Distribution and typology of glucose oxidase activity in the genus Penicillium

Glucose oxidase (GOD) production by 84 strains of the genus Penicillium was studied in order to define the distribution of the activity among and within the species. Plate screening and sub-screening in shake culture showed a rather homogeneous behaviour of strains belonging to the same species. Penicillium chrysogenum, P. expansum, P. italicum and P. variabile had a concomitant good production of GOD and catalase activity; peroxidase and polyphenol oxidase were never present. The capability of the culture filtrates to oxidase substrates other than glucose was also studied.     

Glucose oxidase overproducing mutants of Penicillium variabile (P16)

Abstract Conidia of Penicillium variabile P16 were subjected to mutagenesis and selection for glucose oxidase production on media containing o -dianisidine. Studies of the relationship between dose of UV irradiation and conidial survival and frequency of mutation showed that the best frequency of positive mutation (17%) was obtained in correspondence to a conidial survival of 52%. Out of 54 overproducing mutants tested in shaken flasks, M-80.10 showed the highest level of glucose oxidase activity (127% higher than the wild-type). M-80.10 mutant, transferred every 15 days to fresh medium and tested monthly for 8 months, appeared stable. The time course of growth and enzyme production by the mutant M-80.10 showed an increase of the glucose oxidase activity in the culture medium up to 19 U ml⁻¹ after 96 h of fermentation.     

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.     

Optimization of culture conditions for glucose oxidase production by a Penicillium chrysogenum SRT 19 strain

The enzyme glucose oxidase (GOD) has been used for a variety of biotechnological applications in food and pharmaceutical industries. In this study, the optimization of extracellular GOD production was carried out in a Penicillium chrysogenum SRT 19 strain isolated from contaminated and decaying cheese samples. Maximum GOD production was attained at pH 6 and 20°C in fermentation broth after 72 h of incubation. The effects of metal ions and sugars were screened for the induction of higher GOD production. The results revealed that glucose and lactose give the highest production of enzyme (0.670 and 0.552 U/mL, respectively) as compared with other sugars (sucrose, cellulose, mannitol and fructose). Out of the seven metal ions studied, CaCO₃ (1.123 U/mL) and FeSO₄ (0.822 U/mL) act as modulators, while MgSO₄ (0.535 U/mL), CuSO₄ (0.498 U/mL), HgCl₂ (0.476 U/mL), ZnSO₄ (0.457 U/mL) and BaSO₄ (0.422 U/mL) yield lower production. The study therefore suggests that a strain of P. chrysogenum SRT 19 can be used as a new strain for GOD production.     

Kinetics of glucose oxidase excretion by recombinant Aspergillus niger

The kinetics of glucose oxidase (GOD) excretion by recombinant Aspergillus niger NRRL-3 (GOD3-18) were investigated using enzymatic activity measurements as well as gel electrophoresis techniques. The majority of GOD was produced during rapid growth in the first phase of the cultivation. The high excretion rate during this phase did not prevent the endocellular accumulation of GOD up to 40% of the total soluble cell protein demonstrating that the production rate exceeded the excretion rate of the enzyme into the culture medium. During the second phase of the cultivation, excretion of GOD occurred at a slower rate, although the majority of GOD produced during the first phase was excreted during the second phase of the cultivation. At the end, about 90% of the total GOD produced was recovered from the culture medium. Two-dimensional gel electrophoresis provided evidence that endo- and exocellular GOD were indistinguishable, revealing identical posttranslational modifications (e.g., signal sequence cleavage, glycosylation pattern). The results demonstrate that the initial steps of the secretory pathway are fast and that the excretion of the enzyme into the culture fluid was most likely delayed due to retention by the cell wall. (c) 1996 John Wiley & Sons, Inc.     

