Glucose Oxidase Synthesis Research Articles

Optimization of Aspergillus niger Fermentation for the Production of Glucose Oxidase

A number of nutritional factors influencing glucose oxidase (EC 1.1.3.4) production by Aspergillus niger NCIM 545 were studied. The synthesis of glucose oxidase by A. niger was investigated in two steps using submerged fermentation at 30 ± 2 °C and 180 rpm for 96 h. Primarily, nutritional components were selected by one-factor-at-a-time method, and the significance of each component with respect to glucose oxidase production was identified by Plackett–Burman design (seven variables including six nutritional viz. sucrose, sodium nitrate, peptone, calcium carbonate, magnesium sulfate, and potassium dihydrogen phosphate, and one dummy or unassigned variable were studied with eight experiments). In the second step, concentration of most significant factors and their interaction were studied with response surface methodology (central composite design). Each variable in the design was studied at five different levels, with all variables taken at a central coded value of zero. Considerable amount of glucose oxidase was produced from A. niger species with sucrose as the carbon source, sodium nitrate as the inorganic nitrogen source, and peptone as the organic nitrogen source. Glucose oxidase activity increased remarkably by 28.93 fold (from 0.00993 to 0.29 U ml−1) with CaCO3-supplemented media. The outcome of Plackett–Burman design showed CaCO3, peptone, and MgSO4 as significant parameters. Further optimization using a three-factor central composite design with 20 experiments increased yield of glucose oxidase from 0.29 to 2.05 U ml−1 (sevenfold) with a decrease in cultivation time from 96 to 72 h.

KLONING OF GLUCOSE OXIDASE GENE IN PENICILLLIUM ADAMETZII LF F-2044.1

Vector pNOM102-GOX was engineered to cloning gene gox in P. adametzii LF F-2044.1 and conditions were optimized for production of fungal protoplasts. Electroporation of P. adametzii LF F-2044.1 was conducted and transformants resistant to antibiotic geneticin were selected. Strains P. adametzii LF F-2044.1.17 and P. adametzii LF F-2044.1.18 displayed increased levels of glucose oxidase synthesis - mycelium productivity rose 2 - 2.5 times. It was found that efficient preservation of vectors in the transformants requires maintenance on the media containing antibiotic geneticin.

Spontaneous variability of glucose oxidase-producing fungus Penicillium adametzii LF F-2044

The glucose oxidase-producing fungus Penicillium adametzii LF F-2044 was studied for natural variability. Four variants of the fungus differed in morphological characteristics and glucose oxidase synthesis. The synthesis of extracellular glucose oxidase and the productivity of morphological variants P. adametzii LF F-2044.1 and P. adametzii LF F-2044.2 were 127-146 and 95-159% higher, respectively, than the control. Highly active morphological variants of the fungus were chosen for further selection experiments.

Growth Characteristics and Glucose Oxidase Production in Mutant Penicillium funiculosum Strains

The main parameters of growth and glucose oxidase production by the mutant Penicillium funiculosum strains BIM F-15.3, NMM95.132, and 46.1 were studied. The synthesis of extracellular glucose oxidase by these strains was constitutive and occurred following the phase of exponential growth. The mutant strains also synthesized extracellular invertase and cell-associated catalase and glucose oxidase. The syntheses of invertase, the cell-associated enzymes, and extracellular glucose oxidase were found to be maximum between 14 and 18 h, between 48 and 52 h, and by the 96th h of cultivation, respectively. Among the mutants studied, P. funiculosum 46.1 showed the maximal rates of growth and glucose oxidase synthesis.

Selection of Penicillium funiculosum strains with high glucose oxidase activity

Metabolic inhibitors, riboflavin, and end products of glucose oxidation were shown to hold much promise for the selection of Penicillium funiculosum mutant strains with a high glucose oxidase activity. The incidence of positive mutations was highest in clones resistant to sodium azide, riboflavin, and beta-D-glucono-delta-lactone. Enzyme activity in Penicillium funiculosum mutants was studied under conditions of submerged cultivation. The intensity of glucose oxidase synthesis in seven cultures was 24-56% higher than that in the parent strain of Penicillium funiculosum NMM95.132.

