Department of Chemical and Biochemical Engineering, Technical University of Denmark, Kongens Lyngby, Denmark.
VTT Technical Research Center of Finland Ltd., Finland.
Biotechnol Bioeng. 2018 Dec;115(12):2869-2880. doi: 10.1002/bit.26820. Epub 2018 Oct 16.
Adsorption of cellulases onto lignin is considered a major factor in retarding enzymatic cellulose degradation of lignocellulosic biomass. However, the adsorption mechanisms and kinetics are not well understood for individual types of cellulases. This study examines the binding affinity, kinetics of adsorption, and competition of four monocomponent cellulases of Trichoderma reesei during adsorption onto lignin. TrCel7A, TrCel6A, TrCel7B, and TrCel5A were radiolabeled for adsorption experiments on lignin-rich residues (LRRs) isolated from hydrothermally pretreated spruce (L-HPS) and wheat straw (L-HPWS), respectively. On the basis of adsorption isotherms fitted to the Langmuir model, the ranking of binding affinities was TrCel5A > TrCel6A > TrCel7B > TrCel7A on both types of LRRs. The enzymes had a higher affinity to the L-HPS than to the L-HPWS. Adsorption experiments with dilution after 1 and 24 hr and kinetic modeling were performed to quantify any irreversible binding over time. Models with reversible binding parameters fitted well and can explain the results obtained. The adsorption constants obtained from the reversible models agreed with the fitted Langmuir isotherms and suggested that reversible adsorption-desorption existed at equilibrium. Competitive binding experiments showed that individual types of cellulases competed for binding sites on the lignin and the adsorption data fitted the Langmuir adsorption model. Overall, the data strongly indicate that the adsorption of cellulases onto lignin is reversible and the findings have implications for the development of more efficient cellulose degrading enzymes.
纤维素酶吸附到木质素上被认为是延缓木质纤维素生物质酶解的主要因素。然而，对于单一类型的纤维素酶，其吸附机制和动力学仍不清楚。本研究考察了四种重组木霉纤维素酶（TrCel7A、TrCel6A、TrCel7B 和 TrCel5A）在吸附到木质素丰富的残留物（LRR）上时的结合亲和力、吸附动力学和竞争。TrCel7A、TrCel6A、TrCel7B 和 TrCel5A 分别被放射性标记，用于在水热预处理云杉（L-HPS）和小麦秸秆（L-HPWS）分离的富含木质素的残留物（LRR）上进行吸附实验。根据吸附等温线拟合的 Langmuir 模型，在两种类型的 LRR 上，结合亲和力的排序为 TrCel5A > TrCel6A > TrCel7B > TrCel7A。酶对 L-HPS 的亲和力高于 L-HPWS。进行了 1 和 24 小时后稀释的吸附实验和动力学建模，以量化随时间不可逆结合的任何情况。具有可逆结合参数的模型拟合良好，可以解释所获得的结果。从可逆模型获得的吸附常数与拟合的 Langmuir 等温线一致，并表明在平衡时存在可逆吸附-解吸。竞争结合实验表明，不同类型的纤维素酶竞争木质素上的结合位点，吸附数据拟合 Langmuir 吸附模型。总体而言，数据强烈表明纤维素酶吸附到木质素上是可逆的，这一发现对开发更有效的纤维素降解酶具有重要意义。
