Enhanced Thermostability and Molecular Insights for l-Asparaginase from Bacillus licheniformis via Structure- and Computation-Based Rational Design.

l-Asparaginase has gained much attention for effectively treating acute lymphoblastic leukemia (ALL) and mitigating carcinogenic acrylamide in fried foods. Due to high-dose dependence for clinical treatment and low mitigation efficiency for thermal food processes caused by poor thermal stability, a method to achieve thermostable l-asparaginase has become a critical bottleneck. In this study, a rational design including free energy combined with structural and conservative analyses was applied to engineer the thermostability of l-asparaginase from Bacillus licheniformis (BlAsnase). Two enhanced thermostability mutants D172W and E207A were screened out by site-directed saturation mutagenesis. The double mutant D172W/E207A exhibited highly remarkable thermostability with a 65.8-fold longer half-life at 55 °C and 5 °C higher optimum reaction temperature and melting temperature (Tm) than those of wild-type BlAsnase. Further, secondary structure, sequence, molecular dynamics (MD), and 3D-structure analysis revealed that the excellent thermostability of the mutant D172W/E207A was on account of increased hydrophobicity and decreased flexibility, highly rigid structure, hydrophobic interactions, and favorable electrostatic potential. As the first report of rationally designing l-asparaginase with improved thermostability from B. licheniformis, this study offers a facile and efficient process to improve the thermostability of l-asparaginase for industrial applications.

Thermostability Improvement of L-Asparaginase from Acinetobacter soli via Consensus-Designed Cysteine Residue Substitution.

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 (Tm) 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.

Characterization of a novel and glutaminase-free type II L-asparaginase from Corynebacterium glutamicum and its acrylamide alleviation efficiency in potato chips.

Type II L-asparaginase as a pivotal enzyme agent has been applied to treating for acute lymphoblastic leukemia (ALL) and efficient mitigation of acrylamide formed in fried and baked foods. However, low activity, narrow range of pH stability, as well as undesirable glutaminase activity hinder the applications of this enzyme. In our work, A novel type II L-asparaginase (CgASNase) from Corynebacterium glutamicum with molecular mass of about 35 kDa was chosen to express in E. coli. CgASNase shared only 27 % structural identity with the reported L-asparaginase from Helicobacter pylori. The purified CgASNase showed the highest specific activity of 1979.08 IU mg-1 to L-asparagine, compared with reported type II ASNases in the literature. CgASNase displayed superior stability at a wide pH range from 5.0 to 11.0, and retained about 76 % of its activity at 30 °C for 30 min. The kinetic parameters Km (Michaelis constant), kcat (turnover number), and kcat/Km (catalytic efficiency) values of 4.66 mM, 79,697.40 min-1, and 17,102.45 mM-1 min-1, respectively. More importantly, CgASNase exhibited strict substrate specificity towards L-asparagine, no detectable activity to l-glutamine. To explore its ability to catalyze L-asparagine, CgASNase was supplied in frying potato chips, which produced the fries with 84 % less acrylamide content compared with no supply. These findings suggest that CgASNase presents excellent properties for chemotherapy against diseases and great potential in the food processing industry.

Case report: Prompt response to radiotherapy and chemotherapy combined with crizotinib in gingival sarcomatoid squamous cell carcinoma with MET 14 mutation.

Background: As a kind of squamous cell carcinoma of head and neck (HNSCC), gingival sarcomatoid squamous cell carcinoma (GSSCC) is a rare biphasic malignant neoplasm. To date, surgical resection was often utilized for gingival squamous cell carcinoma (GSCC), while for patients with advanced gingival carcinoma who cannot tolerate surgery, radiotherapy and chemotherapy can be regarded as a treatment strategy. Many molecular-targeted drugs were investigated and approved for the treatment of malignant diseases, including hematologic diseases and solid tumors. Although targeted therapies such as EGFR inhibitors have shown therapeutic efficacy in HNSCC, there are still some patients who cannot benefit from it. New therapeutic targets and strategies should be further explored. Case presentation: An 83-year-old woman was referred to our hospital with left lower gingival mass for more than 1 month in June 2021. Pathologic diagnosis is sarcomatoid squamous cell carcinoma. Due to the large tumor at the time of diagnosis and poor quality of life, the patient was intolerant to surgery, so she was given radiotherapy (RT) combined with concurrent chemotherapy (CT) with albumin bound paclitaxel. According to next-generation sequencing (NGS) results (MET exon 14 skipping mutation-positive), she was treated with crizotinib, a tyrosine kinase inhibitor that targets MET. Through the comprehensive treatment, the patient's condition promptly improved, clinical complete remission (CR) was achieved in 2 months, and 9-month progression-free survival (PFS) was obtained. She finally died from non-cancer-related diseases. Conclusion: Here we report the treatment of a GSSCC patient with MET mutation, who responded to crizotinib promptly and positively. It provides a new reference for understanding MET abnormalities in GSSCC and offers a new idea for the targeted treatment of gingival carcinoma.

[An investigation of the protective effect of prostaglandin E1 liposome on acute lung injury in pig].

OBJECTIVE: To assess the protective effect of prostaglandin E1 liposome (lipo-PGE1) on acute lung injury (ALI) in a porcine model of ALI. METHODS: The ALI model was reproduced by lipopolysaccharide (LPS) intravenous instillation and high tidal volume ventilation. A total of 16 domestic pigs were randomized to receive lipo-PGE1 (n=8) or placebo (n=8). Parameters of haemodynamics and pulmonary gas exchange were monitored through pulmonary artery catheter and blood gas analysis at baseline and 2, 3, 4 and 5 hours after LPS instillation. The inflation quasi-static pressure-volume (P-V) curve was obtained by ventilator occlusion technique, and the P-V data sets were fit with sigmoidal equation in order to define and compare the respiratory mechanics in animals of two groups. Plasma levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: There was a decrease in cardiac index (CI) and mean arterial pressure (MAP) in control group compared with those in the group who received lipo-PGE1. In the group receiving lipo-PGE1 the parameter of oxygenation, including partial pressure of oxygen in arterial blood (PaO(2)), oxygenation index (PaO(2)/FiO(2)), and alveolar-arterial oxygen difference (A-aDO(2)) were significantly improved (all P<0.05) and pulmonary shunt (Qs/Qt) associated with lung injury was also significantly reduced (P<0.05) compared with control group. The respiratory mechanics (including lower and upper inflection point) in the group given lipo-PGE1 were better than those of control group (all P<0.05). Plasma levels of TNF-alpha and IL-8 were found to rise in both groups, but the rise in TNF-alpha and IL-8 in the group in which lipo-PGE1 was given were lower with shorter period compared with control group(all P<0.05). CONCLUSION: Intravenous delivery of lipo-PGE1 significantly attenuate ALI caused by LPS instillation and high tidal ventilation, and it also shows beneficial effects on haemodynamics.