Spores of Aspergillus niger as reservoir of glucose oxidase synthesized during solid-state fermentation and their use as catalyst in gluconic acid production

AIMS: To exploit conidiospores of Aspergillus niger as a vector for glucose oxidase extraction from solid media, and their direct use as biocatalyst in the bioconversion of glucose to gluconic acid. METHODS AND RESULTS: Spores of A. niger (200 h old) were shown to fully retain all the glucose oxidase synthesized by the mycelium during solid-state fermentation (SSF). They acted as catalyst and carried out the bioconversion reaction effectively, provided they were permeabilized by freezing and thawing. Glucose oxidase activity was found retained in the spores even after repeated washings. Average rate of reaction was 1.5 g l(-1) h(-1) with 102 g l(-1) of gluconic acid produced out of 100 g l(-1) glucose consumed after approx. 100 h reaction, which corresponded to a molar yield close to 93%. These results were obtained with permeabilized spores in the presence of a germination inhibitor, sodium azide. CONCLUSIONS: Spores of A. niger served as efficient catalyst in the model bioconversion reaction after permeabilization. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first detailed study on the ability of A. niger spores to act as reservoir of enzyme synthesized during SSF without its release into solid media. Use of this material served as an innovative concept for enzyme extraction and purification from a solid medium. Moreover, this approach could compete efficiently with the conventional use of mycelial form of the fungus in gluconic acid production.     

Purification and thermodynamic characterization of glucose oxidase from a newly isolated strain of Aspergillus niger

An intracellular glucose oxidase (GOD) was isolated from the mycelium extract of a locally isolated strain of Aspergillus niger NFCCP. The enzyme was partially purified to a yield of 28.43% and specific activity of 135 U mg(-1) through ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The enzyme showed high specificity for D-glucose, with a K(m) value of 25 mmol L(-1). The enzyme exhibited optimum catalytic activity at pH 5.5. Optimum temperature for GOD-catalyzed D-glucose oxidation was 40 degrees C. The enzyme displayed a high thermostability having a half-life (t(1/2)) of 30 min, enthalpy of denaturation (H*) of 99.66 kJ mol(-1), and free energy of denaturation (G*) of 103.63 kJ mol(-1). These characteristics suggest that GOD from A. niger NFCCP can be used as an analytical reagent and in the design of biosensors for clinical, biochemical, and diagnostic assays.     

Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy

A simple and direct assay method for glucose oxidase (EC 1.1.3.4) from Aspergillus niger and Penicillium amagasakiense was investigated by Fourier transform infrared spectroscopy. This enzyme catalyzed the oxidation of d-glucose at carbon 1 into d-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide ((2)H(2)O). The intensity of the d-glucono-1,5-lactone band maximum at 1212 cm(-1) due to CO stretching vibration was measured as a function of time to study the kinetics of d-glucose oxidation. The extinction coefficient epsilon of d-glucono-1,5-lactone was determined to be 1.28 mM(-1)cm(-1). The initial velocity is proportional to the enzyme concentration by using glucose oxidase from both A. niger and P. amagasakiense either as cell-free extracts or as purified enzyme preparations. The kinetic constants (V(max), K(m), k(cat), and k(cat)/K(m)) determined by Lineweaver-Burk plot were 433.78+/-59.87U mg(-1) protein, 10.07+/-1.75 mM, 1095.07+/-151.19s(-1), and 108.74 s(-1)mM(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on horseradish peroxidase in aqueous media: 470.36+/-42.83U mg(-1) protein, 6.47+/-0.85 mM, 1187.77+/-108.16s(-1), and 183.58 s(-1)mM(-1) for V(max), K(m), k(cat), and k(cat)/K(m), respectively. Therefore, this spectroscopic method is highly suited to assay for glucose oxidase activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of glucose oxidase activity.     

Purification,characterization, and identification of a novel bifunctional catalase-phenol oxidase from <Emphasis Type="Italic">Scytalidium thermophilum</Emphasis>

A novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. 10-fold with 46% yield and was biochemically characterized. The enzyme contains heme and has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. Catalase and phenol oxidase activities were most stable at pH 7.0. The activation energies of catalase and phenol oxidase activities of the enzyme were found to be 2.7 +/- 0.2 and 10.1 +/- 0.4 kcal/mol, respectively. The pure enzyme can oxidize o-diphenols such as catechol, caffeic acid, and L-DOPA in the absence of hydrogen peroxide and the highest oxidase activity is observed against catechol. No activity is detected against tyrosine and common laccase substrates such as ABTS and syringaldazine with the exception of weak activity with p-hydroquinone. Common catechol oxidase inhibitors, salicylhydroxamic acid and p-coumaric acid, inhibit the oxidase activity. Catechol oxidation activity was also detected in three other catalases tested, from Aspergillus niger, human erythrocyte, and bovine liver, suggesting that this dual catalase-phenol oxidase activity may be a common feature of catalases.     

