Recombinant Glucose Oxidase Research Articles

Production in Nicotiana benthamiana of a thermotolerant glucose oxidase that shows enzybiotic activity against Escherichia coli and Staphylococcus aureus

Glucose oxidase (GOX) catalyzes the FAD-dependent oxidation of α-D-glucose to D-gluconolactone with production of hydrogen peroxide. This enzyme encounters many biotechnological applications from glucose sensors to applications in food, pharma and textile industries. For this purpose, recombinant GOX versions, usually derived from Aspergillus niger, are produced in fermentation systems, frequently in filamentous fungi because other production platforms such as bacteria or yeast have rendered meager results. We wondered whether A. niger GOX, more specifically a mutant version with superior thermotolerant properties, could be efficiently produced in Nicotiana benthamiana plants. To this aim, we used a tobacco mosaic virus-derived vector that is inoculated into plant tissues using Agrobacterium tumefaciens. Results exhibited the efficient production of the recombinant GOX in plants and the facile downstream purification when the recombinant protein is targeted to the apoplast, the space between plasma membranes and cell walls. The plant-made recombinant GOX displayed excellent catalytic properties in broad pH and temperature conditions. In addition to establishing a new strategy to produce recombinant GOX in plants as a green alternative to traditional fungal fermentation, we further investigated the potential application of this protein as an ezybiotic. Results exhibited a remarkable bacteriocide activity against Escherichia coli and Staphylococcus aureus.

Sensitive electrochemical sequential enzyme biosensor for glucose and starch based on glucoamylase- and glucose oxidase-controllably co-displayed yeast recombinant

The sequential enzyme biosensors hold significant importance in measuring species which are usually hard to process with single-enzyme-based biosensors. However, sequential enzyme electrodes experience critical issues such as low catalytic efficiency, insensitivity and poor reproducibility. In this work, yeast surface co-displaying sequential enzymes of glucoamylase (GA) and glucose oxidase (GOx) with controllable ratios through the specific cohesion-dockerin protein interaction was explored, by which starch hydrolyzing by GA into glucose is the rate-limiting step. The modified electrodes were prepared by immobilizing yeast-GA&GOx whole-cell and reduced graphene oxide (RGO) on glassy carbon electrode (GCE), for which the direct electron transfer between the electrode and recombinant GOx was arrived. Interestingly, the current responses of sensors to starch and glucose are dependent on the displayed enzyme composition, of which the yeast-GA&GOx (2:1) exhibited the highest current. Thereafter, sequential enzyme sensor of yeast-GA&GOx (2:1)/RGO/GCE was developed. Based on reduction detection at negative potential without interference, the sensor is stable and capable of assaying glucose (linear range: 2.0–100 mg/L) or starch (linear range, 50–3500 mg/L), separately. Coupled with yeast-GOx/RGO/GCE glucose sensor, both glucose and starch in real samples can be detected satisfactorily. This work provides new ideas for the development of other sequential enzyme electrodes for potential applications.

Statistical optimization of recombinant glucose oxidase production by yeast Yarrowia lipolytica using response surface methodology

مقدمه: گلوکز اکسیداز به عنوان یک آنزیم تجاری در صنایع دارویی، دارویی و غذایی مورد توجه است. بهینه سازی محیط و شرایط تخمیر برای افزایش تولید گلوکز اکسیداز هترولوگ مهم است. هدف از مطالعه حاضر بهینه سازی تولید گلوکز اکسیداز در یک سویه نوترکیب مخمر یارروویا لیپولیتیکا از طریق روش‌های آماری بود.مواد و روش ها: روش سطح پاسخ به عنوان یک روش آماری برای طراحی آزمایش برای بهینه سازی تولید گلوکز اکسیداز نوترکیب توسط مخمر یارروویا لیپولیتیکا استفاده شد. سپس، از شرایط بهینه شده برای تولید گلوکز اکسیداز در کشت بیوراکتور استفاده شد.یافته ها: تولید گلوکز اکسیداز پس از بهینه سازی محیط در کشت ارلن تا 570 واحد در لیتر افزایش یافت که 2 برابر بیشتر از محیط غیربهینه شده است. در کشت بیوراکتور با استفاده از محیط بهینه شده حاوی گلوکز و ساکارز به عنوان منابع کربن، تولید گلوکز اکسیداز به ترتیب پس از دو و شش روز به 720 و 730 واحد در لیتر رسید.بحث و نتیجه گیری: نتایج این مطالعه اطلاعات ارزشمندی در مورد بهینه سازی آماری توسعه فرآیند زیستی برای تولید پروتئین هترولوگ در مخمر یارروویا لیپولیتیکا ارائه می‌دهد.