Effects of agitation and aeration on the production of extracellular glucose oxidase from a recombinant Saccharomycescerevisiae

A recombinant strain of Saccharomyces cerevisiae, GAL-GO2, was developed to facilitate the production of extracellular glucose oxidase (GOD). The recombinant strain secreted 85% (8.7?U/ml) of the total GOD (10.3?U/ml) produced in shake flask culture. For further enhancement of GOD production, optimization of the speed of agitation and the rate of aeration in a stirred tank fermentor was carried out. Response surface methodology with appropriate statistical experimental design was employed for this purpose. The maximal level of extracellular GOD was achieved when the speed of agitation and the rate of aeration were 420?rpm and 0.25?vvm, respectively. The enzyme production was increased by 74% compared to the level obtained under unoptimized conditions.     

Glucose oxidase catalysed oxidation of glucose in a dialysis membrane electrochemical reactor (D-MER)

The purpose of this work was to evaluate the effectiveness of a new Membrane Electrochemical Reactor (MER) for the production of gluconic acid by glucose oxidase (GOD) catalysed glucose oxidation. The GOD was confined against the electrode surface with a dialysis membrane. The role of the electrochemical step was to eliminate by oxidation the hydrogen peroxide that appeared as a by-product of the reaction and strongly inhibited and/or inactivated GOD. The dialysis MER gave a transformation ratio of 30% with an initial glucose concentration of around 300 mM. This result is significantly better than the maximum of 10% obtained when hydrogen peroxide was eliminated by addition of a large excess of catalase in solution, as is generally done. The D-MER also revealed unexpected properties of the enzyme kinetics, such as an oscillatory behaviour, which were discussed.     

Enhancement of glucose oxidase production in batch cultivation of recombinant Saccharomyces cerevisiae: optimization of oxygen transfer condition

AIMS: To obtain an optimal combination of agitation speed and aeration rate for maximization of specific glucose oxidase (GOD) production in recombinant Saccharomyces cerevisiae, and to establish a correlation between kLa vis-à-vis oxygen transfer condition and specific glucose oxidase production. METHODS AND RESULTS: The oxygen transfer condition was manifested indirectly by manipulating the impeller speed and aeration rate in accordance with a Central Composite Rotatory Design (CCRD). The dissolved oxygen concentration and the volumetric oxygen transfer coefficient (kLa) were determined at corresponding combinations of impeller speed and aeration rate. The maximal specific extracellular glucose oxidase production (3.17 U mg-1 dry cell mass) was achieved when the initial dissolved oxygen concentration was 6.83 mg l-1 at the impeller speed of 420 rev min-1 and at the rate of aeration of 0.25 vvm. It was found out that while impeller speed had a direct effect on the production of enzyme, a correlation between kLa and specific GOD production could not be established. CONCLUSION: At the agitation speed of 420 rev min-1 and at 0.25 vvm aeration rate, the degree of turbulence and the dissolved oxygen concentration were thought to be optimal both for cellular growth and production of enzyme. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined effect of agitation and aeration on recombinant glucose oxidase production in batch cultivation has not yet been reported in the literature. Therefore, this study gives an insight into the effect of these two important physical parameters on recombinant protein production. It also suggests that since there is no correlation between kLa and specific production of GOD, kLa should not be used as one of the scale-up parameters.     

Structure and biosensor characteristics of complex between glucose oxidase and plasma-polymerized nanothin film

The structure and biosensor characteristics of complex between glucose oxidase (GOD) and plasma-polymerized nanothin film (PPF), in which the thickness is several nanometers, were investigated by atomic force microscopy (AFM) and electrochemical measurement. The GOD molecules were densely adsorbed onto the PPF surface treated by nitrogen plasma and the individual GOD molecules were observed. Subsequently, the GOD densely packed array on the PPF surface was subsequently treated by plasma polymerization (overcoating). AFM image showed that the thicker film gave the smoother surface, indicating that the GOD adsorbed on the surface was embedded more deeply in PPF. The amperometric biosensor characteristics of the GOD-PPF complex on a platinum electrode showed the current increment due to the enzymatic reaction with glucose addition, indicating that enzyme activity was retained although the enzyme has been exposed to the plasma gas related to diffusion of the substrate. This means that under mild exposure to organic plasma, the enzyme does not become seriously dysfunctional. Amperometric biosensor characteristics were strongly affected by monomer and thickness of PPF overcoating related with the diffusion of the substrate (glucose). Considering that the film deposition was performed using microfabrication-compatible organic plasma, our new method for protein architecture has a great potential of enabling high throughput production of bioelectronic devices.     

