College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
Institute of Food Safety and Nutrition, Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
Int J Mol Sci. 2022 Aug 26;23(17):9663. doi: 10.3390/ijms23179663.
Low catalytic activity is a key factor limiting the widespread application of type II L-asparaginase (ASNase) in the food and pharmaceutical industries. In this study, smart libraries were constructed by semi-rational design to improve the catalytic activity of type II ASNase from . Mutants with greatly enhanced catalytic efficiency were screened by saturation mutations and combinatorial mutations. A quintuple mutant ILRAC was ultimately obtained with specific activity of 841.62 IU/mg and / of 537.15 min·mM, which were 4.24-fold and 6.32-fold more than those of wild-type ASNase. The highest specific activity and / were firstly reported in type II ASNase from . Additionally, enhanced pH stability and superior thermostability were both achieved in mutant ILRAC. Meanwhile, structural alignment and molecular dynamic simulation demonstrated that high structure stability and strong substrate binding were beneficial for the improved thermal stability and enzymatic activity of mutant ILRAC. This is the first time that enzymatic activity of type II ASNase from has been enhanced by the semi-rational approach, and results provide new insights into enzymatic modification of L-asparaginase for industrial applications.
低催化活性是限制 II 型 L-天冬酰胺酶（ASNase）在食品和制药工业中广泛应用的关键因素。在本研究中，通过半理性设计构建了智能文库，以提高 II 型 ASNase 的催化活性。通过饱和突变和组合突变筛选出催化效率大大提高的突变体。最终获得了五重突变体 ILRAC，比野生型 ASNase 的比活和半衰期分别提高了 4.24 倍和 6.32 倍。在 II 型 ASNase 中首次报道了最高的比活和半衰期。此外，突变体 ILRAC 的 pH 稳定性和热稳定性均得到增强。同时，结构比对和分子动力学模拟表明，高结构稳定性和强底物结合有利于提高突变体 ILRAC 的热稳定性和酶活性。这是首次通过半理性方法提高 II 型 ASNase 的酶活性，研究结果为 L-天冬酰胺酶的工业应用提供了酶修饰的新见解。
