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.

Lipoxygenases have proven to be a potential biocatalyst for various industrial applications. However, low catalytic activity, low thermostability, and narrow range of pH stability largely limit its application. Here, a lipoxygenase (LOX) gene from Enterovibrio norvegicus DSM 15893 (EnLOX) was cloned and expressed in Escherichia coli BL21 (DE3). EnLOX showed the catalytic activity of 40.34 U mg⁻¹ at 50 °C, pH 8.0. Notably, the enzyme showed superior thermostability, and wide pH range stability. EnLOX remained above 50% of its initial activity after heat treatment below 50 °C for 6 h, and its melting point temperature reached 78.7 °C. More than 70% of its activity was maintained after incubation at pH 5.0–9.5 and 4 °C for 10 h. In addition, EnLOX exhibited high substrate specificity towards linoleic acid, and its kinetic parameters of V_max, K_m, and K_cat values were 12.42 mmol min⁻¹ mg⁻¹, 3.49 μmol L⁻¹, and 16.86 s⁻¹, respectively. LC-MS/MS analysis indicated that EnLOX can be classified as 13-LOX, due to its ability to catalyze C₁₈ polyunsaturated fatty acid to form 13-hydroxy fatty acid. Additionally, EnLOX could improve the farinograph characteristics and rheological properties of wheat dough. These results reveal the potential applications of EnLOX in the food industry.

Lipoxygenase (EC1.13.11.12, LOX) has been potentially used in the food industry for food quality improvement. However, the low activity, poor thermal stability, narrow range of pH stability, as well as undesirable isoenzymes and off-flavors, have hampered the application of current commercial LOX. In this study, a putative mini-lipoxygenase gene from cyanobacteria, Nostoc sphaeroides (NsLOX), was cloned and expressed in E. coli BL21. NsLOX displayed only 26.62% structural identity with the reported LOX from Cyanothece sp., indicating it as a novel LOX. The purified NsLOX showed the maximum activity at pH 8.0 and 15 °C, with superior stability at a pH range from 6.0 to 13.0, retaining about 40% activity at 40 °C for 90 min. Notably, NsLOX exhibited the highest specific activity of 78,080 U/mg towards linoleic acid (LA), and the kinetic parameters—K_m, k_cat, and k_cat/K_m—attain values of 19.46 μM, 9199.75 s⁻¹, and 473.85 μM⁻¹ s⁻¹, respectively. Moreover, the activity of NsLOX was obviously activated by Ca²⁺, but it was completely inhibited by Zn²⁺ and Cu²⁺. Finally, NsLOX was supplied in steamed bread and contributed even better improved bread quality than the commercial LOX. These results suggest NsLOX as a promising substitute of current commercial LOX for application in the food industry.