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 observe the differences of biological property of glioma stem cells (GSCs) and glioma non-stem cells (nGSCs), and their related protein expressions. Methods The proliferations of GSCs1, GSCs2 and nGSCs1 and nGSCs2 were detected by CCK8 after two, 4, 6, 8, 10 and 12 d of culture in vitro. The sensitivities of the cells to temozolomide (TMZ) were detected by CCK8 after 2 d of culture. The adhesion abilities of cells were tested by adhesion assay. Transwell assay was used to detect the migration and invasion abilities of cells. The activity of matrix metalloproteinase-2 (MMP-2) was detected by gelatin zymography. Western blotting and immunofluorescence staining were used to detect the protein expressions of Notchl and epidermal growth factor receptor (EGFR). Results The survival rate of nGSCs1 was significantly higher than that of GSCs1 and the survival rate of nGSCs2 was significantly higher than that of GSCs2 after 4, 6, 8, 10 and 12 d of culture (P<0.05). The inhibitory concentration (IC)50 of TMZ for GSCs1, nGSCs1, GSCs2 and nGSCs2 was (1536.0±17.67) μmol/L, (514.5±13.44) μmol/L, (2543.0±39.87) μmol/L, (889.6±17.43) μmol/L, respectively (P<0.05). Number of GSCs1 adhering to extracellular matrix proteins Fibronectin and Collagen I was significantly larger than that of nGSCs1, and that of GSCs2 was significantly larger than that of nGSCs2 (P<0.05). The number of migrated GSCs112 and 24 h of cultivation was statistically larger than that of nGSCs1, and that of GSCs2 was statistically larger than that of nGSCs2 (P<0.05). The number of invaded GSCs124 and 36 h of cultivation was larger than that of nGSCs1, and that of invaded GSCs2 was larger than that of nGSCs2, with statistical differences (P<0.05). The activity of MMP2 secreted by GSCs1 was significantly higher than that by nGSCs1, and that of MMP2 secreted by GSCs2 was significantly higher than that by nGSCs2 (P<0.05). Western blotting showed that the relative protein expression level of EGFR/Notch1 in GSCs1 was significantly lower than that in nGSCs1, and that in GSCs2 was significantly lower than that in nGSCs2 (P<0.05). The results of immunofluorescence staining were consistent with those of Western blotting; EGFR protein strongly expressed in nGSCs and weakly expressed in GSCs; Notch1 protein strongly expressed in GSCs and weakly expressed in nGSCs. Conclusion As compared with the high-EGFR-expressing and proliferative primary glioma cells, the high-Notch1-expressing glioma stem cells have higher activity level of MMP-2,stronger abilities of adhesion, migration and invasion, which may be contributed to glioma treatment resistance and its occurrence.

Objective To isolate the glioblastoma multiforme stem cells (GBM-SCs) from GBM specimens and to investigate the biological role ofmiR-153 in GBM-SCs so as to explore the application of gene therapy of GBMs.Methods CD133+ cells were separated using magnetic cell sorting technique (MACS) after primary culture.Immunofluorescence staining was employed to detect the CD133,nestin,glial fibrillary acidic protein (GFAP),microtubule-associated protein 2 (MAP2) expressions; real time-PCR was used to analyze miR-153 mRNA expression in CD 133-cells and CD 133+ cells.Lipofectamine RNAiMAX was used to transfect MiR-153 mimic (miR-153 group) and scrambled control oligonucleotides (NC group) into GBM-SCs; 7 d after that,sphere formation assay was performed to determine the self-renewal ability of GBM-SCs.Real time-PCR and immunofluorescence were carried out to examine the CD133,nestin,GFAP,MAP2 mRNA and protein expressions.At last,the proliferation ability of miR-153 treated GBM-SCs and NC cells was determined by CCK-8 at 24,48,72,96 and 120 h after the transfection and the apoptosis ratio was detected by flow cytometry 3 d after the transfection.Results GBM-SCs isolated from GBM specimens could express stem cell markers CD133 and nestin; after differentiation,the cells could express astrocyte marker GFAP and neuron marker MAP2; miR-153 expression in CD133+ cells was signficantly down-regulated as compared with that in CD133-cells (P＜0.05).Seven d after transfection,the number of spheres in the NC group was significantly larger than that in the miR-153 group (P＜0.05); real time-PCR indicated that the mRNA expressions of CD 133 and nestin in the miR-153 group were significantly decreased,and the GFAP and MAP2 mRNA expressions were statistically increased as compared with those in the NC group (P＜0.05).The results detected by immunofluorescence were in accordance with those by real time-PCR; 48,72,96h after the transfection,cell viability in cells from miR-153 group was statistically significant lower than that in the NC group (P＜0.05); flow cytometry showed that the apoptosis rate of cells from the miR-153 group (9.4 1％±1.98％) was significantlyhigher than that in the NC group (4.28％±0.31％,P＜0.05).Conclusion Reactivation of miR-153 expression suggests novel therapeutic strategy for GBM-SCs.

Objective To summarize experience of the combined treatment of ependymoma in fourth ventricle of the brain of child and to study improvement of effect of the operation plus radiotherapy.Methods 35 cases with ependymoma in fourth cerebral ventricle were treated with microneuro-surgical resection.Among the 35 cases,25 were treated with total brain and spinal cord plus local focus radiotherapy within 2 to 3 weeks after operation;8 only with local focus radiotherapy,2 cases were not done with radiotherapy.Results Total removal of tumors was done in 20 cases,subtotal removal in 15 cases,and no patient died from operation.After operation,20 patients had a good recovery,10 had a light disability,and 5 needed assistance.The 5-year survival rate were 90.0%(18/20),6.6%(1/15),88.0(22/25),62.5%(5/8) and 0%,in different group respectively.Conclusion Surgical treatment is obviously effective to ependymoma in fourth ventricle of the brain and total removal of tumors combined with radiotherapy is aid to extend the patient's survival time.

Objective To discuss the diagnosis and treatment of microneuro-surgery for meningiomas of the lateral ventricle.Methods A retrospe CT analysis was performed on 20 patients with meningomas of the lateral ventricle during a 7-year period.Results 20 cases had total tumor removal.among them 6 cases were completed removal,14 cases were cent piece removal.There were no postoperative deaths.The follow-up period ranged from 0.5 to 7 years.All followed patients went well.Conclusion Lateral ventricular meningiomas can be diagnosed by CT or MRI and fedding vascular of tom or revealed by cerebral vascular angiography.By using the ideal approach,the tumors could be totally removed under microscope.

Objective To study the techniques for treating the intracranial tumor.Methods Through a keyhole approach with endoscope-assisted micro-neurosurgery,30 patients with intracranial tumor were treated.With microneurosurgery,larger part of tumor was resected,then under neuroendo-scope remains of tumors were found out and removed.Results Tumors were totally removed in 22 patients,subtotally removed in 5.In three patients cerebral aneurysm was clipped successfully.Two patients with pituitary adenoma had temporay diuresis and one patient non-bacteria meningitis,and after two weeks treatment all recovered.There was no-mortality in 41 patients,no cerebral hemorrhage,optic nerve injury,internal carotid injury,and other complications occurred.Conclusion Endoscope-assisted microneurosurgery through a keyhole approach can increase the total-resection rate for tumors,reduce the trauma of operation and postoperative reaction.