Enhancing the Catalytic Activity of Type II L-Asparaginase from Bacillus licheniformis through Semi-Rational Design

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 Bacillus licheniformis. 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 k(cat)/K-m of 537.15 min(-1)center dot mM(-1), which were 4.24-fold and 6.32-fold more than those of wild-type ASNase. The highest specific activity and k(cat)/K-m were firstly reported in type II ASNase from Bacillus licheniformis. 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 Bacillus licheniformis has been enhanced by the semi-rational approach, and results provide new insights into enzymatic modification of L-asparaginase for industrial applications.

Automated summary

In this study, smart libraries were constructed using semi-rational design to improve the catalytic activity of type II L-asparaginase from Bacillus licheniformis. Mutants with significantly enhanced catalytic efficiency were obtained through saturation mutations and combinatorial mutations. The quintuple mutant ILRAC showed the highest specific activity and k(cat)/K-m, and also exhibited improved pH stability and thermal stability. Structural alignment and molecular dynamic simulation revealed that the stability and substrate binding of ILRAC contributed to its enhanced thermal stability and enzymatic activity. This study presents a novel semi-rational approach to enhance the enzymatic activity of type II L-asparaginase and provides insights for industrial applications.