Glucose oxidase (GOD) is an oxygen-consuming dehydrogenase that can catalyze the production of gluconic acid hydrogen peroxide from glucose, and its specific mechanism of action makes it promising for applications, while the low catalytic activity and poor thermostability have become the main factors limiting the industrial application of this enzyme. In this study, we used the glucose oxidase AtGOD reported with the best thermostability as the source sequence for phylogenetic analysis to obtain the GOD with excellent performance. Six genes were screened and successfully synthesized for functional validation. Among them, the glucose oxidase AhGODB derived from Aspergillus heteromorphus was expressed in Pichia pastoris and showed better thermostability and catalytic activity, with an optimal temperature of 40 ℃, a specific activity of 112.2 U/mg, and a relative activity of 47% after 5 min of treatment at 70 ℃. To improve its activity and thermal stability, we constructed several mutants by directed evolution combined with rational design. Compared with the original enzyme, the mutant T72R/A153P showcased the optimum temperature increasing from 40 to 50 ℃, the specific activity increasing from 112.2 U/mg to 166.1 U/mg, and the relative activity after treatment at 70 ℃ for 30 min increasing from 0% to 33%. In conclusion, the glucose oxidase mutants obtained in this study have improved catalytic activity and thermostability, and have potential for application.
Glucose Oxidase/chemistry* ; Enzyme Stability ; Aspergillus/genetics* ; Pichia/metabolism* ; Temperature ; Catalysis ; Fungal Proteins/metabolism* ; Hot Temperature

Objective:To investigate the molecular mechanism of antiglioma effect of curcumin.Methods:Cell experiment: (1) U251MG and SHG-44 cells at logarithmic growth phase were treated with 10 μmol/L curcumin (curcumin group) or same volume of dimethyl sulfoxide solution (control group); cells were transfected with negative control small interfering RNA (siRNA) and long non-coding RNA (lncRNA) H19 siRNA (negative control siRNA group and H19 siRNA group); cells were transfected with negative control siRNA and H19 siRNA, respectively, and then, they were treated with 10 μmol/L curcumin (negative control siRNA+curcumin group and H19 siRNA+curcumin group); the H19 siRNA was co-transfected with negative control miR inhibitor or miR-491-5p inhibitor into these cells (H19 siRNA+negative control inhibitor group and H19 siRNA+miR-4915p inhibitor group); H19 siRNA+negative control miR inhibitor or H19 siRNA+miR-491-5p inhibitor were co-transfected into the cells, and then, they were treated with 10 μmol/L curcumin (H19 siRNA+negative control inhibitor+curcumin group and H19 siRNA+miR-491-5p inhibitor+curcumin group); the cells were co-transfected with miR-491-5p mimic+blank plasmid or miR-491-5p mimic+HOXA9 overexpression plasmid, and then they were treated with 10 μmol/L curcumin (miR-491-5p mimic+blank plasmid+curcumin group and miR-491-5p mimic+HOXA9 overexpression plasmid+curcumin group); real-time fluorescent quantitative PCR (qRT-PCR) was used to detect the mRNA expressions of H19, miR-491-5p, and HOXA9; CCK-8 assay was used to detect the cell proliferation; flow cytometry was used to detect the cell apoptosis; plate cloning method was employed to detect the number of cell clone formation; Transwell assay was used to detect the cell migration; and the HOXA9 protein expression was measured by Western blotting. (2) The 293T cells at the logarithmic growth phase were chosen; the negative control miRNA mimics or miR-491-5p mimics combined with wild-type H19, wild-type HOXA9 3'-UTR plasmid vectors were co-transfected into the cells, respectively (negative control mimic+wild type H19 group and miR-491-5p mimic+wild type H19 group, negative control mimic+wild type HOXA9 3'-UTR group and miR-491-5p mimic+wild type HOXA9 3'-UTR group); the luciferase activity was detected by dual luciferase reporter experiment. (3) Thirty specimens from glioma patients (glioma group) underwent surgical resection and pathologically confirmed in our hospital from May 2017 to May 2019 and 30 normal brain tissue specimens obtained during decompression (normal group) at the same period were chosen; the mRNA expressions of H19, miR-491-5p, and