Optimization of glucose oxidase production by Aspergillus niger using genetic-and process-engineering techniques

Wild-type Aspergillus niger NRRL-3 was transformed with multiple copies of the glucose oxidase structural gene (god). The gene was placed under the control of the gpd A promoter of A. nidulans. For more efficient secretion the -amylase signal peptide from A oryzae was inserted in front of god. Compared to the wild type, the recombinant strain NRRL-3 (GOD3-18) produced up to four times more extracellular glucose oxidase under identical culture conditions. Addition of yeast extract (2 g l^–1) to a mineral salts medium containing only glucose as carbon source increased volumetric and specific extracellular glucose oxidase activities by 130% and 50% respectively. With the same medium composition and inoculum size, volumetric and specific extracellular glucose oxidase activities increased more than ten times in bioreactor cultivations compared to shake-flask cultures.     

High-level expression of the <Emphasis Type="Italic">Penicillium notatum</Emphasis> glucose oxidase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using codon optimization

The glucose oxidase (GOD) gene from Penicillium notatum was expressed in Pichia pastoris. The 1,815 bp gene, god-w, encodes 604 amino acids. Recombinant GOD-w had optimal activity at 35–40°C and pH 6.2 and was stable, from pH 3 to 7 maintaining >75% maximum activity after incubation at 50°C for 1 h. GOD-w worked as well as commercial GODs to improve bread making. To achieve high-level expression of recombinant GOD in P. pastoris, 272 nucleotides involving 228 residues were mutated, consistent with the codon bias of P. pastoris. The optimized recombinant GOD-m yielded 615 U ml⁻¹ (2.5 g protein l⁻¹) in a 3 l fermentor—410% higher than GOD-w (148 U ml⁻¹), and thus is a low-cost alternative for the bread baking industry.     

Calorimetric Glucose Determination by Glucose Oxidase —Methylene Blue

The reaction between glucose and methylene blue, catalyzed by glucose oxidase (GOD)was analysed calorimetrically. The amount of heat produced under saturating methylene blue concentrations ( > 10^?2 mol/1)was measured with glucose concentration and time as parameters (kinetic procedure) Kinetic constants (pseudo one substrate kinetics) were derived from the experimental data: K_M(glucose)= 1.18 × 10^?3 mol/l and V_max = 0.085 J/mg GOD min (3.89 · 10^?6 mol/mg GOD min) Comparison of caloric with optical measurements gave an enthalpy of reaction of 22.52 kJ/mol. Considering the observed substrate inhibition, glucose determinations are possible up to glucose concentrations of 0.1 mol/l.     

Cloning,deletion, and overexpression of a glucose oxidase gene in <Emphasis Type="Italic">Aureobasidium</Emphasis> sp. P6 for Ca<Superscript>2+</Superscript>-gluconic acid overproduction

This study aimed to overexpress a glucose oxidase gene (GOD1) in Aureobasidium sp. P6 to achieve Ca²⁺-gluconic acid (GA) overproduction. The GOD1 gene was cloned, deleted, and overexpressed. A protein deduced from the GOD1 gene of Aureobasidium sp. P6 strain had 1824 bp that encoded a protein with 606 amino acids, with a conserved NADB-ROSSMAN domain and a GMC-oxred domain. Deleting the GOD1 gene made the disruptant GOK1 completely lose the ability to produce GA and GOD1 activity, whereas overexpressing the GOD1 gene rendered the transformant GOEX8 to produce considerably more Ca²⁺-GA (160.5?±?5.6 g/L) and higher GOD1 activity (1438.6?±?73.2 U/mg of protein) than its parent P6 strain (118.7?±?4.3 g/L of Ca²⁺-GA and 1100.0?±?23.6 U/mg of GOD1 protein). During a 10-L fermentation, the transformant GOEX8 grown in the medium containing 160.0 g/L of glucose produced 186.8?±?6.0 g/L of Ca²⁺-GA, the yield was 1.2 g/g of glucose, and the volumetric productivity was 1.7 g/L/h. Most of the produced GOD1 were located in the yeast cell wall. The purified product was identified to be a GA. The transformant GOEX8 overexpressing the GOD1 gene could produce considerably more Ca²⁺-GA (186.8?±?6.0 g/L) than its wild-type strain P6.     