Enhanced in vitro anticancer activity of yeast expressed recombinant glucose oxidase versus commercial enzyme.

Cancer treatments continue to have many disadvantages. Reactive oxygen species, such as H2O2, in high concentrations, can cause cytotoxicity to cells, being even greater in cancer cells. One of the H2O2-producing enzymes is glucose oxidase; its application in cancer treatment should be explored. In this work, the extracellular expression of the mutated recombinant enzyme glucose oxidase was carried out in the eukaryotic expression system Pichia pastoris SMD1168, through the modification and optimization of the gox gene of Aspergillus niger to improve its expression in yeast and its purification. Also, the secretion signal of the alpha-mating factor from Saccharomyces cerevisiae was added to the gene for extracellular expression, and it was inserted into the expression vector pPIC3.5k. The extracellular expression of the enzyme facilitated purification by anion exchange chromatography; the purification was corroborated by SDS-PAGE, with a molecular weight of its subunit between 63 kDa and 100 kDa. The mutated recombinant enzyme glucose oxidase showed greater anticancer activity compared to the commercial glucose oxidase and could have potential for cancer treatment. KEY POINTS: • Pichia pastoris is an excellent eukaryotic expression system for proteins that need post-translational modifications. • Extracellular expression facilitates protein purification. • Glucose oxidase has potential application in cancer treatment.

In silico and in vivo analysis of signal peptides effect on recombinant glucose oxidase production in nonconventional yeast Yarrowia lipolytica.

Signal peptide (SP) is an important factor and biobrick in the production and secretion of recombinant proteins. The aim of this study was in silico and in vivo analysis of SPs effect on the production of recombinant glucose oxidase (GOX) in Yarrowia lipolytica. Several in silico softwares, namely SignalP4, Signal-CF, Phobius, WolfPsort 0.2, SOLpro and ProtParam, were used to analyse the potential of 15 endogenous and exogenous SPs for the secretion of recombinant GOX in Y. lipolytica. According to in silico results, the SP of GOX was predicted as suitable in terms of high secretory potential and of protein solubility and stability which is chosen for in vivo analysis. The recombinant Y. lipolytica strain produced 280U/L of extracellular GOX after 7days in YPD medium. The results show that the SP of GOX can be applied to efficient production of extracellular heterologous proteins and metabolic engineering in Y. lipolytica.

Expression and Characterization of Glucose Oxidase from Aspergillus niger in Yarrowia lipolytica.

Glucose oxidase (GOX) is currently used in clinical, pharmaceutical, food and chemical industries. The aim of this study was expression and characterization of Aspergillus niger glucose oxidase gene in the yeast Yarrowia lipolytica. For the first time, the GOX gene of A. niger was successfully expressed in Y. lipolytica using a mono-integrative vector containing strong hybrid promoter and secretion signal. The highest total glucose oxidase activity was 370U/L after 7days of cultivation. An innovative method was used to cell wall disruption in current study, and it could be recommended to use for efficiently cell wall disruption of Y. lipolytica. Optimum pH and temperature for recombinant GOX activity were 5.5 and 37°C, respectively. A single band with a molecular weight of 80kDa similar to the native and pure form of A. niger GOX was observed for the recombinant GOX in SDS-PAGE analysis. Y. lipolytica is a suitable and efficient eukaryotic expression system to production of recombinant GOX in compered with other yeast expression systems and could be used to production of pure form of GOX for industrial applications.

Expression of Aspergillus niger glucose oxidase in yeast Pichia pastoris SMD1168

ABSTRACTThe aim of this study was to establish a system for high expression of Aspergillus niger glucose oxidase (GOD) with glyceraldehyde-3-phosphate dehydrogenase gene promoter (pGAP) in Pichia SMD1168. The GOD gene from A. niger accc30161 was inserted into pGAPZαA plasmid carrying a pGAP promoter and was expressed in yeast Pichia pastoris. The expression vector, pGAPZαA-GOD, was validated by colony polymerase chain reaction (PCR), agarose gel electrophoresis, restriction enzyme analysis and sequencing methods. The pGAPZαA-GOD vector was transformed into yeast P. pastoris SMD1168 by electroporation and the positive strain was validated by PCR. The expression and enzyme activity of the recombinant GOD were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and enzymatic detection. The recombinant GOD in yeast was purified by ion exchange chromatography, and its biochemical properties (dynamics, thermal and pH stability) were analysed. The obtained results showed that the expression vector, pGAPZαA-GOD, was successfully constructed to express A. niger accc30161 GOD protein. After transformation, the pGAPZαA-GOD DNA fragment had been integrated into the P. pastoris SMD1168-GOD genome. High expression of GOD was achieved in SMD1168-GOD, and the enzyme activity of GOD reached 107.18 U/mL in the supernatant of the culture medium at 30 °C and pH 6. The activity of recombinant GOD protein in yeast was 1.35-fold higher than the commercial A. niger GOD, and its affinity to glucose, thermal stability and pH stability are similar to commercial GOD. Using pGAP promoter, A. niger GOD protein is efficiently expressed in recombinant P. pastoris SMD1168-GOD with defective protease.

