A thermostable glucose oxidase from Aspergillus heteromophus CBS 117.55 with broad pH stability and digestive enzyme resistance.

In this study, the heterologous expression of an engineered thermostablle glucose oxidase from Aspergillus heteromophus CBS 117.55 was achieved in P. pastoris. This recombinant GoxAh was thermostable, with an optimal temperature range 25 °C-65 °C, and it was capable of retaining greater than 90% of its initial activity following a 10-min incubation at 75 °C. This enzyme had an optimum pH of 6.0, and it could retain above 80% of its initial activity following a 2-h incubation at a broad pH range (2.0-8.0). Moreover, GoxAh displayed excellent pepsin and trypsin resistance, and highly resistant to a range of tested metal ions and chemical reagents. These good properties make GoxAh a promising candidate for feed additive. The Km and kcat/Km values of GoxAh were 187 mM and 1.09/mM/s, which limited its widespread application to some degree. However, due to its excellent characteristics, GoxAh is still of potential economic value for high value-added areas, as well as a good initial enzyme for developing applicable feed enzyme by protein engineering.

A High-Throughput Screening System Based on Droplet Microfluidics for Glucose Oxidase Gene Libraries.

Glucose oxidase (GOx) is an important industrial enzyme that can be optimized for specific applications by mutagenesis and activity-based screening. To increase the efficiency of this approach, we have developed a new ultrahigh-throughput screening platform based on a microfluidic lab-on-chip device that allows the sorting of GOx mutants from a saturation mutagenesis library expressed on the surface of yeast cells. GOx activity was measured by monitoring the fluorescence of water microdroplets dispersed in perfluorinated oil. The signal was generated via a series of coupled enzyme reactions leading to the formation of fluorescein. Using this new method, we were able to enrich the yeast cell population by more than 35-fold for GOx mutants with higher than wild-type activity after two rounds of sorting, almost double the efficiency of our previously described flow cytometry platform. We identified and characterized novel GOx mutants, the most promising of which (M6) contained a combination of six point mutations that increased the catalytic constant kcat by 2.1-fold compared to wild-type GOx and by 1.4-fold compared to a parental GOx variant. The new microfluidic platform for GOx was therefore more sensitive than flow cytometry and supports comprehensive screens of gene libraries containing multiple mutations per gene.

An automated food protein isolation approach on preparative scale by two-dimensional liquid chromatography with active modulation interface.

In this work, an automated 2D-LC approach for protein isolation from egg samples on preparative scale is proposed. The method is based on the use of a C18 guard column installed in a switching valve to focus the proteins coming from the first dimension column, before their elution in the second column. For the first dimension separation, a size-exclusion column, packed with 3 µm ultrapure silica particles was used. An RP column based on core-shell technology was used for the second dimension separation. A standard mixture of BSA, ß-lactoglobulin, and glucose oxidase, chosen as a protein model system, was used to optimize the chromatographic separation conditions. The fully automated workflow allowed to isolate, in a single-chromatographic analysis, a protein amount of 50 µg for each peak fraction, with a total time of 15 min for the first separation and additional 30 min of the second separation for each trapped protein. The final aim was the development of proper analytical tools for protein isolation from foodstuffs to be used for the molecular identification by MS, as well as for biotherapeutic uses, allergy testing, and large-scale investigations in biological systems.

How a multimeric macromolecule is affected by divalent salts? Experimental and simulation study.