One-step "green" preparation of graphene nanosheets and carbon nanospheres mixture by electrolyzing graphite rob and its application for glucose biosensing

The graphene nanosheets and carbon nanospheres mixture (GNS–CNS) was prepared by electrolyzing graphite rob in KNO₃ solution under constant current, which was characterized by TEM, AFM, SEM, FT-IR, XRD, XPS, TGA and UV–vis. The nano-mixture can keep stable in water for more than one month. Based on this kind of mixture material, a novel electrochemical biosensing platform for glucose determination was developed. Cyclic voltammetry of glucose oxidase (GOD) immobilized on GNS–CNS/GCE exhibited a pair of well-defined quasi-reversible redox peaks at −0.488 V (E_pa) and −0.509 V (E_pc) by direct electron transfer between the protein and the electrode. The charge-transfer coefficient (α) was 0.51, the electron transfer rate constant was 2.64 s⁻¹ and the surface coverage of HRP was 3.18 × 10⁻¹⁰ mol cm⁻². The immobilized GOD could retain its bioactivity and catalyze the reduction of dissolved oxygen. The glucose biosensor has a linear range from 0.4 to 20 mM with detection limit of 0.1 mM. Moreover, the biosensor exhibits acceptable reproducibility and storage stability. The fabricated biosensor was further used to determine glucose in human plasma sample with the recoveries from 96.83% to 105.52%. Therefore, GOD/GNS–CNS/GCE could be promisingly applied to determine blood sugar concentration in the practical clinical analysis.     

Screening of <Emphasis Type="Italic">Aspergillus</Emphasis>-derived FA<Emphasis Type="SmallCaps">D</Emphasis>-glucose dehydrogenases from fungal genome database

Aspergillus-derived FAD-dependent glucose dehydrogenases (FADGDHs) were screened from fungal genomic databases, primarily by searching for putative homologues of the Aspergillus niger-derived glucose oxidase (GOD). Focusing on a GOD active-site motif, putative proteins annotated as belonging to the glucose methanol choline (GMC) oxidoreductase family were selected. Phylogenetic analysis of these putative proteins produced a GOD clade, which includes the A. niger and Penicillium amagasakiens GODs, and a second clade made up of putative proteins showing 30–40% homology with GOD. The genes encoding the proteins from the second clade were functionally expressed in Escherichia coli, resulting in dye-mediated glucose dehydrogenase (GDH) activity but not GOD activity. These results suggest that the putative proteins belonging to the second clade are FADGDHs. The 3D structure models of these FADGDHs were compared with the 3D structure of GOD.     

Screening, mutagenesis and protoplast fusion of Aspergillus niger for the enhancement of extracellular glucose oxidase production

Various strains of Aspergillus niger were screened for extracellular glucose oxidase (GOD) activity. The most effective producer, strain FS-3 (15.9 U mL^–1), was mutagenized using UV-irradiation or ethyl methane sulfonate. Of the 400 mutants obtained, 32 were found to be resistant to 2-deoxy d-glucose, and 17 of these exhibited higher GOD activities (from 114.5 to 332.1%) than the original FS-3 strain. Following determination of antifungal resistance of the highest producing mutants, four mutants were selected and used in protoplast fusions in three different intraspecific crosses. All fusants showed higher activities (from 285.5 to 394.2%) than the original strain. Moreover, of the 30 fusants isolated, 19 showed higher GOD activity than their corresponding higher-producing parent strain.     

Glucose-sensitivity of liposomes incorporating conjugates of glucose oxidase and poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecylacrylate)

Liposomes, which release their contents in response to the concentration of glucose, were prepared by modifying the liposomal surface with the conjugate of poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecylacrylate) (P(NIPAM-co-MAA-co-ODA)) and glucose oxidase (GOD). The maximum enzymatic activity of copolymer conjugated GOD (Polym-GOD) was observed around pH 5.0 and the value was about 40% of that of native GOD. Nine lysine residues per GOD molecule, on average, were found to be covalently attached to the copolymers. Egg phosphatidylcholine liposomes bearing Polym-GOD released their contents in response to the concentration of glucose and the sensitivity was higher than dipalmitoylphosphatidylcholine liposomes.     