Glucose oxidase synthesis by Aspergillus niger GIV-10 on starch.

The effect of various kinds of starch, as the sole source of organic carbon, on the biosynthesis of glucose oxidase by A. niger GIV-10 was examined. A. niger grown on 6% wheat starch medium provided extracellular and intracellular glucose oxidase with the highest enzymatic activities. A new method of intracellular glucose oxidase extraction (without disruption of mycelium), developed and discussed in this paper, increased 2 to 3.8-times glucose oxidase yield, as compared to that described earlier.     

Mediation of glycosylated and partially-deglycosylated glucose oxidase of Aspergillus niger by a ferrocene-derivatised detergent.

A ferrocene-derivatised detergent, (11-ferrocenylundecyl) trimethylammonium bromide (FTMAB), when oxidised to the corresponding ferricinium ion, was found by electrochemical studies to be an effective electron acceptor for reduced glucose oxidase of Aspergillus niger (EC 1.13.4) and thus acts as a electron-transfer mediator between glucose oxidase and a working electrode held at a potential sufficiently positive to reoxidise reduced FTMAB. An increase in mediating activity was produced when FTMAB was present in concentrations above its critical micelle concentration. An 'enzyme electrode' was formed by adsorption of glucose oxidase and FTMAB surfactant on a graphite rod. The electrode functioned as an amperometric biosensor for glucose in phosphate-buffered saline solution. A mixed micelle of glucose oxidase and FTMAB, probably adsorbed on the electrode surface, appears to be advantageous for the amperometric determination of glucose. Additionally, glucose oxidase was treated with alpha-mannosidase. When this partially-deglycosylated glucose oxidase was incorporated in an enzyme electrode, a 100-fold increase in the second-order rate constant (k) for electron transfer between the enzyme and FTMAB was observed, together with increased current densities, with respect to the equivalent values for FTMAB and commercial glucose oxidase. The use of deglycosylated enzymes in biosensors is suggested.     

Effects of immobilization on the kinetics of enzyme-catalyzed reactions. I. Glucose oxidase in a recirculation reactor system.

Glucose oxidase from Aspergillus niger was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed-column recycle reactor. The optimum pH of the immobilized enzyme was the same as that of soluble enzyme; however, immobilized glucose oxidase showed a sharper pH-activity profile than that of the soluble enzyme. The kinetic behavior of immobilized glucose oxidase at optimum pH and 25 degrees C was similar to that of the soluble enzyme, but the immobilized material showed increased temperature sensitivity. Immobilized glucose oxidase showed no loss in activity on storage at 4 degrees C for nearly ten weeks. On continuous use for 60 hr, the immobilized enzyme showed about a 40% loss in activity but no change in the kinetic constant.     

Expression of an Aspergillus niger glucose oxidase in saccharomyces cerevisiae and its use to optimize fructo-oligosaccharides synthesis

Fructo-oligosaccharides (FOS) represent the most abundantly supplied and utilized group of nondigestible oligosaccharides as food ingredients. These prebiotics can be produced from sucrose using the transglycosylating activity of beta-fructofuranosidases (EC 3.2.1.26) at high concentrations of the starting material. The main problem during FOS synthesis is that the activity of the enzyme is inhibited by the glucose generated during the reaction, and therefore the maximum FOS content in commercial products reaches up to 60% on a dry substance basis. The glucose oxidase (gox) gene from Aspergillus niger BT18 was cloned and integrated, as part of an expression cassette, into the ribosomal DNA of a Saccharomyces cerevisiae host strain. One of the recombinant strains with a high copy number of the gox gene and showing a high GOX specific activity was used to produce the enzyme. Addition of the extracellular glucose oxidase to the FOS synthesis reaction helped to remove the glucose generated, avoiding the inhibition of the fungal beta-fructofuranosidase. As a result, a final syrup containing up to 90% of FOS was obtained.     

