An integrated upstream process was developed for the high-level production of a low-immunogenic L-asparaginase mutant (EcaII P40S/S206C) in Escherichia coli, combining high-cell-density fed-batch cultivation with controlled induction and post-induction nutrient feeding. Biomass accumulation was achieved by exponential substrate feeding, followed by pH-stat-based nutrient supply during the expression phase. Induction protocols were optimized using biomass-specific IPTG dosing (µmol IPTG/gcell), applied either as a pulse or via exponential feeding. This combined strategy resulted in a maximum volumetric enzyme activity of 134,516 U/L and a specific productivity of 3387 U/gcell, corresponding to 91-fold and 24-fold increases, respectively, compared with standard batch cultivation. Overall, these results indicate that the coordinated control of feeding strategy and induction conditions substantially improves both volumetric and cell-specific productivity of recombinant L-asparaginase and provides a robust upstream framework for the efficient manufacture of improved enzyme variants.

Lipoxygenases (LOXs) are enzymes that catalyze the regioselective dioxygenation of polyunsaturated fatty acids (PUFAs), leading to the formation of fatty acid hydroperoxides (FAHPs). In addition to dioxygenase activity, some eukaryotic LOXs exhibit hydroperoxide isomerase (HPI) activity under specific conditions, resulting in the production of structurally diverse compounds such as epoxy alcohols and ketones. Until now, the presence of HPI activity in bacterial LOXs has not been documented. In this study, we investigated the HPI activity of LOX from Burkholderia thailandensis (Bt-LOX) and examined the effects of reaction conditions on its catalytic profile using three different C18 PUFA substrates. The results demonstrated that Bt-LOX exhibits significant HPI activity, especially at high enzyme concentrations, with ketone formation showing strong substrate dependence. Oxygen level was identified as a critical factor in directing the catalytic performance of Bt-LOX: HPI activity was inhibited under O₂-saturated conditions and enhanced under O₂-limited conditions. These findings establish Bt-LOX as the first bacterial LOX reported to exhibit pronounced HPI activity, and highlights its expanded potential for biocatalytic applications.