Direct electrochemistry of glucose oxidase and biosensing for glucose based on boron-doped carbon nanotubes modified electrode

Due to their unique physicochemical properties, doped carbon nanotubes are now extremely attractive and important nanomaterials in bioanalytical applications. In this work, selecting glucose oxidase (GOD) as a model enzyme, we investigated the direct electrochemistry of GOD based on the B-doped carbon nanotubes/glassy carbon (BCNTs/GC) electrode with cyclic voltammetry. A pair of well-defined, quasi-reversible redox peaks of the immobilized GOD was observed at the BCNTs based enzyme electrode in 0.1M phosphate buffer solution (pH 6.98) by direct electron transfer between the protein and the electrode. As a new platform in glucose analysis, the new glucose biosensor based on the BCNTs/GC electrode has a sensitivity of 111.57 microA mM(-1)cm(-2), a linear range from 0.05 to 0.3mM and a detection limit of 0.01mM (S/N=3). Furthermore, the BCNTs modified electrode exhibits good stability and excellent anti-interferent ability to the commonly co-existed uric acid and ascorbic acid. These indicate that boron-doped carbon nanotubes are the good candidate material for the direct electrochemistry of the redox-active enzyme and the construction of the related enzyme biosensors.     

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。     

Coimmobilization of gluconolactonase with glucose oxidase for improvement in kinetic property of enzymatically induced volume collapse in ionic gels

The object of this paper is to provide an enzymatic means to attain faster swelling or shrinking kinetics of polyelectrolyte gels that undergo volume phase transition as an immobilized enzyme reaction sets in. For this, we studied the coimmobilization of gluconolactonase (GL) with glucose oxidase (GOD). A gel used was in the shape of a small cylinder (several hundred micrometers in diameter) and composed of a lightly cross-linked copolymer of N-isopropylacrylamide and acrylic acid. GL was isolated from Aspergillus niger and purified about 100-fold. It was found that in a substrate solution containing glucose, the gel with the coimmobilized GL and GOD shrinks very rapidly. The shrinking rate was identical to that of the enzyme-free gel that undergoes a shrinking transition in response to a sudden pH change of the outer medium from 7 to 5. This indicates the rate-limiting step in the shrinking process to be diffusion of the networks, but not the enzyme reaction. In the gel with singly immobilized GOD, a very slow shrinking was observed because the process is governed by the enzyme reaction. These results were discussed in full in connection with an enzymatically induced decrease in pH within and in the vicinity of the gel phase. As a result, it has become apparent that the faster shrinking kinetics in the coimmobilized enzyme system is attained by the GL-catalyzed hydrolysis of D-glucono-delta-lactone resulting from the oxidation of glucose with GOD.     

A glucose oxidase electrode based on polypyrrole with polyanion/PEG/enzyme conjugate dopant