Salts exist in any cell and living organism in contact with biological macromolecules. How these salts affect biomolecules such as enzyme is important from both basic sciences and practical technologies. It was observed that divalent salts can change structure and function of protein at higher concentrations. Here, we investigated the effect of divalent salt on the behavior of a multimeric enzyme. We treated glucose oxidase as dimer-active enzyme in different CaCl2 concentration and seen that the enzyme become inactive at high concentration of salt. These experimental results are in agreement with recently published researches. To find a possible mechanism, a series of molecular dynamics simulation of the enzyme were performed at different salt concentration. According to the MD simulation, the conformational changes at the active site and FAD-binding site support the hypothesis of enzyme inactivation at high CaCl2 concentration. MD simulations also showed that enzyme has an unstable conformation at higher salt concentration which is in agreement with our experimental data. Detailed structural properties of the enzyme have been analyzed under different conditions. To the best of our knowledge, this is the first study that bears detailed structural mechanism about the salt effects on multimeric macromolecules.

Direct comparison of gluco-oligosaccharide oxidase variants and glucose oxidase: substrate range and H2O2 stability.

Glucose oxidase (GO) activity is generally restricted to glucose and is susceptible to inactivation by H2O2. By comparison, the Y300A variant of gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum showed broader substrate range and higher H2O2 stability. Specifically, Y300A exhibited up to 40 times higher activity on all tested sugars except glucose, compared to GO. Moreover, fusion of the Y300A variant to a family 22 carbohydrate binding module from Clostridium thermocellum (CtCBM22A) nearly doubled its catalytic efficiency on glucose, while retaining significant activity on oligosaccharides. In the presence of 200 mM of H2O2, the recombinant CtCBM22A_Y300A retained 80% of activity on glucose and 100% of activity on cellobiose, the preferred substrate for this enzyme. By contrast, a commercial glucose oxidase reported to contain ≤0.1 units catalase/ mg protein, retained 60% activity on glucose under the same conditions. GOOX variants appear to undergo a different mechanism of inactivation, as a loss of histidine instead of methionine was observed after H2O2 incubation. The addition of CtCBM22A also promoted functional binding of the fusion enzyme to xylan, facilitating its simultaneous purification and immobilization using edible oat spelt xylan, which might benefit the usage of this enzyme preparation in food and baking applications.

A benzoboroxole-based affinity ligand for glycoprotein purification at physiological pH.

Developing ligands capable of carbohydrate recognition has become increasingly important as the essential roles of glycoproteins and glycolipids in a diverse array of cellular signaling, pathophysiology, and immune response mechanisms are elucidated. Effective ligands for the glycan portion of glycoproteins and glycolipids are needed for pre-enrichment proteomics strategies, as well as for the purification of individual glycoproteins from complex biological milieu encountered both in biochemistry research and bio-pharmaceutical development. In this work, we developed a carbohydrate specific affinity ligand for glycoprotein purification using a one-pot, multi-component synthesis reaction (Ugi synthesis) and an amine-functionalized benzoboroxole moiety immobilized on agarose beads. Benzoboroxoles are unique boronic acid derivatives that have recently been found to bind specifically to the cis-diol groups of carbohydrates at physiological pH, with superior affinity to any other Wulff-type boronic acid. The solid-phase affinity ligand developed herein specifically binds the carbohydrate moiety of the glycoprotein glucose oxidase, as well as a fluorescein isothiocyanate-dextran, as shown through deglycosylation binding studies. Additionally, the ligand is able to purify glucose oxidase from crude Escherichia coli lysate, at physiological pH, equitably to commercially available boronic acid-functionalized agarose beads that required alkaline pH conditions. Thus, this affinity ligand is a marked improvement on current, commercially available boronic acid-based glycoprotein enrichment matrices and has the potential to exhibit high individual glycoprotein specificity because of the additional functional groups available for variation on the Ugi scaffold.

Purification, biochemical characterization and antifungal activity of a novel Aspergillus tubingensis glucose oxidase steady on broad range of pH and temperatures.

