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·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.

To extend the application range of L-asparaginase in food pre-processing, the thermostability improvement of the enzyme is essential. Herein, two non-conserved cysteine residues with easily oxidized free sulfhydryl groups, Cys8 and Cys283, of Acinetobacter soli L-asparaginase (AsA) were screened out via consensus design. After saturation mutagenesis and combinatorial mutation, the mutant C8Y/C283Q with highly improved thermostability was obtained with a half-life of 361.6 min at 40 °C, an over 34-fold increase compared with that of the wild-type. Its melting temperature (T_m) value reaches 62.3 °C, which is 7.1 °C higher than that of the wild-type. Molecular dynamics simulation and structure analysis revealed the formation of new hydrogen bonds of Gln283 and the aromatic interaction of Tyr8 formed with adjacent residues, resulting in enhanced thermostability. The improvement in the thermostability of L-asparaginase could efficiently enhance its effect on acrylamide inhibition; the contents of acrylamide in potato chips were efficiently reduced by 86.50% after a mutant C8Y/C283Q treatment, which was significantly higher than the 59.05% reduction after the AsA wild-type treatment. In addition, the investigation of the mechanism behind the enhanced thermostability of AsA could further direct the modification of L-asparaginases for expanding their clinical and industrial applications.

To explore the characteristic volatile compounds of ‘Hujing Milu’ peaches from different growing regions, headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME-GC-MS) and headspace gas chromatography–ion mobility spectrometry (HS-GC-IMS) were employed to analyze volatile components in samples from six production areas. A total of 73 and 56 volatile compounds were identified by HS-SPME-GC-MS and HS-GC-IMS, respectively. Quantitative analysis revealed that esters, aldehydes, and alcohols were the main contributors to the aroma profile, accounting for over 70% of the total relative content. Combined with chemometric analysis (VIP > 1 and OAV/ROAV > 1), 17 potential biomarkers were identified that can distinguish ‘Hujing Milu’ peaches from different regions, including ethyl acetate, hexanol, (E)-2-nonenal, and dihydro-β-ionone. Moreover, soil properties of these regions and their correlation with volatile compounds were analyzed to elucidate the formation mechanisms of characteristic aromas. The results showed that ethyl acetate exhibited a significant positive correlation with soil pH (r = 0.530, p < 0.05), whereas dihydro-β-ionone showed a significant positive correlation with soil organic matter (r = 0.587, p < 0.05) and available potassium (r = 0.830, p < 0.05). This study identified characteristic volatile compounds of ‘Hujing Milu’ peaches from different regions, providing a reliable technical basis for origin traceability and the enhancement of aroma quality in ‘Hujing Milu’ peaches.