Ariaeenejad Shohreh, Lanjanian Hossein, Motamedi Elaheh, Kavousi Kaveh, Moosavi-Movahedi Ali A, Hosseini Salekdeh Ghasem
Department of Systems and Synthetic Biology, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research Education and Extension Organization (AREEO), Karaj, 31359, Iran.
Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, 13145, Iran.
Bioconjug Chem. 2020 Sep 16;31(9):2158-2171. doi: 10.1021/acs.bioconjchem.0c00361. Epub 2020 Aug 11.
While polysaccharide-based superabsorbent hydrogels (SHs) have attracted increasing interest as proficient carriers in the enzyme immobilization, the nature of the favored interactions between the SHs and enzymes is still unclear. Herein, a combined experimental and computational study was employed to investigate the dominant parameters affecting on the stabilization of two metagenomic xylanases on the SHs. The thermostable enzymes (PersiXyn3 and PersiXyn4) with similar domains were screened, cloned, expressed, and purified from cattle rumen metagenome. Then, the enzymes were immobilized on the carboxymethyl cellulose--poly(acrylic acid--acrylamide) hydrogel which resulted in increasing their activity and stability. The carboxymethyl cellulose (CMC)-based characteristic of the hydrogel provided high numbers of H-bondings/ionic bridges, causing an improvement in the stability, hydrolysis performance, and reusability of the immobilized enzymes. More specifically, enzyme immobilization resulted in ∼40% increase in the content of the reducing sugars released after treatment of paper pulp. After 16 reuse cycles, the immobilized PersiXyn4 displayed 35.9% activity, but the immobilized PersiXyn3 retained just 8.2% of its initial activity. The comparative investigations illustrated that a higher number of positively charged amino acids in the binding site of the enzyme provided stronger electrostatic attractions between it and negative functionalities of the hydrogel. This was suggested as the main reason for the higher affinity of PersiXyn4 toward hydrogel and explained the better hydrolysis performance and reusability of the immobilized PersiXyn4 on the SH. These findings are essential for designing novel innovative SH carriers and the successful engineering of optimal enzyme assemblies through the prediction of the immobilized enzyme's stabilities.
虽然基于多糖的超吸水性水凝胶（SHs）作为酶固定化的有效载体已引起越来越多的关注，但SHs与酶之间有利相互作用的本质仍不清楚。在此，采用实验与计算相结合的研究方法，来探究影响两种宏基因组木聚糖酶在SHs上稳定性的主要参数。从牛瘤胃宏基因组中筛选、克隆、表达并纯化了具有相似结构域的热稳定酶（PersiXyn3和PersiXyn4）。然后，将这些酶固定在羧甲基纤维素-聚（丙烯酸-丙烯酰胺）水凝胶上，这提高了它们的活性和稳定性。水凝胶基于羧甲基纤维素（CMC）的特性提供了大量的氢键/离子桥，从而提高了固定化酶的稳定性、水解性能和可重复使用性。更具体地说，酶固定化使纸浆处理后释放的还原糖含量增加了约40%。经过16次重复使用循环后，固定化的PersiXyn4显示出35.9%的活性，但固定化的PersiXyn3仅保留了其初始活性的8.2%。对比研究表明，酶结合位点中带正电荷的氨基酸数量越多，其与水凝胶的负官能团之间的静电吸引力就越强。这被认为是PersiXyn4对水凝胶具有更高亲和力的主要原因，并解释了固定化的PersiXyn4在SH上具有更好的水解性能和可重复使用性。这些发现对于设计新型创新的SH载体以及通过预测固定化酶的稳定性成功构建最佳酶组装体至关重要。