This study was carried out to evaluate the in vitro and in vivo antifungal efficiency of Aspergillus tubingensis CTM 507 glucose oxidase (GOD) against plant pathogenic fungi. GOD displayed a wide inhibitory spectrum toward different fungi at a concentration of 20 AU. The GOD had a strong inhibitor effect on mycelia growth and spore germination of Pythium ultimum. Interestingly, the GOD exhibited a potent in vivo antifungal effect against P. ultimum responsible for potato plants disease. The antifungal GOD was purified 13-fold with 27 % yield and a specific activity of 3435 U/mg. The relative molecular mass of the GOD was 180 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The GOD activity was optimum at pH 4.5 and 60 °C. It was found to be stable over a large pH range (3-9). It also displayed a marked thermostability with a 50-min half-life at 65 °C. The 10 residues of the N-terminal sequence of the purified GOD (S-K-G-S-A-V-T-T-P-D) showed no homology to the other reported GOD, identifying a novel GOD. FTIR spectroscopic analysis revealed the presence of C-O and C=O groups corresponding to a D-glucono-lactone. The findings indicated that GOD is the first A. tubingensis-produced fungicide ever reported to exhibit such promising biological properties. It could become a natural alternative to synthetic fungicides to control certain important plant microbial diseases.

Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases.

Glucose oxidase (GOx) catalyzes the oxidation of glucose to form gluconic acid and hydrogen peroxide, a reaction with important applications in food preservation, the manufacture of cosmetics and pharmaceuticals, and the development of glucose monitoring devices and biofuel cells. We expressed Aspergillus niger wild type GOx and the B11 mutant, which has twice the activity of the wild type enzyme at pH 5.5, as C-terminal fusions with the Saccharomyces cerevisiae Aga2 protein, allowing the fusion proteins to be displayed on the surface of yeast EBY100 cells. After expression, we extracted the proteins from the yeast cell wall and purified them by ion-exchange chromatography and ultrafiltration. This produced a broad 100-140kDa band by denaturing SDS-PAGE and a high-molecular-weight band by native PAGE corresponding to the activity band revealed by zymography. The wild type and B11 fusion proteins had kcat values of 33.3 and 61.3s(-1) and Km values for glucose of 33.4 and 27.9mM, respectively. The pH optimum for both enzymes was 5.0. The kinetic properties of the fusion proteins displayed the same ratio as their native counterparts, confirming that yeast surface display is suitable for the high-throughput directed evolution of GOx using flow cytometry for selection. Aga2-GOx fusion proteins in the yeast cell wall could also be used as immobilized catalysts for the production of gluconic acid.

Safety evaluation of glucose oxidase from Penicillium chrysogenum.

Glucose oxidase (ß-d-glucose:oxygen 1-oxidoreductase; EC 1.1.2.3.4) is used in the food and beverage industry as a preservative and stabilizer and is commonly derived from the fungus Aspergillus niger. Although the safety of glucose oxidase preparations from A. niger is well-established, the use of preparations derived from other fungal species is of interest; however, an assessment of their safety is warranted. Here, we report on the safety of a glucose oxidase preparation derived from the fungus Penicillium chrysogenum (designated as PGO) for commercial use in food processing, as well as an ingredient in food. In a repeated dose 90-day oral toxicity study conducted in rats, PGO was without compound-related adverse effects at doses of up to 15,600U/kg body weight/day, equivalent to 193mg total organic solids/kg body weight/day. In addition, PGO was non-genotoxic in a series of genotoxicity tests, including a bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and a combined in vivo mammalian erythrocyte micronucleus test and comet assay. The results of these studies support the safe use of PGO in food for human consumption.

Fabrication of nanoindented electrodes for glucose detection.

