Expression and Characterization of Glucose Oxidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

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 370 U/L after 7 days 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 80 kDa 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.     

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

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

A Penicillium expansum glucose oxidase-encoding gene, GOX2, is essential for gluconic acid production and acidification during colonization of deciduous fruit

Penicillium expansum, the causal agent of blue mold rot, causes severe postharvest maceration of fruit through secretion of total, d-gluconic acid (GLA). Two P. expansum glucose oxidase (GOX)-encoding genes, GOX1 and GOX2, were analyzed. GOX activity and GLA accumulation were strongly related to GOX2 expression, which increased with pH to a maximum at pH 7.0, whereas GOX1 was expressed at pH 4.0, where no GOX activity or extracellular GLA were detected. This differential expression was also observed at the leading edge of the decaying tissue, where GOX2 expression was dominant. The roles of the GOX genes in pathogenicity were further studied through i) development of P. expansum goxRNAi mutants exhibiting differential downregulation of GOX2, ii) heterologous expression of the P. expansum GOX2 gene in the nondeciduous fruit-pathogen P. chrysogenum, and iii) modulation of GLA production by FeSO(4) chelation. Interestingly, in P. expansum, pH and GLA production elicited opposite effects on germination and biomass accumulation: 26% of spores germinated at pH 7.0 when GOX activity and GLA were highest whereas, in P. chrysogenum at the same pH, when GLA did not accumulate, 72% of spores germinated. Moreover, heterologous expression of P. expansum GOX2 in P. chrysogenum resulted in enhanced GLA production and reduced germination, suggesting negative regulation of spore germination and GLA production. These results demonstrate that pH modulation, mediated by GLA accumulation, is an important factor in generating the initial signal or signals for fungal development leading to host-tissue colonization by P. expansum.     

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

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

Secretory expression and purification of Aspergillus niger glucose oxidase in Saccharomyces cerevisiae mutant deficient in PMR1 gene

The gene encoding glucose oxidase (GOD) from Aspergillus niger was expressed as a secretory product in the yeast Saccharomyces cerevisiae. Six consecutive histidine residues were fused to the C-terminus of GOD to facilitate purification. The recombinant GOD-His(6) secreted by S. cerevisiae migrated as a broad diffuse band on SDS-PAGE, with an apparent molecular weight higher than that in natural A. niger GOD. To investigate the effects of hyperglycosylation on the secretion efficiency and enzyme properties, GOD-His(6) was expressed and secreted in a S. cerevisiae mutant in which the PMR1 gene encoding Ca(++)-ATPase was disrupted. The pmr1 null mutant strain secreted an amount of GOD-His(6) per unit cell mass higher than that in the wild-type strain. In contrast to the hyperglycosylated GOD-His(6) secreted in the wild-type strain, the pmr1 mutant strain secreted GOD-His(6) in a homogeneous form with a protein band pattern similar to that in natural A. niger GOD, based on SDS-PAGE. The hyperglycosylated and pmr1Delta mutant-derived GOD-His(6) enzymes were purified to homogeneity by immobilized metal ion-affinity chromatography and their specific activities and stabilities were compared. The specific activity of the pmr1Delta mutant-derived GOD-His(6) on a protein basis was very similar to that of the hyperglycosylated GOD-His(6), although its pH and thermal stabilities were lower than those of the hyperglycosylated GOD-His(6).     