GpdA-promoter-controlled production of glucose oxidase by recombinant Aspergillus niger using nonglucose carbon sources.

The gpdA-promoter-controlled exocellular production of glucose oxidase (GOD) by recombinant Aspergillus niger NRRL-3 (GOD3-18) during growth on glucose and nonglucose carbon sources was investigated. Screening of various carbon substrates in shake-flask cultures revealed that exocellular GOD activities were not only obtained on glucose but also during growth on mannose, fructose, and xylose. The performance of A. niger NRRL-3 (GOD3-18) using glucose, fructose, or xylose as carbon substrate was compared in more detail in bioreactor cultures. These studies revealed that gpdA-promoter-controlled GOD synthesis was strictly coupled to cell growth. The gpdA-promoter was most active during growth on glucose. However, the unfavorable rapid GOD-catalyzed transformation of glucose into gluconic acid, a carbon source not supporting further cell growth and GOD production, resulted in low biomass yields and, therefore, reduced the advantageous properties of glucose. The total (endo- and exocellular) specific GOD activities were lowest when growth occurred on fructose (only a third of the activity that was obtained on glucose), whereas utilization of xylose resulted in total specific GOD activities nearly as high as reached during growth on glucose. Also, the portion of GOD excreted into the culture fluid reached similar high levels (approximately equal to 90%) by using either glucose or xylose as substrate, whereas growth on fructose resulted in a more pelleted morphology with more than half the total GOD activity retained in the fungal biomass. Finally, growth on xylose resulted in the highest biomass yield and, consequently, the highest total volumetric GOD activity. These results show that xylose is the most favorable carbon substrate for gpdA-promoter-controlled production of exocellular GOD.

Synthesis of glucose oxidase and catalase by Aspergillus niger in resting cell culture system.

The synthesis of glucose oxidase and catalase by Aspergillus niger was investigated using a resting cell culture system without growth being established. Calcium carbonate induced the synthesis of both enzymes and calcium chloride inhibited it. The optimal pH for the biosynthesis of glucose oxidase and catalase was 6.0 and 5.7, respectively. The effects of other bivalent cations, reductive compounds and metabolic products on enzyme synthesis were also tested. The biosynthesis of glucose oxidase and catalase was promoted by MnCO3, thioglycolic acid, pyroracemic acid and gluconic acid.

Glucose oxidase biosynthesis in relation to biochemical mutations in Aspergillus niger

AbstractThe production of extracellular glucose oxidase in a submerged culture by a number of auxotrophic, 2‐deoxy‐D‐glucose resistant and protease‐less mutants of Aspergillus niger was evaluated. Among the auxotrophic strains, no evident dependence was found between the kind of the nutritional requirements and the level of the glucose oxidase activity. However, the majority of auxotrophs, requiring serine or niacin, showed a higher enzyme activity (from 16 to 680%) than the parent strain. The dynamics of the glucose oxidase synthesis by the free and immobilized mycelium of the most active niacin− mutant of A. niger was also investigated.

Intensification of glucose oxidase synthesis by multistage mutagenesis of Aspergillus niger. Scientific note.

Intensification of glucose oxidase synthesis by multistage mutagenesis of Aspergillus niger. Scientific note.

Expression of the glucose oxidase gene from Aspergillus niger in Hansenula polymorpha and its use as a reporter gene to isolate regulatory mutations