BACKGROUND: The objective of this article was to design, fabricate, and evaluate a novel type of glucose biosensors based on the use of atomic force microscopy to create nanoindented electrodes (NIDEs) for the selective detection of glucose. METHODS: Atomic force microscopy nanoindentation techniques were extended to covalently immobilized glucose oxidase on NIDEs via composite hydrogel membranes composed of interpenetrating networks of inherently conductive poly(3,4-ethylenedioxythiophene) tetramethacrylate grown within ultraviolet cross-linked hydroxyethylmethacrylate-based hydrogels to produce an in vitro amperometric NIDE biosensor for the long-term monitoring of glucose. RESULTS: The calibration curve for glucose was linear from 0.25 to 20 mM. Results showed that the NIDE glucose biosensor has a much higher detection sensitivity of 0.32 microA/mM and rapid response times (<5 seconds). There was no interference from the competing interferent (fructose) present; the only interference was from species that react with H(2)O(2) (ascorbic acid). The linear equation was B(response) (microA) = 0.323 [glucose] (mM) + 0.634 (microA); n = 24, r(2) = 0.994. CONCLUSION: Results showed that the resultant NIDE glucose biosensor increases the dynamic range, device sensitivity, and response time and has excellent detecting performance for glucose.

Cloning and heterologous expression of glucose oxidase gene from Aspergillus niger Z-25 in Pichia pastoris.

A gene of glucose oxidase (GOD) from Aspergillus niger Z-25 was cloned and sequenced. The entire open reading frame (ORF) consisted of 1,818 bp and encoded a putative peptide of 605 amino acids. The gene was fused to the pPICZalphaA plasmid and overexpressed in Pichia pastoris SMD1168. The recombinant GOD (rGOD) was secreted into the culture using MF-alpha factor signal peptide under the control of the AOX1 promoter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that rGOD exhibited a single band at around 94 kDa. The maximal GOD activity of approximately 40 U/mL was achieved in shake flask by induction under optimal conditions after 7 days. rGOD was purified by ammonium sulfate precipitate leading to a final specific activity of 153.46 U/mg. The optimum temperature and pH of the purified enzyme were 40 degrees C and 6.0, respectively. Over 88% of maximum activity was maintained below 40 degrees C. And the recombinant enzyme displayed a favorable stability in the pH range from 4.0 to 8.0. The Lineweaver-Burk plotting revealed that rGOD exhibited a K (m) value of 16.95 mM and a K (cat) value of 484.26 s(-1).

An improved glucose/O2 membrane-less biofuel cell through glucose oxidase purification.

A key objective in any bioelectrochemical systems is to improve the current densities and mass transport limitation. Most of the work is focused on increasing the specific surface of the electrodes or improving the electron transfer between enzymes and electrodes. However, nothing is said about the comparison of purified and non-purified enzyme and their effects on the biosensor efficiency. To illustrate the effect of the enzyme purity, we studied the widely used commercial Glucose Oxidase (GOx) from Aspergillus niger that we are using in our miniature membrane-less biofuel cell. Our results indicate that even if additional compounds contained in the lyophilized enzyme powder do not interfere with its intrinsic catalytic properties, they could prevent a good electron transfer between the enzyme and the electrode surface. By introducing a purified glucose oxidase into a bioelectrocatalyst immobilized on an electrode surface, we show that we can increase the interaction between the enzyme and the redox polymer, forming a better homogenous, leather like gel. At 5mM glucose concentration and under oxygen atmosphere, the current is three-fold higher when using a purified enzyme than it is when using a non-purified enzyme. Built with this novel anode, we showed that a miniature implantable membrane-less glucose-O(2) biofuel cell could produce, under air, twice the power density that is usually obtained when using a non-purified GOx.

Trehalose-mediated thermal stabilization of glucose oxidase from Aspergillus niger.