黑曲霉葡萄糖氧化酶基因的克隆及其在酵母中的高效表达

将黑曲霉葡萄糖氧化酶(GOD)基因重组进大肠杆菌酵母穿梭质粒Ppic9,转化甲基营养酵母Pichia pastoris GS115,构建出GOD的高产酵母工程菌株。在酵母αFactor及AOX1基因启动子和终止信号的调控下,黑曲霉GOD在甲基酵母中大量表达并分泌至胞外,经甲醇诱导3～4d,发酵液中的GOD活力可达30～40u/mL。SDS-PAGE证实GOD在培养物上清中的含量显著高于其它杂蛋白,约占胞外蛋白总量的60%～70%,经Q SepharoseTMFast Flow离子交换柱一步纯化即达电泳纯。重组酵母GOD比活达426.63u/mg蛋白,是商品黑曲霉GOD的1.6倍。动力学性质分析表明,重组酵母GOD的K_m和K_cat分别为38.25mmol/L和3492.66s-1,与商品黑曲霉GOD相比,具有更高的催化效率。重组酵母GOD的高活力特性可有效提高葡萄糖传感器的线性检测范围。     

Growth inhibition by glucose oxidase system of enterotoxic Escherichia coli and Salmonella derby: in vitro studies

The antibacterial effect of different glucose oxidase (GOX)/glucose combinations was studied on two food-poisoning organisms, enterotoxic Escherichia coli PM 015 and Salmonella derby BP 177. Growth of E. coli in nutrient broth (NB) was clearly inhibited by 4.0 mg/ml glucose after 24 h when combined with 2.0 U/ml GOX and after 48 h when combined with 0.5 or 1.0 U/ml GOX. Salmonella derby was more resistant than E. coli, but showed clear growth inhibition only after 48 h when inoculated in tubes where 2 mg glucose/ml and 2 U GOX/ml (or 4 mg glucose/ml and 1 U GOX/ml) were combined. In order to understand if the enzyme effect on microbial growth can be attributed to hydrogen peroxide or to pH decrease as a result of the production of gluconic acid, catalase (CAT) was added to GOX/glucose system. Since CAT decomposes H₂O₂ to H₂O and O₂, the antibacterial effect was ascribed to a pH decrease as a result of gluconic acid in the system.     

A glucose biosensor based on microporous polyacrylonitrile synthesized by single rare-earth catalyst

A glucose biosensor was constructed by electrochemical adsorption of glucose oxidase (GOD) in microporous polyacrylonitrile (PAN), which was synthesized by single rare-earth catalyst-Y(OAr)(3.) The biosensor shows a fast response, good operational stability and reproducibility. The influences of molecular weight of polymer, pH, operating potential and temperature on the biosensor response were explored by amperometric method. The apparent activation energy and optimum pH of enzyme-catalyzed reaction are 35.9 kJ mol(-1) and 6.2, respectively.     

The glucose oxidase of Penicillium variabile P16: gene cloning, sequencing and expression

AIMS: Isolation and characterization of the glucose oxidase (GOX)-encoding gene from a Penicillium variabile strain (P16) having a high level of GOX activity and comparison of its expression with that of another strain of P. variabile (NRRL 1048) characterized by low GOX activity. METHODS AND RESULTS: The gene, isolated by PCR consisted of 1818 bp encoding 605 amino acid residues. Gene expression was analysed by Northern blotting and compared with that of P. variabile NRRL 1048. The higher GOX activity of strain P16 appeared likely because of de novo mRNA synthesis. Southern blotting analyses of the genomic DNA showed that the hybridization pattern of the two strains differed for the size of hybridizing fragment detected by the probe and slightly for their signal intensity. CONCLUSIONS: The GOX-encoding gene of P. variabile P16 was isolated and characterized to identify the molecular bases of its high level of expression and in view of improving enzyme production by developing a process based on heterologous expression. SIGNIFICANCE AND IMPACT OF THE STUDY: GOX-encoding genes can be subjected to high difference in their expression levels. The P16 strain of P. variable producing large amount of GOX as well as its encoding gene might be exploited for industrial applications.     

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

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

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.     