Transparent and Capacitive Bioanode Based on Specifically Engineered Glucose Oxidase

AbstractHere we detail an optimized transparent capacitive glucose oxidizing bioanode, capable of supplying current densities of 10 µA cm−2 at applied potentials of 0.1 V–0.2 V versus saturated calomel electrode, when continuously performing in a simple phosphate buffer, pH 7.4 and artificial human tears, both with a glucose concentration of 0.05 mM only. When operating in pulse mode, the bioanode was able to deliver current densities as high as 21 µA cm−2 at the beginning of the pulse with 571 µC cm−2 total charges stored. The biogenic part of the enzymatic device was a recombinant glucose oxidase mutant from Penicillium amagasakiense with high catalytic efficiency towards glucose, up to 14.5⋅104 M−1 s−1. The non‐biogenic part of the anodic system was based on a poly(3,4‐ethylenedioxythiophene)‐graphene nanocomposite, as a highly capacitive component with a capacitance density in the 1 mF cm−2 range, multi‐walled carbon nanotubes, as an additional nanostructuring element, and a conductive organic complex, as an electron shuttle between the redox enzyme and the electrode surface. The bioanode could potentially serve as a prototype of a double‐function enzymatic anode for hybrid electric power biodevices, energizing smart contact lenses.

Enhanced enzymatic hydrolysis of sugarcane bagasse hemicellulose using recombinant glucose oxidase expressed by Pichia pastoris

To demonstrate the role of glucose oxidase (GOD) on the enzyme-catalyzed production of xylose from SCB hemicellulose, a variant of the GOD gene, which contains two amino acid substitutions, was cloned from Aspergillus niger CICC40179. Three-dimension structure modeling showed that the two variable amino acid residues were positioned on the surface of the protein molecule. The GOD gene was expressed in Pichia pastoris. Approximately 16.31U/mL of GOD was obtained after 3 days of fermentation in the optimized medium. The catalytic characteristic of GOD was considered to apply in the production of xylose from hemicellulose. The pH value, temperature and the loading of GOD were optimized using the Box–Behnken design to improve the yield of xylose. The xylose yield was increased by 46% in 16h of hydrolysis by adding GOD when the reaction was conducted under the optimal conditions (pH 5.1, temperature 44.9°C and 31.68U GOD per gram substrate). Glucose was not detected in the hydrolysis system supplemented with GOD. This is the first study demonstrating the application of glucose oxidase (GOD) on the enzyme-catalyzed production of xylose from SCB hemicellulose. The newly developed enzymatic hydrolysis system can contribute to the industrial production of xylose.

Production and characterization of recombinant glucose oxidase from Aspergillus niger expressed in Pichia pastoris

Recombinant glucose oxidase from Aspergillus niger expressed in Pichia pastoris by fed-batch fermentation was purified and assessed with 1·26 purification fold to homogeneity using Q-Sepharose F.F. chromatography. The enzyme was determined by SDS-PAGE and gradient PAGE, which showed a dimeric form of 150 kDa. The purified rGOD was proved to be a glycoprotein, and the content of which was estimated to be 36·7 and 25·14% by phenol-sulfuric acid and anthrone-sulfuric acid methods. Characteristics demonstrated that the highest activity was in pH 6·0 at 40°C and was stable at a broad pH range from 4·0 to 9·0 at 55°C or below. The optimum substrate for this enzyme was d-glucose, and the Km was 21·06 mmol l(-1) as well as the Vmax was 359 μmol min(-1) mg(-1). rGOD possessed high resistance to various chemicals except for Hg(2+), Fe(2+), Ag(+), Cu(2+), 1,4-dithiothreitol, sodium dodecyl sulfate and ascorbic acid. In addition, the inhibitors also exhibited intensive fluorescence quenching effect on rGOD. Glucose oxidase is a very important enzyme produced by several species. However, large-scale applications have always been postponed by its complexity in fermentation and purification. Our research focused on developing new purification strategy of recombinant GOD from A. niger expressed in P. pastoris. Here, we described this novel one-step purification method and subsequent research in the characteristics of rGOD which showed different results from previous work. These can open new opportunities to increase its application.

Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H

Aspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZαA vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k cat of 189.4 s⁻¹ and K(m) of 28.26 mM while M12 GOx had k cat of 352.0 s⁻¹ and K m of 13.33 mM for glucose at pH 5.5. Specificity constants k(cat)/K(m) of wt (6.70 mM⁻¹ s⁻¹) and M12 GOx (26.7 mM⁻¹ s⁻¹) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells.

Yeast Surface Displaying Glucose Oxidase as Whole-Cell Biocatalyst: Construction, Characterization, and Its Electrochemical Glucose Sensing Application

The display of glucose oxidase (GOx) on yeast cell surface using a-agglutinin as an anchor motif was successfully developed. Both the immunochemical analysis and enzymatic assay showed that active GOx was efficiently expressed and translocated on the cell surface. Compared with conventional GOx, the yeast cell surface that displayed GOx (GOx-yeast) demonstrated excellent enzyme properties, such as good stability within a wide pH range (pH 3.5-11.5), good thermostability (retaining over 94.8% enzyme activity at 52 °C and 84.2% enzyme activity at 56 °C), and high d-glucose specificity. In addition, direct electrochemistry was achieved at a GOx-yeast/multiwalled-carbon-nanotube modified electrode, suggesting that the host cell of yeast did not have any adverse effect on the electrocatalytic property of the recombinant GOx. Thus, a novel electrochemical glucose biosensor based on this GOx-yeast was developed. The as-prepared biosensor was linear with the concentration of d-glucose within the range of 0.1-14 mM and a low detection limit of 0.05 mM (signal-to-noise ratio of S/N = 3). Moreover, the as-prepared biosensor is stable, specific, reproducible, simple, and cost-effective, which can be applicable for real sample detection. The proposed strategy to construct robust GOx-yeast may be applied to explore other oxidase-displaying-system-based whole-cell biocatalysts, which can find broad potential application in biosensors, bioenergy, and industrial catalysis.

High-level expression of the Penicillium notatum glucose oxidase gene in Pichia pastoris 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.

Recombinant glucose oxidase from Penicillium amagasakiense for efficient bioelectrochemical applications in physiological conditions

GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions. The present work describes the production of a recombinant glucose oxidase from Penicillium amagasakiense (yGOXpenag) displaying a more efficient glucose catalysis ( k cat/ K M(glucose) = 93 μM −1 s −1) than the native GOX from Aspergillus niger (nGOXaspng), which is the most industrially used ( k cat/ K M(glucose) = 27 μM −1 s −1). Expression in Pichia pastoris allowed easy production and purification of the recombinant active enzyme, without overglycosylation. Its biotechnological interest was further evaluated by measuring kinetics of ferrocinium–methanol (FM ox) reduction, which is commonly used for electron transfer to the electrode surface. Despite their homologies in sequence and structure, pH-dependant FM ox reduction was different between the two enzymes. At physiological pH and temperature, we observed that electron transfer to the redox mediator is also more efficient for yGOXpenag than for nGOXaspng( k cat/ K M(FM ox) = 27 μM −1 s −1 and 17 μM −1 s −1 respectively). In our model system, the catalytic current observed in the presence of blood glucose concentration (5 mM) was two times higher with yGOXpenag than with nGOXaspng. All our results indicated that yGOXpenag is a better candidate for industrial development of efficient bioelectrochemical devices used in physiological conditions.

Identification and functional characterization of the HpALG11 and the HpRFT1 genes involved in N-linked glycosylation in the methylotrophic yeast Hansenula polymorpha

