥远的绑定模块的功能差异。因此,底物结合的分配系数变化了4倍,而最大周转率(kcat)则显示了2倍的差异。野生型酶在所有测试的底物上显示出最高的纤维素亲和力和最高的催化转化率。来自印度Serendipita的CBM大大增强了酶形成具有底物表面位点的生产性复合物的能力,但显示出较低的复合物周转率。我们得出结论,煤层气对GH6野生型酶之间的功能差异起着重要作用。野生型酶在所有测试的底物上显示出最高的纤维素亲和力和最高的催化转化率。来自印度Serendipita的CBM大大增强了酶形成具有底物表面位点的生产性复合物的能力,但显示出较低的复合物周转率。我们得出结论,煤层气对GH6野生型酶之间的功能差异起着重要作用。野生型酶在所有测试的底物上显示出最高的纤维素亲和力和最高的催化转化率。来自印度Serendipita的CBM大大增强了酶形成具有底物表面位点的生产性复合物的能力,但显示出较低的复合物周转率。我们得出结论,煤层气对GH6野生型酶之间的功能差异起着重要作用。
更新日期:2020-02-26
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