L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.
Asparaginase/genetics* ; Bacillus subtilis/genetics* ; Industrial Microbiology ; Protein Engineering ; Rhizomucor/enzymology* ; Sequence Alignment

Cutaneous mucormycosis is a rare disease caused by zygomycetes such as Rhizomucor, Mucor, Absidia, and Rhizopus. The disease usually occurs in immunocompromised individuals, and the organism is rarely pathogenic in an immunocompetent host. Herein, we report a 77-year-old female patient who had multiple erythematous papules and pustules on the left 3rd finger. She had received systemic steroid therapy prior to the occurrence of the skin lesions. The histopathological examination of Periodic Acid Schiff stained section showed chronic granulomatous inflammation and fungal hyphae. Rhizopus species was isolated on the fungal culture of the tissue specimen. The patient was finally diagnosed with cutaneous mucormycosis and was treated with itraconazole.
Absidia ; Aged ; Female ; Fingers ; Humans ; Hyphae ; Inflammation ; Itraconazole ; Mucor ; Mucormycosis* ; Periodic Acid ; Rare Diseases ; Rhizomucor ; Rhizopus ; Skin