This study investigated a new glucose sensor prepared by electrochemical polymerization of pyrrole with polyanion/poly(ethylene glycol) (PEG)/glucose oxidase (GOD) conjugate dopants. GOD was coupled to a strong polyanion, poly(2-acrylamido-2-methylpropane sulfonic acid) (AMPS) via PEG spacer to effectively and reproducibly immobilize GOD within a polypyrrole matrix onto a Pt electrode surface. PEGs with four different chain lengths (1000, 2000, 3000, and 4000) were used as spacers to study the spacer length effect on enzyme immobilization and electrode function. After conjugation, more than 90% of the GOD bioactivity was preserved and the bioactivity of the conjugated GOD increased with longer PEG spacers. The resulting polyanion/PEG/GOD conjugate was used as a dopant for electropolymerizing pyrrole. The activity of the immobilized enzyme on the electrode ranged from 119 to 209 mU cm(-2) and the bioactivity increased with the use of longer PEG spacers. The amperometric response of the enzyme electrode was linear up to 20 mM glucose concentration with a sensitivity ranging from 180 to 270 nA mM(-1) cm(-2). The kinetic parameters Michaelis-Menten constant (K(M)(app)) and maximum current density (j(max)) depended on the amount of active enzyme, level of substrate diffusion, and PEG spacer length. An increase in the electrical charge passed during polymerization (thus, increasing polypyrrole thickness) to 255 mC cm(-2) increased the sensitivity of the enzyme electrode because of the greater amount of incorporated enzyme. However, although the amount of incorporated GOD continued to increase when the charge increased above 255 mC cm(-2), the sensitivity began to decline gradually. The condition for preparing the enzyme electrode was optimized at 800 mV potential with a dopant concentration of 1 mg ml(-1).     

Co-immobilization of glucose oxidase and hexokinase on silicate hybrid sol-gel membrane for glucose and ATP detections

The co-immobilization of glucose oxidase (GOD) and hexokinase/glucose-6-phosphate dehydrogenase (HEX) in the silica hybrid sol-gel film for development of amperometric biosensors was investigated. The silica hybrid film fabricated by hydrolysis of the mixture of tetraethyl orthosilicate and 3-(trimethoxysiyl)propyl methacrylate possessed a three-dimension vesicle structure and good uniformity and conformability, and was ready for enzyme immobilization. The electrochemical and spectroscopic measurements showed that the silica hybrid sol-gel provided excellent matrice for the enzyme immobilization and that the immobilized enzyme retained its bioactivity effectively. The immobilized GOD could catalyze the oxidation of glucose, which could be used to determine glucose at +1.0 V without help of any mediator. The competition between GOD and HEX for the substrate glucose involving ATP as a co-substrate led to a decrease of the glucose response, which allowed us to develop an ATP sensor with a good stability. The fabricated silica hybrid sol-gel matrice offered a stage for further study of immobilization and electrochemistry of proteins.     

Genetic modification of glucose oxidase for improving performance of an amperometric glucose biosensor

Glucose oxidase (GOD) was genetically modified by adding a poly-lysine chain at the C-terminal with a peptide linker inserted between the enzyme and poly-lysine chain. The poly-lysine chain was added in order to anchor more electron transfer mediator, ferrocenecarboxylic acid, to GOD for the purpose of improving sensitivity and stability of glucose biosensors. The modified GOD had similar K(m) and K(cat) to those of the wild type enzyme. After interacted with the electron transfer mediator, the modified enzyme retained 90.01% of its native activity, while the commercial GOD and the wild type GOD (Aspergillus niger) retained only 22.43 and 22.17%, respectively. Screen-printed electrodes coated with the modified GOD, wild type yeast-derived GOD or the commercial GOD were tested in glucose solution of different concentrations. Experimental results showed that the biosensor based on the modified GOD gave the largest signal among the three. In addition, the linear range of the biosensor prepared by the modified GOD could extend to 45 mM, while they were about 20 mM for the biosensors based on the wild type yeast-derived enzyme and the commercial enzyme.     

蔗糖-葡萄糖双功能酶传感器的研究

结合蔗糖转化酶(INV)酶管与葡萄糖氧化酶(GOD)-葡萄糖变旋酶(MUT)双酶电极构成一种新的蔗糖传感器。该传感器可以分别用于蔗糖及葡萄糖的测定。蔗糖经酶管作用产生α-D-葡萄糖,再用COD-MUT双酶电极定糖。若是样品中蔗糖和葡萄糖共存,比较样品流经不同路径(Ws和Wg)时传感器的响应值,可以排除葡萄糖对蔗糖测定的干扰。传感器的最适pH和温度范围分别为:5.0—6.5和30—40℃。在稳态法实验中,传感器的线性范围为:2.5×10~(-4)—5×10~(-3)mol/L。传感器的重复性很好,CV<1％。该传感器在用于测定发酵培养基(含葡萄糖)的蔗糖含量,平均回收率为97.9％。传感器与糖度计法测定的相关系数为0.997。传感器至少可以稳定使用8天以上。     