一株海洋低温葡萄糖氧化酶菌株的筛选、鉴定及部分酶学性质

【目的】从海洋样品中分离筛选出产葡萄糖氧化酶菌株。【方法】采用双层平板筛选法进行初筛、复筛确定一株酶活较好的菌株,命名为GOD2(Glucose oxidase)。通过形态学、生理生化特征及16S rRNA基因序列分析研究其分类地位,并对其产生的葡萄糖氧化酶进行分离纯化和部分酶学性质的研究。【结果】细菌GOD2为产葡萄糖氧化酶菌株且遗传稳定,初步鉴定该菌株为假单胞杆菌(Pseudomonas migulae),其所产酶最适反应温度为20°C,热稳定性较差,40°C剩余相对酶活80%;超过40°C酶活力迅速下降。【结论】GOD2是一株极具研究价值的产低温葡萄糖氧化酶菌株。目前没有关于利用该菌生产葡萄糖氧化酶的报道。     

A bienzyme channeling glucose sensor with a wide concentration range based on co-entrapment of enzymes in SBA-15 mesopores

A novel bienzyme-channeling sensor was constructed by entrapping glucose oxidase (GOD) and horseradish peroxidase (HRP) in the mesopores of well-ordered hexagonal mesoporous silica structures (SBA-15). The SBA-15 mesoporous materials accelerated the electron transfer between the entrapped HRP and electrode. Both HRP and GOD retained their catalytic activities in the bienzyme-entrapped SBA-15 film. In presence of glucose the enzymatic reaction of GOD-glucose-dissolved oxygen system generated hydrogen peroxide in the bienzyme-entrapped mesopores, which was immediately reduced at -0.40 V by an electrocatalytic reaction with the HRP entrapped in the same mesopore to lead to a sensitive and fast amperometric response. Thus the bienzyme channeling could be used for the detection of glucose with excellent performance without the addition of any mediator. Optimization of the experimental parameters was performed with regard to pH, operating potential and temperature. The detection limit was down to 2.7 x 10(-7)M with a very wide linear range from 3.0 x 10(-6) to 3.4 x 10(-2)M. The constructed bienzyme channeling provided a strategy for amperometric detection of oxidase substrates by co-entrapping the corresponding oxidase and HRP in the mesoporous materials.     

Inner epidermis of onion bulb scale: As natural support for immobilization of glucose oxidase and its application in dissolved oxygen based biosensor

Inner epidermal membrane of the onion bulb scales was studied as a natural polymer support for immobilization of the glucose oxidase (GOD) enzyme for biosensor application. Onion epidermal membrane was used for immobilization of glucose oxidase and was associated with dissolved oxygen (DO) probe for biosensor reading. Glucose was detected on the basis of depletion of oxygen, when immobilized GOD oxidizes glucose into gluconolactone. A wide detection range between 22.5 and 450 mg/dl was estimated from calibration plot. A single membrane was reused for 127 reactions with retention of approximately 90% of its initial enzyme activity. Membrane was stable for 45 days ( approximately 90% activity) when stored in buffer at 4 degrees C. Surface structure studies of the immobilized membranes were carried under SEM. To our knowledge, this is the first report on employing inner epidermal membrane of onion bulb scales as the solid support for immobilization of enzyme.     

Improvement of the stability of glucose oxidase via encapsulation in sodium alginate–cellulose sulfate–poly(methylene-co-guanidine) capsules

Encapsulation of glucose oxidase (GOD) in polyelectrolyte complex capsules and its influence on properties of the enzyme is reported. The immobilization of GOD in the capsules made of sodium alginate (SA), cellulose sulfate (CS), poly(methylene-co-guanidine) (PMCG), CaCl₂ and NaCl (GOD–SA–CS/PMCG capsules) was achieved using a one-step highly reproducible encapsulation protocol which was monitored by a Electrospray Ionization-Mass Spectrometry (ESI-MS). A leakage of the enzyme from the capsules was negligible. Encapsulated GOD exhibited higher thermostability, wider range of pH optimum and improved storage stability in comparison with free GOD. The 92% retained activity by the encapsulated GOD after 45 biooxidation cycles was markedly higher than that of the GOD entrapped in calcium pectate gel beads showing no activity after 12 cycles. Optimization of conditions of oxygen supplementation resulted in increased oxygen availability within the GOD–SA–CS/PMCG capsules. Oxygen supplementation was accompanied with a mild decrease in the mechanical resistance of the SA–CS/PMCG capsules.     