A mediator-free glucose biosensor based on glucose oxidase/chitosan/α-zirconium phosphate ternary biocomposite

A novel glucose oxidase/chitosan/α-zirconium phosphate (GOD/chitosan/α-ZrP) ternary biocomposite was prepared by co-intercalating glucose oxidase (GOD) and chitosan into the interlayers of α-zirconium phosphate (α-ZrP) via a delamination–reassembly procedure. The results of X-ray diffraction, infrared spectroscopy, circular dichroism, and ultraviolet spectrum characterizations indicated not only the layered and hybrid structure of the GOD/chitosan/α-ZrP ternary biocomposite but also the recovered activity of the intercalated GOD improved by the co-intercalated chitosan. By depositing the GOD/chitosan/α-ZrP biocomposite film onto a glassy carbon electrode, the direct electrochemistry of the intercalated GOD was achieved with a fast electron transfer rate constant, k_s, of 7.48 ± 3.52 s⁻¹. Moreover, this GOD/chitosan/α-ZrP biocomposite modified electrode exhibited a sensitive response to glucose in the linear range of 0.25–8.0 mM (R = 0.9994, n = 14), with a determination limit of 0.076 mM.     

Effect of aPMR1 disruption on the processing of heterologous glycoproteins secreted in the yeastSaccharomyces cerevisiae

TheSaccharomyces cerevisiae PMR1 gene encodes a Ca²⁺-ATPase localized in the Golgi. We have investigated the effects ofPMR1 disruption inS. cerevisiae on the glycosylation and secretion of three heterologous glycoproteins, human α₁-antitrypsin (α₁-AT), human antithrombin III (ATHIII), andAspergillus niger glucose oxidase (GOD). Thepmr1 null mutant strain secreted larger amounts of ATHIII and GOD proteins per a unit cell mass than the wild type strain. Despite a lower growth rate of thepmr1 mutant, two-fold higher level of human ATHIII was detected in the culture supernatant from thepmr1 mutant compared to that of the wild-type strain. Thepmr1 mutant strain secreted α₁-AT and the GOD proteins mostly as core-glycosylated forms, in contrast to the hyperglycosylated proteins secreted in the wild-type strain. Furthermore, the core-glycosylated forms secreted in thepmr1 mutant migrated slightly faster on SDS-PAGE than those secreted in themnn9 deletion mutant and the wild type strains. Analysis of the recombinant GOD with anti-α1,3-mannose antibody revealed that GOD secreted in thepmr1 mutant did not have terminal α1,3-linked mannoses unlike those secreted in themnn9 mutant and the wild type strains. The present results indicate that thepmr1 mutant, with the super-secretion phenotype, is useful as a host system to produce recombinant glycoproteins lacking high-mannose outer chains.     

Potentiometric glucose determination in human serum samples with glucose oxidase biosensor based on iodide electrode

Glucose potentiometric biosensor was prepared by immobilizing glucose oxidase on iodide-selective electrode. The hydrogen peroxide formed after the oxidation of glucose catalysed by glucose oxidase (GOD) was oxidized by sodium molybdate (SMo) at iodide electrode in the presence of dichlorometane. The glucose concentration was calculated from the decrease of iodide concentration determined by iodide-selective sensor. The sensitivity of glucose biosensor towards iodide ions and glucose was in the concentration ranges of 1.0 × 10^?1–1.0 × 10^?6 M and 1.0 × 10^?2?1.0 × 10^?4 M, respectively. The characterization of proposed glucose biosensor and glucose assay in human serum were also investigated.     

Statistical optimization of glucose oxidase production from Aspergillus niger NRC9 under submerged fermentation using response surface methodology

Response surface methodology (RSM), employing the fractional factorial design (FFD) was used to optimize the fermentation medium for the production of glucose oxidase (GOD) from a marine isolate (NRC9) of Aspergillus niger under submerged fermentation. The design was employed by selecting glucose, CaCO_3, ammonium phosphate and MgSO₄ concentrations as model factors by ‘one variable at a time’ experiment. A second-order quadratic model and response surface method showed that the optimum concentrations (g/l) glucose, 100; CaCO₃, 25; (NH₄)₂HPO₄, 1.8 and 0.4 of MgSO₄, resulted in an improvement of GOD production (170?±?0.88 U/ml) as compared to the initial level (109.81?±?1.38 U/ml) after four days of incubation at 200 rpm and 30 °C, whereas its predicted value obtained by the quadratic model was 164.36 U/ml. Analysis of variance (ANOVA) showed a high coefficient of determination value (R ²) of 0.967, ensuring a satisfactory adjustment of the quadratic model with the experimental data. This is the first report on production of glucose oxidase from a marine fungal isolate, Aspergillus niger NRC9, using statistical experimental design and response surface methodology in optimization of its production under submerged fermentation.     

