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

Abstract

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 kcat/Km of 537.15 min−1·mM−1, which were 4.24-fold and 6.32-fold more than those of wild-type ASNase. The highest specific activity and kcat/Km 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.

Desirable L-asparaginases for treating cancer and current research trends

“…Structure-based rational design is a potent protein engineering strategy to enhance the enzymatic capabilities of ASNases. Using bioinformatics servers (HotSpot Wizard and EVcoupling), rational design approach first screens active center residues using structural analysis, compares sequences, and identifies distal residues, allowing the construction of smart libraries for performing mutations at specific sites with less screening work ( Zhou et al, 2022 ). Zhou et al (2022) increased the type II ASNase from Bacillus licheniformis’s catalytic activity by using semi-rational design ( Zhou et al, 2022 ).…”

“…Using bioinformatics servers (HotSpot Wizard and EVcoupling), rational design approach first screens active center residues using structural analysis, compares sequences, and identifies distal residues, allowing the construction of smart libraries for performing mutations at specific sites with less screening work ( Zhou et al, 2022 ). Zhou et al (2022) increased the type II ASNase from Bacillus licheniformis’s catalytic activity by using semi-rational design ( Zhou et al, 2022 ). According to their findings, the mutant’s K m value was 1.45 mM instead of 2.33 mM, and its K cat value was 778.87 min-1 as opposed to 197.95 min −1 in the wild type.…”

Production, characterization, and applications of a novel thermo-acidophilic L-asparaginase of Pseudomonas aeruginosa CSPS4

“…L-asparaginase (EC 3.5.1.1) hydrolyzes the amide group of Lasparagine and liberates aspartic acid and ammonia (Qeshmi et al 2022;Zhou et al 2022). The enzyme finds several biotechnological applications related to human health in the food and pharmaceutical industries (Batool et al 2016;Brumano et al 2019).…”