The initial steps in N-linked glycosylation involve the synthesis of a lipid-linked core oligosaccharide followed by the transfer of the core glycan to nascent polypeptides in the endoplasmic reticulum (ER). In this study, we have identified two genes, HpALG11and HpRFT1, in the metylotrophic yeast Hansenula polymorpha. Detailed analysis of the glycan structures of the N-linked glycans of secreted recombinant glucose oxidase in mutant strains Hpalg3Δ, Hpalg11Δ, and Hpalg3Δalg11Δ with the assistance of over-expression of RFT1 was performed by linkage-specific mannosidase digestion. The results suggest that HpALG11 and HpRFT1 were responsible for catalyzing the sequential transfer of terminal α-1,2-Man residues to form the Man(5)GlcNAc(2)-PP-Dol intermediate at the cytosolic side of the ER before flipping to the luminal side and encoding an evolutionarily conserved protein required for the translocation of Man(5)GlcNAc(2)-PP-Dol from the cytoplasmic to the lumenal leaflet of the ER membrane, respectively. Deletion of the HpALG11 gene leads to poor growth and temperature-sensitive lethality, whereas over-expression of HpRft1p can improve growth of the Hpalg11Δ and Hpalg3Δalg11Δ strains. Furthermore, deletion of the HpALG11 gene in the Hpalg3Δ strain resulted in the secretion of glycoproteins with a predicted structure mainly containing trimannosyl core N-linked glycans (Man(3)GlcNAc(2)).

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).

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 2 L) was designed for the purposes of characterization of rGOx mutants during directed protein evolution. The Aspergillus niger GOx was cloned into a pYES2-αMF-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.

Secretory and continuous expression of Aspergillus niger glucose oxidase gene in Pichia pastoris

We proposed a yeast transformant cell incorporating the Aspergillus niger glucose oxidase gene (GOX gene), which is capable of constitutively as well as secretory expression. The GOX gene has been cloned in this study. This conclusion is based on the following: first, the ligated DNA determined by electrophoresis, was a 1489–1882 bp fragment, close to the size of glucose oxidase (GOD), which is 1818 bp. Secondly, the single open reading frame encoded a protein of 605 amino acids. Thirdly, secreted GOD recombinant proteins in the culture supernatants of the GOX gene transformant migrated as a single band in SDS–PAGE with an apparent molecular mass of between 75,000 and 100,000 Da, which is glycosylated GOD by the Pichia pastoris X-33 host machinery during the secretion process. Finally, the clones were cultured and secreted a protein, which possessed the GOD activity of catalyzing β- d-glucose oxidation. With regard to the pH characteristics, the activity was more than 80% of the maximum activity in the range between pH 5 and pH 7. As for the temperature characteristics, the activity was not less than 92% of the maximum in the temperature range between 10 and 45 °C. The GOX gene transformant was able to maintain the GOD enzyme activity and produce recombinant GOD continuously for at least 2 weeks.

Expression of Penicillium variabile P16 glucose oxidase gene in Pichia pastoris and characterization of the recombinant enzyme

Glucose oxidase (GOX) is a glycoprotein that finds wide application in food industry and clinical analysis. The gene encoding the GOX from Penicillium variabile P16 was expressed in Pichia pastoris X 33 using the methanol inducible AOX1 promoter. Among 11 transformants resistant toward high zeocin concentrations, six Mut + strains were screened in shaken flasks and the strain X33 c9, producing 0.33 U ml −1 of heterologous GOX after 11 days of fermentation, was selected. Recombinant GOX (ca. 50 U ml −1) was produced in a 3-l fermenter under not optimized conditions, recovered and purified in order to characterize and to compare it with the native one. The GOX from P. pastoris had a molecular weight of 82 kDa. Comparison of carbohydrate moieties showed a slight over-glycosylation of the GOX from Pichia over the native enzyme (17 and 14%, respectively). pH behavior of the recombinant enzyme, in terms of both activity and stability, was similar to that of the native one; on the other hand, a certain difference was observed in optimal temperature for activity and in thermal stability. P. pastoris appears to be a good expression system for GOX production.

PH control of the production of recombinant glucose oxidase in Aspergillus nidulans.

Recombinant Aspergillus nidulans sVAL040, capable of synthesizing and secreting glucose oxidase derived from Aspergillus niger was used to study the influence of pH and carbon source on enzyme production. Glucose oxidase gene (goxC) was expressed under transcriptional regulation by using the promoter of A. nidulans xlnB gene (encoding an acidic xylanase). A maximum specific glucose oxidase activity of approx. 10 U mg(-1) protein and a maximum volumetric productivity of 29.9 U l(-1) h(-1) were obtained at pH 5.5, after 80 h of growth by using xylose as inducer. Enzyme volumetric productivity increased when xylans were used instead of xylose; however, specific glucose oxidase activity did not differ significantly. Specific GOX activity obtained at pH 5.5 are two to three times more than those previously described for goxC multicopy transformants of A. nidulans. Xylans were a more powerful inducer than xylose although fungal growth was lower when the polymers were used. The obtained results by using xlnB promoter in A. nidulans could be useful in improving heterologous enzyme production by using genetic- and process-engineering strategies.