瑞氏木霉表达黑曲霉葡萄糖氧化酶

利用高表达分泌纤维素酶的真菌瑞氏木霉表达重组的黑曲霉葡萄糖氧化酶。在大肠杆菌DH5α中构建瑞氏木霉纤维素酶CBHI启动子和CBHI信号肽基因黑曲霉葡萄糖氧化酶基因瑞氏木霉纤维素酶CBHI终止子构巢曲霉的甘油醛3磷酸脱氢酶启动子大肠杆菌抗潮霉素B磷酸转移酶基因构巢曲霉色氨酸C终止子pUC19(命名为pCBHGOD)质粒,线性化后用瑞氏木霉纤维素酶CBHI启动子和CBHI信号肽基因黑曲霉葡萄糖氧化酶基因瑞氏木霉纤维素酶CBHI终止子构巢曲霉的甘油醛3磷酸脱氢酶启动子大肠杆菌抗潮霉素B磷酸转移酶基因构巢曲霉色氨酸C终止子(命名为CBHGOD)核酸片段转化瑞氏木霉QM9414原生质体。用PCR扩增方法筛选出同源重组葡萄糖氧化酶基因的瑞士木霉突变株。用麦杆诱导瑞氏木霉突变株,生产黑曲霉葡萄糖氧化酶,Westernblot分析重组的葡萄糖氧化酶分子量与Sigma公司的天然黑曲霉葡萄糖氧化酶一致,生产的重组酶活性25umL,相当于Sigma公司葡萄糖氧化酶标准品的产量为0.5gL。瑞氏木霉可用于生产黑曲霉葡萄糖氧化酶。     

Intracellular glucose oxidase from Penicillium funiculosum 46.1

A method for isolating extracellular glucose oxidase from the fungus Penicillium funiculosum 46.1, using ultrafiltration membranes, was developed. Two samples of the enzyme with a specific activity of 914-956 IU were obtained. The enzyme exhibited a high catalytic activity at pH above 6.0. The effective rate constant of glucose oxidase inactivation at pH 2.6 and 16 degrees C was 2.74 x 10(-6) s-1. This constant decreased significantly as pH of the medium increased (4.0-10.0). The temperature optimum for glucose oxidase-catalyzed beta-D-glucose oxidation was in the range 30-65 degrees C. At temperatures below 30 degrees C, the activation energy for beta-D-glucose oxidation was 6.42 kcal/mol; at higher temperatures, this parameter was equal to 0.61 kcal/mol. Kinetic parameters of glucose oxidase-catalyzed delta-D-glucose oxidation depended on the initial concentration of the enzyme in the solution. Glucose oxidase also catalyzed the oxidation of 2-deoxy-D-glucose, maltose, and galactose.     

Glucose oxidase from Aspergillus niger. Cloning, gene sequence, secretion from Saccharomyces cerevisiae and kinetic analysis of a yeast-derived enzyme

The gene for Aspergillus niger glucose oxidase (EC 1.1.3.4) has been cloned from both cDNA and genomic libraries using oligonucleotide probes derived from the amino acid sequences of peptide fragments of the enzyme. The mature enzyme consists of 583 amino acids and is preceded by a 22-amino acid presequence. No intervening sequences are found within the coding region. The enzyme contains 3 cysteine residues and 8 potential sites for N-linked glycosylation. The protein shows 26% identity with alcohol oxidase of Hansenuela polymorpha, and the N terminus has a sequence homologous with the AMP-binding region of other flavoenzymes such as p-hydroxybenzoate hydroxylase and glutathione reductase. Recombinant yeast expression plasmids have been constructed containing a hybrid yeast alcohol dehydrogenase II-glyceraldehyde-3-phosphate dehydrogenase promoter, either the yeast alpha-factor pheromone leader or the glucose oxidase presequence, and the mature glucose oxidase coding sequence. When transformed into yeast, these plasmids direct the synthesis and secretion of between 75 and 400 micrograms/ml of active glucose oxidase. Analysis of the yeast-derived enzymes shows that they are of comparable specific activity and have more extensive N-linked glycosylation than the A. niger protein.     