Effects of glucose oxidase and its combination with B. amyloliquefaciens SC06 on intestinal microbiota,immune response and antioxidative capacity in broilers

Glucose oxidase (GOD) is an aerobic dehydrogenase, which catalyses the oxidation of β-D-glucose to gluconic acid and hydrogen peroxide. This study aimed to investigate the effects of dietary glucose oxidase and its combined effects with Bacillus amyloliquefaciens SC06 (BaSC06) on the intestinal microbiota, immune function and antioxidant capacity of broilers. One-day-old male Lingnan yellow-feathered broilers (n = 720) were randomly assigned to four treatment groups: Control group (basal diet), Anti group (basal diet supplemented with 200 mg/kg enramycin), GOD group (basal diet supplemented with 75 U/kg GOD), and combination of GOD and BaSC06 (GB) group (GOD diet (75 U/kg) supplemented with 1 × 10⁸ colony-forming units BaSC06/kg feed), with six replicates per group and 30 birds per replicate. The experiment was conducted over 52 days. The results indicated a significant decrease in α-diversity (Observed species, Chao1, PD_whole_tree and Shannon) with GOD treatment, compared with the control group. GB treatment also significantly decreased the Shannon index of cecal microbiota. GOD treatment significantly decreased the α-diversity, whereas GB treatment significantly increased these indices except for the Chao1 index, compared with the Anti group. Compared with the control group, the relative abundance of Bacteroides in the GOD and GB groups was significantly increased, whereas a decrease in Firmicutes was observed. Compared with the Anti group, GOD treatment significantly increased the relative abundances of Bacteroides and Lactobacillales, while GB treatment significantly increased Lactobacillales and decreased Proteobacteria levels. In addition, GOD treatment significantly decreased interleukin-10 and interferon-γ levels, compared with the control group. In contrast, GB treatment significantly downregulated interferon-γ levels and upregulated secretory immunoglobulin A, transforming growth factor-β and interleukin-2 expression in the jejunal mucosa. GOD treatment significantly decreased transforming growth factor-β and interleukin-10 levels, whereas GB treatment markedly increased interferon-γ expression in the jejunal mucosa compared with the Anti group. Furthermore, GB treatment significantly increased the total antioxidant capability levels and the total superoxide dismutase (T-SOD) and catalase (CAT) activities compared with the control group. Meanwhile, GOD treatment significantly increased glutathione peroxidase (GSH-Px) activity in the jejunal mucosa. Total superoxide dismutase, GSH-Px and CAT activities in the Anti group were higher than in the GOD and GB groups. The malondialdehyde levels in the control group were the highest among all groups. In conclusion, our results indicated that supplementation with GOD alone and its combination with BaSC06 in diet could increase antioxidant capacity, immune function and improve the intestinal microbiota composition of broilers. Combination treatment with GOD with BaSC06 exerted stronger effects than GOD treatment only.     

Wheat Dehydrin DHN-5 Exerts a Heat-Protective Effect on β-Glucosidase and Glucose Oxidase Activities

Group-2 late embryogenesis abundant (LEA) proteins, also known as dehydrins, are claimed to stabilize macromolecules against damage caused by freezing, dehydration, ionic or osmotic stresses. However, their precise function remains unknown. Here, we investigated the effect of wheat dehydrin (DHN-5) protein on the activity and thermostability of two distinct enzymes, β-glucosidase (bglG) and glucose oxidase/peroxidase (GOD/POD) in vitro. The purified DHN-5 protein had the capacity to preserve and stabilize the activity of bglG subjected to heat treatment. In addition, DHN-5 stabilized oxidizing enzymes, as it improved reliability in measuring glucose concentrations with a glucose oxidase/peroxidase (GOD/POD) kit while the temperature increased from 37 to 70 °C. All together the data presented provide evidence that DHN-5 is a dehydrin able to preserve enzyme activities in vitro from adverse effects induced by heating.     

Direct electrochemistry and electrocatalysis of glucose oxidase immobilized on glassy carbon electrode modified by Nafion and ordered mesoporous silica-SBA-15

In this paper, it was found that glucose oxidase (GOD) has been stably immobilized on glassy carbon electrode modified by ordered mesoporous silica-SBA-15 and Nafion. The sorption behavior of GOD immobilized on SBA-15 matrix was characterized by transmission electron microscopy (TEM), ultraviolet–visible (UV–vis), FTIR, respectively, which demonstrated that SBA-15 can facilitate the electron exchange between the electroactive center of GOD and electrode. The direct electrochemistry and electrocatalysis behavior of GOD on modified electrode were characterized by cyclic voltammogram (CV) which indicated that GOD immobilized on Nafion and SBA-15 matrices displays direct, nearly reversible and surface-controlled redox reaction with an enhanced electron transfer rate constant of 3.89 s⁻¹ in 0.1 M phosphate buffer solution (PBS) (pH 7.12). Furthermore, it was also discovered that, in the absence of O₂, GOD immobilized on Nafion and SBA-15 matrices can produce a wide linear response to glucose in the positive potential range. Thus, Nafion/GOD-SBA-15/GC electrode is hopeful to be used in the third non-mediator's glucose biosensor. In addition, GOD immobilized on SBA-15 and Nafion matrices possesses an excellent bioelectrocatalytic activity for the reduction of O₂. The Nafion/GOD-SBA-15/GC electrode can be utilized as the cathode in biofuel cell.     

