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l-Asparaginase, which catalyzes the hydrolysis of l-asparagine, is an important enzyme in both the clinical and food industry. Exploration of efficient l-asparaginase with high substrate specificity, especially high chiral selectivity, is essential for extending its use. Herein, various crystal structures of type I l-asparaginase from Bacillus licheniformis (BlAsnase) have been resolved, and we found that there are two additional tyrosines in BlAsnase, contributing to the binding and catalysis of d-asparagine. Strikingly, the substitution of Tyr278 with methionine impaired the interaction with d-asparagine via water molecules due to the small hydrophobic side chain of methionine, which forced the ligand to the deep side of the active site toward the catalytic residues and thus resulted in the loss of hydrolyzing function. Our investigation of the substrate recognition mechanism of BlAsnase is significant for both a better understanding of l-asparaginase and its rational design to achieve high specificity for clinical and industrial applications.

l -Asparaginases have the potential to inhibit the formation of acrylamide, a harmful toxin formed during high temperature processing of food. A novel bacterium which produces l -asparaginase was screened. Type I l -asparaginase gene from Acinetobacter soli was cloned and expressed in Escherichia coli. The recombinant l -asparaginase had an activity of 42.0 IU mL−1 and showed no activity toward l -glutamine and d -asparagine. The recombinant l -asparaginase exhibited maximum catalytic activity at pH 8.0 and 40°C. The enzyme was stable in the pH ranging from 6.0 to 9.0. The activity of the recombinant enzyme was substantially enhanced by Ba2+, dithiothreitol, and β-mercaptoethanol. The Km and Vmax values of the l -asparaginase for the l -asparagine were 3.22 mmol L−1 and 1.55 IU μg−1, respectively. Moreover, the recombinant l -asparaginase had the ability to mitigate acrylamide formation in potato chips. Compared with the untreated group, the content of acrylamide in samples treated with the enzyme was effectively decreased by 55.9%. These results indicate that the novel type I l -asparaginase has the potential for application in the food processing industry.

Directed evolution methodologies have been used as promising strategies for improving the catalytic properties of many existing enzymes. In the presented work, this approach was applied to improve the enzyme activity of l -asparaginase I obtained from Bacillus megaterium H-1. After two rounds of error-prone polymerase chain reaction (epPCR) and two generations of sequential DNA shuffling, all of 5 different mutants showed a significant increase in the enzyme activity of l -asparaginase I, ranging from 6.27 to 22.78 IU/mL. Among these mutants, D-9B and DD-12G displayed the relatively high catalytic activity, which were 20.22-fold and 21.33-fold higher than the wild-type enzyme (WT), respectively. Furthermore, the catalytic efficiency (kcat/Km) of D-9B and DD-12G were also improved, which were 132.73 min−1mM−1 and 146.39 min−1mM−1, respectively, in comparison to that of WT (3.39 min−1mM−1). In addition, mutant DD-12G showed tolerance toward wider range of pH values and higher temperatures than its WT counterpart. Homology modeling of above two mutants reflected a reduction of hydrogen bonds and an introduction of flexible residues in the loops near the active catalytic site Thr15. These changes contributed to the flexibility of loops, which may lead to further enhancement in catalytic efficiency. Results also showed that approximately 88.5% (0.978 mg/kg) acrylamide could be removed from mutant DD-12G pre-treated fried potato chips. This study clearly shows that directional evolution methods can indeed be utilized to improve the activity of l -asparaginase, which could also provide research basis for future application in food industry.