Rational redesign of glucose oxidase for improved catalytic function and stability

Glucose oxidase (GOx) is an enzymatic workhorse used in the food and wine industries to combat microbial contamination, to produce wines with lowered alcohol content, as the recognition element in amperometric glucose sensors, and as an anodic catalyst in biofuel cells. It is naturally produced by several species of fungi, and genetic variants are known to differ considerably in both stability and activity. Two of the more widely studied glucose oxidases come from the species Aspergillus niger (A. niger) and Penicillium amagasakiense (P. amag.), which have both had their respective genes isolated and sequenced. GOx from A. niger is known to be more stable than GOx from P. amag., while GOx from P. amag. has a six-fold superior substrate affinity (K(M)) and nearly four-fold greater catalytic rate (k(cat)). Here we sought to combine genetic elements from these two varieties to produce an enzyme displaying both superior catalytic capacity and stability. A comparison of the genes from the two organisms revealed 17 residues that differ between their active sites and cofactor binding regions. Fifteen of these residues in a parental A. niger GOx were altered to either mirror the corresponding residues in P. amag. GOx, or mutated into all possible amino acids via saturation mutagenesis. Ultimately, four mutants were identified with significantly improved catalytic activity. A single point mutation from threonine to serine at amino acid 132 (mutant T132S, numbering includes leader peptide) led to a three-fold improvement in k(cat) at the expense of a 3% loss of substrate affinity (increase in apparent K(M) for glucose) resulting in a specify constant (k(cat)/K(M)) of 23.8 (mM(-1) · s(-1)) compared to 8.39 for the parental (A. niger) GOx and 170 for the P. amag. GOx. Three other mutant enzymes were also identified that had improvements in overall catalysis: V42Y, and the double mutants T132S/T56V and T132S/V42Y, with specificity constants of 31.5, 32.2, and 31.8 mM(-1) · s(-1), respectively. The thermal stability of these mutants was also measured and showed moderate improvement over the parental strain.     

Oxalate accumulation from citrate by Aspergillus niger. I. Biosynthesis of oxalate from its ultimate precursor.

Carbon-14 was incorporated from citrate-1,5-14C, glyoxylate-14C(U), or glyoxylate-1-14C into oxalate by cultures of Aspergillus niger pregrown on a medium with glucose as the sole source of carbon. Glyoxylate-14C(U) was superior to glyoxylate-1-14C and citrate-1,5-14C as a source of incorporation. By addition of a great amount of citrate the accumulation of oxalate was accelerated and its maximum yield increased. In a cell-free extract from mycelium forming oxalate from citrate the enzyme oxaloacetate hydrolase (EC3.7.1.1) was identified. Its in vitro activity per flask exceeded the rate of in vivo accumulation of oxalate. Glyoxylate oxidizing enzymes (glycolate oxidase, EC1.1.3.1; glyoxylate oxidase, EC1.2.3.5;NAD(P)-dependent glyoxylate dehydrogenase; glyoxylate dehydrogenase, CoA-oxalylating, EC1.2.1.7) could not be detected in cell-free extracts. It is concluded that in cultures accumulating oxalate from citrate after pregrowth on glucose, oxalate arises by hydrolytic cleavage of oxaloacetate but not by oxidation of glyoxylate.     

Screening and mutagenesis of moulds for the improvement of glucose oxidase production

The strain of Aspergillus niger G most effective for producing glucose oxidase (see β- -glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) was selected out of 110 moulds belonging to 15 different species by the method of test-tube microculture. Conidia of the selected strain were further subjected to mutagenesis with both u.v. and N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and the products were analysed for glucose oxidase activity with our own diffusion plate method. Among 960 strains isolated after mutagenesis only 12 showed higher activity (from 1.5 to 18%) than the starting strain A. niger G.     

Crystallization and preliminary X-ray diffraction studies of a deglycosylated glucose oxidase from Aspergillus niger

Deglycosylation was shown to be an important prerequisite step for the crystallization of glucose oxidase from Aspergillus niger. Whereas the glycosylated enzyme could not be crystallized, crystals of the deglycosylated enzyme suitable for X-ray diffraction analysis were reproducibly grown in the presence of 1.6 M-ammonium sulphate and octanetriol at pH 5.3 to 5.6. The crystals belong to the space group P3(1)21 or P3(2)21 with refined lattice constants of a = 66.5 A and c = 214.4 A, indicating a cell content of one monomer per asymmetric unit of the crystal. Crystals diffract to at least 2.5 A resolution. Cleavage of 95% of its carbohydrate moiety affected the kinetics of glucose oxidation, stability at low pH and some electrophoretic properties of glucose oxidase, such as molecular mass and the number of isoelectric forms. However, other properties, such as thermal stability, pH and temperature optima of activity were not affected.     