Multifunctional carbon nanotubes for direct electrochemistry of glucose oxidase and glucose bioassay

Polydopamine (Pdop) has recently been shown to adsorb to a wide variety of surfaces and serves as an adhesion layer to immobilize biological molecules. In this work, the multifunctional carbon nanotube (CNT) composites were prepared though the oxidation of dopamine at room temperature and subsequent electroless silver deposition by mildly stirring. The stable immobilization and direct electron transfer of glucose oxidase were achieved on the composite film modified glassy carbon electrode. The resulting electrode gave a well-defined redox peaks with a formal potential of about −482 mV (vs. SCE) in pH 7.0 buffer. The electron transfer rate constant was estimated to be 3.6 s⁻¹, due to the combined contribution of Pdop, CNTs and Ag nanoparticles with the help of Nafion. Furthermore, the method for detecting of glucose was proposed based on the decrease of oxygen caused by the enzyme-catalyzed reaction between glucose oxidase (GOD) and glucose. The linear response to glucose ranging from 50.0 μM to 1.1 mM (R² = 0.9958), with a calculated detection limit of 17.0 μM at a signal-to-noise ratio of 3. The low calculated apparent Michaelis–Menten constant was 5.46 mM, implying the high enzymatic activity and affinity of immobilized GOD for glucose. It can reasonably be expected that this observation might hold true for other noble metal nanostructure-electroactive protein systems, providing a promising platform for the development of biosensors and biofuel cells.     

Development of a disposable glucose biosensor using electroless-plated Au/Ni/copper low electrical resistance electrodes

This paper presents a glucose biosensor, which was developed using a Au/Ni/copper electrode. Until now, research regarding the low electrical resistance and uniformity of this biosensor electrode has not been conducted. Glucose oxidase (GOD) immobilized on the electrode effectively plays the role of an electron shuttle, and allows glucose to be detected at 0.055 V with a dramatically reduced resistance to easily oxidizable constituents. The Au/Ni/copper electrode has a low electrical resistance, which is less than 0.01 Ω, and it may be possible to mass produce the biosensor electrode with a uniform electrical resistance. The low electrical resistance has the advantage in that the redox peak occurs at a low applied potential. Using a low operating potential (0.055 V), the GOD/Au/Ni/copper structure creates a good sensitivity to detect glucose, and efficiently excludes interferences from common coexisting substances. The GOD/Au/Ni/copper sensor exhibits a relatively short response time (about 3 s), and a sensitivity of 0.85 μA mM⁻¹ with a linear range of buffer to 33 mM of glucose. The sensor has excellent reproducibility with a correlation coefficient of 0.9989 (n = 100 times) and a total non-linearity error of 3.17%.     

Optimization of culture conditions for glucose oxidase production by a Penicillium chrysogenum SRT 19 strain

The enzyme glucose oxidase (GOD) has been used for a variety of biotechnological applications in food and pharmaceutical industries. In this study, the optimization of extracellular GOD production was carried out in a Penicillium chrysogenum SRT 19 strain isolated from contaminated and decaying cheese samples. Maximum GOD production was attained at pH 6 and 20°C in fermentation broth after 72 h of incubation. The effects of metal ions and sugars were screened for the induction of higher GOD production. The results revealed that glucose and lactose give the highest production of enzyme (0.670 and 0.552 U/mL, respectively) as compared with other sugars (sucrose, cellulose, mannitol and fructose). Out of the seven metal ions studied, CaCO₃ (1.123 U/mL) and FeSO₄ (0.822 U/mL) act as modulators, while MgSO₄ (0.535 U/mL), CuSO₄ (0.498 U/mL), HgCl₂ (0.476 U/mL), ZnSO₄ (0.457 U/mL) and BaSO₄ (0.422 U/mL) yield lower production. The study therefore suggests that a strain of P. chrysogenum SRT 19 can be used as a new strain for GOD production.