HOXA9 were detected by qRT-PCR, and the HOXA9 protein expression level in these specimens was detected by Western blotting. (4) Twenty-four nude mice were randomly divided into negative control short hairpin RNA (shRNA) group, H19 shRNA group, negative control shRNA+curcumin group, and H19 shRNA+curcumin group ( n=6); U251MG cells stably transfected with negative control shRNA or H19 shRNA were intraperitoneally injected, respectively, into the mice; and 60 mg/kg curcumin was injected on the next d; the tumor volume was measured on the 7 th, 11 th, 15 th, 19 th, 23 rd, and 27 th d of rearing; and the H19, miR-491-5p and HOXA9 mRNA expressions in the tumor tissues were detected by qRT-PCR; the HOXA9 protein expression was detected by Western blotting. Results:(1) When curcumin group comparing with control group, and H19 siRNA group comparing with negative control siRNA group, U251MG and SHG-44 cells had significantly decreased miR-491-5p mRNA and protein expressions, and significantly increased miR-491-5p mRNA expression ( P<0.05); as compared with that in the H19 siRNA+negative control inhibitor group, the HOXA9 mRNA and protein expressions in U251MG and SHG-44 cells of H19 siRNA+miR-491-5p inhibitor group were significantly higher ( P<0.05). When curcumin group comparing with control group, H19 siRNA group comparing with negative control siRNA group, H19 siRNA+curcumin group comparing with negative control siRNA+curcumin group, the U251MG and SHG-44 cells after 72 h of culture had significantly decreased cell proliferation rate, significantly increased apoptosis rate, significantly reduced number of cell clone formation, and significantly reduced cell migration number ( P<0.05). When H19 siRNA+miR-491-5p inhibitor+curcumin group comparing with H19 siRNA+negative control inhibitor+curcumin group, miR-491-5p mimic+HOXA9 overexpression plasmid+curcumin group comparing with miR-491-5p mimic+blank plasmid+curcumin group, the U251MG and SHG-44 cells after 72 h of culture had significantly increased cell proliferation rate, significantly reduced apoptosis rate, significantly increased number of cell clone formation, and significantly increased cell migration number ( P<0.05). (2) When miR-491-5p mimic+wild-type H19 group comparing with negative control mimic+wild-type H19 group, miR-491-5p mimic+wild-type HOXA9 3'-UTR group comparing with negative control mimic+wild-type HOXA9 3'-UTR group, the cell luciferase activity was significantly reduced ( P<0.05). (3) As compared with those in the normal group, the H19 and HOXA9 mRNA expressions and HOXA9 protein expression in the glioma group were significantly increased, and the miR-491-5p mRNA expression was significantly reduced ( P<0.05). (4) On the 27 th d of rearing, when H19 shRNA group comparing with negative control shRNA group, and H19 shRNA+curcumin group comparing with negative control shRNA+curcumin group, the tumor volume was significantly reduced, the miR-491-5p mRNA expression in the tumor tissues was significantly increased, and the H19 mRNA, HOXA9 mRNA and protein expressions were significantly reduced ( P<0.05). Conclusion:Curcumin may inhibit the cell proliferation and migration and promote the apoptosis of glioma cells through lncRNA H19/miR-491-5p/HOXA9 axis.

Objective To study the effect of nucha electroacupuncture (NEA) on 5-hydroxytryptamine (5-HT) in cerebrospinal fluid (CSF) and blood of acute cerebral infarction (ACI) patients. Methods 72 cases with first ACI were randomly divided into NEA group (n= 38) and control group (n=34). Their 5-HT levels in CSF and blood were determined before and 4 weeks after treatment. Results Before treatment, the 5-HT in the CSF was (0.67±0.13) μmol/L in the NEA group and (0.71±0.11) μmol/L in the control group (P>0.05), while that in the blood was (0.44±0.19) μmol/L in the NEA group and (0.41±0.10) μmol/L in the control group (P>0.05). After treatment, the 5-HT in the CSF was (1.12±0.32) μmol/L in the NEA group and (0.83±0.15) μmol/L in the control group (P<0.05), while that in the blood was (0.87± 0.14) μmol/L in the NEA group and (0.63±0.07) μmol/L in the control group (P<0.05). Conclusion NEA can increase the 5-HT in both CSF and blood after ACI, which may facilitate to reduce post-stroke depression.