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Enhancing the Thermostability of Highly Active and Glucose-Tolerant β-Glucosidase Ks5A7 by Directed Evolution for Good Performance of Three Properties
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2018-11-29 00:00:00 , DOI: 10.1021/acs.jafc.8b05662 Lichuang Cao 1 , Shuifeng Li 1 , Xin Huang 1 , Zongmin Qin 1 , Wei Kong 1 , Wei Xie 2 , Yuhuan Liu 1
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2018-11-29 00:00:00 , DOI: 10.1021/acs.jafc.8b05662 Lichuang Cao 1 , Shuifeng Li 1 , Xin Huang 1 , Zongmin Qin 1 , Wei Kong 1 , Wei Xie 2 , Yuhuan Liu 1
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A high-performance β-glucosidase for efficient cellulose hydrolysis needs to excel in thermostability, catalytic efficiency, and resistance to glucose inhibition. However, it is challenging to achieve superb properties in all three aspects in a single enzyme. In this study, a hyperactive and glucose-tolerant β-glucosidase Ks5A7 was employed as the starting point. Four rounds of random mutagenesis were then performed, giving rise to a thermostable mutant 4R1 with five amino acid substitutions. The half-life of 4R1 at 50 °C is 8640-fold that of Ks5A7 (144 h vs 1 min). Meanwhile, 4R1 had a higher specific activity (374.26 vs 243.18 units·mg–1) than the wild type with a similar glucose tolerance. When supplemented to Celluclast 1.5L, the mutant significantly enhanced the hydrolysis of pretreated sugar cane bagasse, improving the released glucose concentration by 44%. With excellent performance in thermostability, activity, and glucose tolerance, 4R1 will serve as an exceptional catalyst for industrial applications.
中文翻译：
通过定向进化增强高活性和葡萄糖耐受性的β-葡萄糖苷酶Ks5A7的热稳定性，以实现三种性能
用于高效纤维素水解的高性能β-葡萄糖苷酶需要在热稳定性，催化效率和抗葡萄糖抑制性方面表现出色。然而，在单一酶中在所有三个方面都实现极好的性能是具有挑战性的。在这项研究中，以高活性和葡萄糖耐量的β-葡萄糖苷酶Ks5A7为起点。然后进行四轮随机诱变，产生具有五个氨基酸取代的热稳定突变体4R1。在50°C下4R1的半衰期是Ks5A7的半衰期的8640倍（144小时对1分钟）。同时，4R1具有更高的比活（374.26 vs 243.18单位·mg –1）比具有相似的葡萄糖耐量的野生型。当添加Celluclast 1.5L时，该突变体显着增强了预处理的甘蔗渣的水解，使释放的葡萄糖浓度提高了44％。4R1具有出色的热稳定性，活性和葡萄糖耐量性能，将成为工业应用的出色催化剂。
更新日期：2018-11-29
中文翻译：
通过定向进化增强高活性和葡萄糖耐受性的β-葡萄糖苷酶Ks5A7的热稳定性，以实现三种性能
用于高效纤维素水解的高性能β-葡萄糖苷酶需要在热稳定性，催化效率和抗葡萄糖抑制性方面表现出色。然而，在单一酶中在所有三个方面都实现极好的性能是具有挑战性的。在这项研究中，以高活性和葡萄糖耐量的β-葡萄糖苷酶Ks5A7为起点。然后进行四轮随机诱变，产生具有五个氨基酸取代的热稳定突变体4R1。在50°C下4R1的半衰期是Ks5A7的半衰期的8640倍（144小时对1分钟）。同时，4R1具有更高的比活（374.26 vs 243.18单位·mg –1）比具有相似的葡萄糖耐量的野生型。当添加Celluclast 1.5L时，该突变体显着增强了预处理的甘蔗渣的水解，使释放的葡萄糖浓度提高了44％。4R1具有出色的热稳定性，活性和葡萄糖耐量性能，将成为工业应用的出色催化剂。
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