The glucose oxidase gene (god) from Aspergillus niger was expressed in Hansenula polymorpha using the methanol oxidase promoter and transcription termination region and the MF-alpha leader sequence from Saccharomyces cerevisiae to direct secretion. The expression cassette was cloned into the S. cerevisiae vector YEp13 and used to transform H. polymorpha strain A16. In the initial transformants plasmid replication was unstable, but was stabilized by a growth regime consisting of alternating cycles of selective and non-selective growth. The stabilized strain was grown to high cell density by fed-batch fermentation. Upon induction of the MOX promoter, glucose oxidase synthesis was initiated. At the end of the fermentation, the culture density was 76 g dry weight/1 and 108 IU/ml (0.5 g/1 or 0.65% dry weight) glucose oxidase was found in the culture medium; a further 86 IU/ml (0.43 g/1 or 0.56% dry weight) was recovered from the cell lysate. A plate assay was used to monitor glucose oxidase levels in individual colonies. This was then used to isolate mutants which showed abnormal regulation of god expression or which showed an altered pattern of secretion. One mutant, which showed increased production of glucose oxidase, was grown to high cell density by fed-batch fermentation (100.6 g/l) and produced 445 IU/ml(2.25 g/l or 2.2% dry weight) extracellularly and 76 IU/ml (0.38 g/l or 0.4% dry weight) intracellularly. The mutant thus not only increased total production but exported 83% of the total enzyme made compared to 55% in the parent strain.

Intensification of glucose oxidase synthesis with aspergillus niger by the way of multistage mutagenization

AbstractConidia of glucose oxidase active Aspergillus niger G 13 strain were subjected to the fourth‐stage mutagenization using different combinations of mutagens in each stage (stage I – UV radiation + nitrosomethylurea, stage II – UV radiation + ethyleneimine, Stage III – acryflavine, stage IV – UV radiation + N‐methyl‐N′‐nitrosoguanidine). In all, 400 strains which showed higher glucose oxidase activity with our own diffusion data method (were isolated out of 5208 colonies after mutagenization). Nine most active mutants were tested in the submerged culture determining the activity of glucose oxidase in the post‐culture liquid which increased from 10.7 to nearly 30%.

Optimization of glucose oxidase synthesis in submerged cultures of Aspergillus niger G-13 mutant

Both shaken flask cultures and aerated fermenter cultures of the previously selected Aspergillus niger G-13 mutant were optimized for glucose oxidase production (β- d- glucose: oxygen 1-oxidoreductase, EC 1.1.3.4). The enzyme synthesis was strongly influenced by glucose and calcium carbonate. Optimum results were obtained after about 36 h of fermenter culture when the concentrations of both ingredients were respectively 8 and 3.5%. The crude enzyme preparate obtained by gel filtration Sephadex G-25 column and lyophilization was highly specific for glucose and 2-deoxyglucose, but the latter substrate was oxidized about three times more slowly. The heat stability and pH sensitivity of the enzyme were also studied.

Gluconate accumulation and enzyme activities with extremely nitrogen-limited surface cultures of Aspergillus niger.

Batch cultures of Aspergillus niger grown from conidia on a medium with high C/N ratio accumulated gluconate from glucose with a yield of 57%. During almost the whole time of accumulation there was no net synthesis of total protein in the mycelium but the activity per flask and the specific activity of glucose oxidase (EC 1.1.3.4) in mycelial extracts increased whereas both values decreased for glucose dehydrogenase (EC 1.1.99.10) 'gluconate 6-phosphatase' (cf. EC 3.1.3.1, 3.1.3.2), gluconokinase (EC 2.7.1.12), glucose 6-phosphate and phosphogluconate dehydrogenases (EC 1.1.1.49, EC 1.1.1.44), phosphoglucomutase (EC 2.7.5.1), and most enzymes of the Embden-Meyerhof pathway and the tricarboxylic acid cycle. Gluconate dehydratase (EC 4.2.1.39), gluconate dehydrogenase (EC 1.1.99.3) and enzymes of the Entner-Doudoroff pathway could not be detected. By cycloheximide the increase of glucose oxidase activity was inhibited. It is concluded that the high yield of gluconate was due mainly to the net (de novo) synthesis of glucose oxidase which occurred during protein turnover after the exhaustion of the nitrogen source, and which was not accompanied by a net synthesis of the other enzymes investigated. Some gluconate may also have been formed by hydrolytic cleavage of gluconate 6-phosphate.