Thermal inactivation and enzyme kinetics of glucose oxidase (a FAD dependent enzyme) were studied in the absence and presence of trehalose. The inactivation rate constant decreased by up to 50% at temperatures between 50 and 70 degrees C in the presence of 0.6M trehalose; as a consequence the glucose oxidase half-life increased. Intrinsic fluorescence spectra showed a maximum center of spectral mass (CSM) red shift of 6.5nm. Therefore, major structural changes seem to be related to glucose oxidase thermal inactivation. Trehalose decreased the rate constant for unfolding as monitored by CSM red shift kinetics indicating that this disaccharide favors the most compact folded state. The E(a) for unfolding was increased from 204 to 221kJ mol(-1). It is proposed that FAD dissociation is preceded by the exposition of hydrophobic regions, while the presence of trehalose was able to hinder the release of FAD. Enzyme kinetics analysis showed that trehalose does not affect V(max) but instead decreases K(m); as a result enzyme efficiency was increased. The stabilizing effect of trehalose in a cofactor-dependent enzyme has not been tested to date. In addition, glucose oxidase has an enormous commercial importance and therefore, the use of trehalose to stabilize glucose oxidase in its multiple applications seems to be promising.

Activation of lactoperoxidase system in milk by glucose oxidase immobilized in electrospun polylactide microfibers.

In this study, glucose oxidase (GOX) was immobilized in polylactide (PLA) fibers that were used to activate the lactoperoxidase (LP) system in milk. The GOX-containing microfibers were electrospun from emulsions prepared by dispersing aqueous GOX in PLA dissolved in a chloroform and N,N-dimethylformamide blend, using sorbitan monopalmitate as an emulsifier. The enzymatic activity of GOX-in-PLA fibers (1100 +/- 400 nm diameter) was more than 19 times higher than that of the GOX-in-PLA membrane formed by direct casting, due to the larger surface area of the electrospun fibers. The activation of LP in model solutions using GOX-in-PLA fibers provided a more sustained generation of antimicrobial OSCN(-) than direct activation using H(2)O(2). Preliminary evaluation on milk samples showed that the electrospun GOX-in-PLA microfibers are capable of activating the naturally present LP system, indicating that they may be promising for active food packaging applications to extend the shelf life of milk.

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.

Purification of glucose oxidase from complex fermentation medium using tandem chromatography.

A fast and efficient purification method for recombinant glucose oxidase (rGOx) for flask fermentation scale (up to 2L) was designed for the purposes of characterization of rGOx mutants during directed protein evolution. The Aspergillus niger GOx was cloned into a pYES2-alphaMF-GOx construct and expressed extracellularly in yeast Saccharomyces cerevisiae. Hydrophobic interaction (HIC)/size exclusion (SEC)-tandem chromatographic system was designed for direct purification of rGOx from a conditioned complex expression medium with minimum preceding sample preparation (only adjustments to conductivity, pH and coarse filtering). HIC on Butyl 650s (50 mM ammonium acetate pH 5.5 and 1.5 M ammonium sulphate) absorbs GOx from the medium and later it is eluted by 100% stepwise gradient with salt free buffer directly into SEC column (Sephadex 200) for desalting and final polishing separation. The electrophoretic and UV-vis spectrophotometric analyses have proven enzyme purity after purification.

A new fast method for nanoLC-MALDI-TOF/TOF-MS analysis using monolithic columns for peptide preconcentration and separation in proteomic studies.

A new fast method for identification and characterization of proteolytic digests of proteins by monolithic liquid chromatography coupled with mass spectrometry has been developed. The advantages of the monolithic columns are a high-pressure stability and low back pressure resulting in higher flow rates for capillary or nanosize columns simplifying the system handling. As was shown in several publications, such monolithic stationary phases are highly qualified for the analysis of peptides and proteins, but so far, only small volumes could be injected into the system, which might hamper the sample preparation leading to protein precipitation and partial loss of sample. To overcome the problem of small injection volumes, we established a system including a short monolithic trap column to allow preconcentration of the peptides. The injected sample is flushed at higher flow rates onto the trap column, bound to the stationary phase, and in this way concentrated in a few nanoliters before starting the separation. The expanded system was optimized and tested using different reference protein samples. Eluting peptides were detected by MALDI-TOF/TOF-MS and identified by database searching. The system is now a permanent part for proteome analysis in our lab, and as such, it was successfully applied for the detection of post-translational modifications and the analysis of membrane proteins. One example for these analyses is also included in this paper.

