Protein Engineering Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, West Bengal, 741246, India.
Center for the Advanced Functional Materials, Indian Institute of Science Education and Research Kolkata, Mohanpur, West Bengal, India.
Appl Microbiol Biotechnol. 2020 May;104(9):3935-3945. doi: 10.1007/s00253-020-10515-0. Epub 2020 Mar 10.
In a previous study, we reported an alkaliphilic and thermostable endoglucanase (BsGH7-3) of glycoside hydrolase family 7 (GH7) from the hemibiotrophic plant pathogen Bipolaris sorokiniana. However, the catalytic efficiency of the enzyme was lower than for some other endoglucanases of the GH7 family reported in the literature. To engineer a more active enzyme, we identified conserved residues in the substrate-binding tunnel and on the surface of the protein that could play a role in charge-charge interaction and stabilize the structure. The mutants D257W and Q225H in the substrate-binding tunnel and Y222R and Q401N on the protein surface showed a 2-fold increase in specific activity and a 1.5-fold increase in turnover number and were active over a broader range of pH. The mutants also showed a higher tolerance to NaCl. The rational design of the BsGH7-3 mutants helped in increasing the catalytic efficiency of the thermostable enzyme and may be useful in combination with other cellulases like cellobiohydrolase and β-glucosidase towards complete saccharification of cellulose into glucose.
在之前的一项研究中，我们报道了一种来自半生物营养型植物病原菌旋孢腔菌的嗜碱耐热内切葡聚糖酶（BsGH7-3），属于糖苷水解酶家族 7（GH7）。然而，该酶的催化效率低于文献中报道的一些其他 GH7 家族的内切葡聚糖酶。为了工程改造出一种更具活性的酶，我们鉴定了在底物结合隧道内和蛋白质表面上的保守残基，这些残基可能在电荷-电荷相互作用中发挥作用，并稳定酶的结构。在底物结合隧道中的 D257W 和 Q225H 以及在蛋白质表面上的 Y222R 和 Q401N 这两个突变体的比活性提高了 2 倍，转换数提高了 1.5 倍，在更宽的 pH 范围内具有活性。这些突变体还表现出对 NaCl 更高的耐受性。对 BsGH7-3 突变体的合理设计有助于提高耐热酶的催化效率，并且可能与其他纤维素酶（如纤维二糖水解酶和β-葡萄糖苷酶）结合使用，以将纤维素完全糖化生成葡萄糖。