Permeabilization and inhibition of the germination of spores of Aspergillus niger for gluconic acid production from glucose

In this study, the role of citral to permeabilize the spores of Aspergillus niger and replace sodium azide in the bioconversion medium was studied. Further, characterization of glucose oxidase of spores was carried out by exposing both permeabilized and unpermeabilized spores to different pressures (1, 2, 2.7 kb) and temperatures (60, 70, 80, 90 degrees C). Unpermeabilized spores after exposure to high temperatures were permeabilized by freezing before using as catalyst in the bioconversion reaction. Results showed that citral permeabilized the spores and could inhibit spore germination in the bioconversion medium. Rate of reaction was significantly increased from 1.5 to 4.35 g/Lh which was higher than the commercial glucose oxidase 2g/Lh). Glucose oxidase activity of A. niger was resistant to pressure. However, pressure treatment could not permeabilize them. Behaviour of fresh and permeabilized spores to temperature varied significantly. Glucose oxidase activity of fresh spores exposed to high temperature was unaffected at 70 degrees C till 15 min and 84% of relative activity was retained even after 1h at 70 degrees C while permeabilized spore got inactivated at 70 degrees C for 15 min, which followed the same pattern as commercial glucose oxidase. Cellular membrane integrity was lost due to permeabilization by freezing which resulted in heat-inactivation of glucose oxidase when spores were permeabilized before heat treatment. Thus, glucose oxidase of spore remains heat stable when unpermeabilized and active while permeabilized and its reaction rate is higher than the commercial glucose oxidase.     

Selective isolation of Aspergillus niger mutants with enhanced glucose oxidase production

After mutagenization and selection, mutant Aspergillus niger strains resistant to certain agents were obtained. Seven of the mutants showed increased extracellular glucose oxidase (GOD), the level for individual cases ranged widely from 8.8 to over 138.5% in comparison with the parental strain. Studies of the relationship between method of selection and frequency of mutation showed that the highest frequency of positive mutations (15.8% and 17.3%) was obtained from mutants resistant to ethidium bromide (1 mmol 1^-1) and sodium gluconate (45%), respectively. The time course of growth and enzyme production by the most active mutant AM-11 showed intra- and extracellular GOD activities to have increased about 2.2- and 2.4-fold, respectively, compared with the parental strain.     

Characterization of the distribution of glucose oxidase in Penicillium sp. CBS 120262 and Aspergillus niger NRRL-3 cultures and its effect on integrated product recovery

Glucose oxidase (GO) is an important industrial enzyme typically purified from Penicillium and Aspergillus sp. As GO distribution within the cultures influences process design for maximal product recovery, distribution of GO activity in Penicillium sp. CBS 120262 and Aspergillus niger NRRL-3, during mid-exponential and stationary phases, is compared. On progression from mid-exponential to stationary phase, the percentage GO activity in the cytoplasm decreased 1.6- and 1.3-fold in Penicillium sp. and A. niger respectively. In Penicillium sp., a concomitant 1.8- and 1.9-fold decrease in the percentage GO activity in the cell envelope and slime mucilage respectively, translated into a 2.0-fold increase in the extracellular fluid. In A. niger, decreasing cytoplasmic GO activity was accompanied by 1.3-fold increases in the cell envelope and slime mucilage, with a 1.3-fold decrease in the extracellular fluid. Similar trends were observed in specific GO activities. As final GO activity recovered is governed by the purification program, recovery from the extracellular fluid plus cell extract or from the extracellular fluid only were compared through simulating processes of varying complexity. A critical yield for each purification stage was identified above which recovery from the extracellular fluid plus cell extract exceeded that from extracellular fluid alone. These results highlight the influence of microorganism, harvest time and efficiency of downstream process on GO activity delivered. In the systems studied, Penicillium sp. is the organism of choice and should be harvested during stationary phase. The purification process chosen should be informed by both enzyme distribution and individual purification stages yields.     

Effects of carbohydrate depletion on the structure, stability and activity of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger was purified to homogeneity by hydrophobic interaction and ion-exchange chromatography. Approx. 95% of the carbohydrate moiety was cleaved from the protein by incubation of glucose oxidase with endoglycosidase H and alpha-mannosidase. Cleavage of the carbohydrate moiety effected a 24-30% decrease in the molecular weight and a reduction in the number of isoforms of glucose oxidase. No significant changes were observed in the circular dichroism spectra of the deglycosylated enzyme. Other properties, such as thermal stability, pH and temperature optima of glucose oxidase activity and substrate specificity were not affected. However, removal of the carbohydrate moiety marginally affected the kinetics of glucose oxidation and stability at low pH. From these results it appears that the carbohydrate chain of glucose oxidase does not contribute significantly to the structure, stability and activity of glucose oxidase.