Glucose oxidase-immobilized glass disks for imaging of D-glucose in acute brain slices.

A biotinylated glucose oxidase (bGOD)-immobilized glass disk was prepared for visualizing D-glucose fluxes in acute brain slices. A mouse hippocampal slice was placed on the bGOD disk and stimulated with a stimulant solution containing horseradish peroxidase (HRP) and a substrate DA-64, followed by capturing digital images of Bindschedler's Green (BG), an oxidized form of DA-64, with a CCD camera. The bGOD membranes responded proportionally to D-glucose, ranging from 2.0 to 5.0 mM. Sucrose, GABA, L-glutamic acid, L-aspartic acid, glycine, acetylcholine and L-ascorbic acid at 10 mM did not cause any responses. The D-glucose fluxes in mouse hippocampal slices stimulated by a hypoxia solution were neuronal region-dependent, i.e., dentate gyrus (DG), cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3), while those stimulated by KCl was independent of the neuronal regions. The response of bGOD disks is discussed in terms of the principle, concentration dependence and selectivity.

Isolation, purification and characterization of a novel glucose oxidase from Penicillium sp. CBS 120262 optimally active at neutral pH.

A novel glucose oxidase (GOX), a flavoenzyme, from Penicillium sp. was isolated, purified and partially characterised. Maximum activities of 1.08U mg(-1)dry weight intracellular and 6.9U ml(-1) extracellular GOX were obtained. Isoelectric focussing revealed two isoenzymes present in both intra- and extracellular fractions, having pI's of 4.30 and 4.67. GOX from Penicillium sp. was shown to be dimeric with a molecular weight of 148kDa, consisting of two equal subunits with molecular weight of 70k Da. The enzyme displayed a temperature optimum between 25 and 30 degrees C, and an optimum pH range of 6-8 for the oxidation of beta-d-glucose. The enzyme was stable at 25 degrees C for a minimum of 10h, with a half-life of approximately 30 min at 37 degrees C without any prior stabilisation. The lyophilized enzyme was stable at -20 degrees C for a minimum of 6 months. GOX from Penicillium sp. Tt42 displayed the following kinetic characteristics: Vmax, 240.5U mg(-1); Km, 18.4mM; kcat, 741 s(-1) and kcat/Km, 40 s(-1)mM(-1). Stability at room temperature, good shelf-life without stabilisation and the neutral range for the pH optimum of this GOX contribute to its usefulness in current GOX-based biosensor applications.

Innovative modular membrane adsorber system for high-throughput downstream screening for protein purification.

To develop the most efficient strategy for the purification of proteins, two types of adsorber membrane devices with different functionalities were designed and tested: 8-strips and single spin columns. The most suitable type of membrane adsorber and the optimal chromatographic loading/elution conditions for several target proteins from different biological matrices could be determined simultaneously in microliter scale. Ion exchange (IEX), metal chelate (MC), and Concanavalin A (Con A) modified membrane types were tested in the devices. Bovine serum albumin (BSA) and lysozyme were used as model proteins for investigations of the binding capacity and protein recovery percentage of the 8-strip anion exchange and the cation exchange membrane. The isolation of His(6)-tagged proteins, Bgl-His and GFP-His from fermentation broth and lysate, respectively, was performed using an 8-strip metal chelate affinity membrane loaded with different metal ions. Separation behavior of a ternary protein mixture (BSA, lysozyme, and Bgl-His) was studied in 8-strips IEX and metal chelate membrane chromatography. The Con A affinity devices were developed on the basis of metal chelate membrane spin columns loaded with Cu(2+) ions and investigated using glucose oxidase (GOD) as model protein. In summary, the advantages of the membrane adsorber technology, such as fast processing and easy scale-up, were utilized. The devices made it possible to load the membrane directly with preclarified fermentation broth or cell lysate and separate the protein of interest often in a single step.