Cloning and characterization of a single chain antibody to glucose oxidase from a murine hybridoma

Glucose oxidase (GOD) is an oxidoreductase catalyzing the reaction of glucose and oxygen to peroxide and gluconolacton (EC 1.1.3.4.). GOD is a widely used enzyme in biotechnology. Therefore the production of monoclonal antibodies and antibody fragments to GOD are of interest in bioanalytics and even tumor therapy. We describe here the generation of a panel of monoclonal antibodies to native and heat inactivated GOD. One of the hybridomas, E13BC8, was used for cloning of a single chain antibody (scFv). This scFv was expressed in Escherichia coli XL1-blue with the help of the vector system pOPE101. The scFv was isolated from the periplasmic fraction and detected by western blotting. It reacts specifically with soluble active GOD but does not recognize denatured GOD adsorbed to the solid phase. The same binding properties were also found for the monoclonal antibody E13BC8.     

双碳源流加对重组毕赤酵母高效表达葡萄糖氧化酶的影响

葡萄糖氧化酶(GOD)是一种具有广泛应用前景的工业酶.为了实现葡萄糖氧化酶的高效生产,提高重组毕赤酵母生产GOD的产量和增强生产强度,对重组毕赤酵母诱导阶段的初始菌体浓度和甲醇浓度进行了优化.在此基础上,诱导期采用了双碳源(甘油、山梨醇和甘露醇)与甲醇混合流加的模式.研究发现,最佳诱导前初始菌体浓度和甲醇浓度分别为100 g/L和18 g/L,此时GOD产量为427.6 U/mL.在诱导阶段采用甘油、山梨醇和甘露醇与甲醇的混合添加均可以提高GOD产量,其中甘露醇与甲醇的混合流加效果最为显著.当甲醇与甘露醇混合流加的比例为20∶1(W/W)时,诱导156h GOD产量和生产强度分别可达711.3 U/mL和4.60 U/(mL·h),比甲醇单一流加策略结果分别提高了66.3％和67.9％.此外采用合适的甘露醇混合流加策略不但不会抑制AOX1启动子的表达,甚至有一定促进作用,AOX酶活性为8.8 U/g(对照为5.2 U/g).双碳源流加方式还能推广到毕赤酵母其他表型中,为该系统高效表达外源蛋白提供一种新策略.     

Glucose oxidase as label in histological immunoassays with enzyme-amplification in a two-step technique: coimmobilized horseradish peroxidase as secondary system enzyme for chromogen oxidation

Summary A sensitive staining procedure for glucose oxidase (GOD) as marker in immunohistology is described. The cytochemical procedure involves a two-step enzyme method in which GOD and horseradish peroxidase (HRP) are coimmobilized onto the same cellular sites by immunological bridging or by the principle of avidin-biotin interaction. In this coupled enzyme technique, H₂O₂ generated during GOD reaction is the substrate for HRP and is utilized for the oxidation of chromogens such as 3,3-diaminobenzidine or 3-amino-9-ethylcarbazole. Due to the immobilization of the capture enzyme HRP in close proximity to the marker enzyme (GOD), more intense and specific staining is produced than can be obtained with soluble HRP as coupling enzyme in the substrate medium. Indirect antibody labelled and antibody bridge techniques including the avidin (streptavidin)-biotin principle have proven the usefulness of this GOD labelling procedure for antigen localization in paraffin sections. Antigens such as IgA in tonsil, alpha-feroprotein in liver and tissue polypeptide antigen in mainmary gland served as models. The immobilized twostep enzyme procedures have the same order of sensitivity and specificity as comparable immunoperoxidase methods. The coupled GOD-HRP principle can be superior to conventional immunoperoxidase labelling for the localization of biomolecules in tissue preparations rich in endogenous peroxidase activities.     

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.