Liquid-liquid extraction of commercial glucose oxidase by reversed micelles

Aim of this work was to establish the optimum operating conditions for the extraction and recovery by cationic reversed micelles of commercial glucose oxidase (GOX) from Aspergillus niger, in view of possible application to raw cell homogenates. The influence of pH, temperature, electric conductivity and solvent/co-solvents ratio on the extraction was investigated by a fractional factorial design of 2(3-1) type, conjugated with a mixture experimental design, using the residual enzyme activity to evaluate the results. The best conditions for GOX extraction were ensured using isooctane as solvent and hexanol and butanol as co-solvents at 76/6/18 volume ratio, pH 6.0, 0.2 M cetyl trimethylammonium bromide (CTAB) as cationic surfactant, and electrical conductivity (kappa) of 4.8 mS cm-1. The highest yield of GOX activity recovery (about 90%) was in fair accordance with the value predicted by the model.     

Cloning and analysis of CUT1, a cutinase gene from Magnaporthe grisea

Summary A gene from Magnaporthe grisea was cloned using a cDNA clone of the Colletotrichum gloeosporioides cutinase gene as a heterologous probe; the nucleotide sequence of a 2 kb DNA segment containing the gene has been determined. DNA hybridization analysis shows that the M. grisea genome contains only one copy of this gene. The predicted polypeptide contains 228 amino acids and is homologous to the three previously characterized cutinases, showing 74% amino acid similarity to the cutinase of C. gloeosporioides. Comparison with previously determined cutinase sequences suggests that the gene contains two introns, 115 and 147 bp in length. The gene is expressed when cutin is the sole carbon source but not when the carbon source is cutin and glucose together or glucose alone. Levels of intracellular and extracellular cutinase activity increase in response to growth in the presence of cutin. The activity level is higher in a transformant containing multiple copies of the cloned gene than in the parent strain. Non-denaturing polyacrylamide gels stained for esterase activity show a single major band among intracellular and extracellular proteins from cutin-grown cultures that is not present among intracellular and extracellular proteins prepared from glucose-grown or carbon-starved cultures. This band stains more intensely in extracts from the multicopy transformant than in extracts from the parent strain. We conclude that the cloned DNA contains a M. grisea gene for cutinase, which we have named CUT1.     

Diet factors responsible for the change of the glucose oxidase activity in labial salivary glands of Helicoverpa armigera

We investigated the change of the glucose oxidase (GOX) activity in labial salivary glands of Helicoverpa armigera larvae fed with the artificial diet or host plant tobacco and the major factors responsible for such a change. Throughout larval development, the labial salivary GOX activities in caterpillars reared on the artificial diet were remarkably higher than those fed with the plant. After fifth-instar plant-fed caterpillars were transferred to the artificial diet, their labial salivary GOX activity increased quickly, which was closely correlated with the time spent feeding on the artificial diet. The total sugar content of the artificial diet was 68 times higher than that of the tobacco leaves. We hypothesized that sugars and secondary metabolites are the possible causes of induction of GOX activity. When fifth-instar caterpillars were fed with tobacco leaves coated with glucose or sucrose, their labial salivary GOX activity was significantly higher than those fed with leaves without sugar coating. Following native PAGE, 1 single band of the labial salivary GOX was observed in all the caterpillars fed with different diets, implying that only the activity of the isoenzyme was changed in response to different diets. Furthermore, the labial salivary GOX activity was determined after caterpillars were fed with artificial diets containing chlorogenic acid, rutin, and quercetin. The results showed that all these phenolic compounds had no effect on the GOX activity. We conclude that sugar in diets was a major factor influencing the labial salivary GOX activity of the larvae. Arch. Insect Biochem. Physiol. 2008.