Uzuner Ugur, Canakci Sabriye, Bektas Kadriye Inan, Sapmaz Merve Tuncel, Belduz Ali Osman
Department of Molecular Biology and Genetics, Faculty of Science, Karadeniz Technical University, 61080, Trabzon, Turkey; Institute for Plant Genomics and Biotechnology, Texas A&M University, 2123 TAMU, College Station, TX, 77840, USA.
Department of Biology, Faculty of Science, Karadeniz Technical University, 61080, Trabzon, Turkey.
Biochimie. 2017 Jun;137:174-189. doi: 10.1016/j.biochi.2017.03.017. Epub 2017 Mar 27.
Thermoalkaliphilic xylanases are highly desired and of great importance due to their vast potential in paper pulp and bleaching processes. Here, we report rapid, cost-effective, and result-oriented combinatorial potential of in silico DNA swapping strategy to engineer the pH optimum of industrially crucial enzymes, particularly engineering of Geobacillus sp. TF16 endoxylanase for alkaline environments. The 3D structures of Geobacillus sp. TF16 and donor Bacillus halodurans C-125 endoxylanases were firstly predicted, analyzed, and compared for their similarities before any in silico design of mutants. Reasonably, to improve its alkaline pH tolerance, the corresponding regions in Geobacillus sp.TF16 endoxylanase were further engineered by swapping with negatively-charged amino acid-rich regions from B. halodurans C-125 endoxylanase. Through only two of four in silico-designed mutants, the optimum pH of GeoTF16 endoxylanase was improved from 8.5 to 10.0. Moreover, as compared to GeoTF16 parental enzyme, both GeoInt3 and GeoInt4 mutants revealed (i) enhanced biobleaching performance, (ii) improved adaptability to alkaline conditions, and (iii) better activity for broader pH range. Unlike GeoTF16 losing activity at pH 11.0 completely, GeoInt4 retained 60% and 40% of its activity at pH 11.0 and 12.0, respectively. Thus, GeoInt4 stands out as a more competent biocatalyst that is suitable for alkaline environments of diverse industrial applications. The current study represents an efficient protein engineering strategy to adapt industrial catalysts to diverse processing conditions. Further comprehensive and fine-tuned research efforts may result in biotechnologically more promising outcomes.
由于嗜热嗜碱木聚糖酶在纸浆和漂白工艺中具有巨大潜力，因此备受关注且极为重要。在此，我们报告了一种快速、经济高效且以结果为导向的计算机辅助DNA交换策略的组合潜力，该策略用于改造工业关键酶的最适pH值，特别是针对嗜热栖热放线菌TF16内切木聚糖酶进行碱性环境工程改造。在对嗜热栖热放线菌TF16和供体嗜碱芽孢杆菌C-125内切木聚糖酶进行任何计算机辅助突变设计之前，首先预测、分析并比较了它们的三维结构，以找出相似之处。合理的做法是，为提高嗜热栖热放线菌TF16内切木聚糖酶对碱性pH的耐受性，通过与嗜碱芽孢杆菌C-125内切木聚糖酶中富含带负电荷氨基酸的区域进行交换，对嗜热栖热放线菌TF16内切木聚糖酶中的相应区域进行进一步改造。通过仅四个计算机辅助设计的突变体中的两个，GeoTF16内切木聚糖酶的最适pH值从8.5提高到了10.0。此外，与GeoTF16亲本酶相比，GeoInt3和GeoInt4突变体均表现出：（i）生物漂白性能增强；（ii）对碱性条件的适应性提高；（iii）在更宽pH范围内具有更好的活性。与GeoTF16在pH 11.0时完全失去活性不同，GeoInt4在pH 11.0和12.0时分别保留了60%和40%的活性。因此，GeoInt4是一种更具竞争力的生物催化剂，适用于各种工业应用的碱性环境。当前的研究代表了一种使工业催化剂适应不同加工条件的有效蛋白质工程策略。进一步全面且经过微调的研究工作可能会带来更具生物技术前景的成果。
