Glucose Oxidase Gene Research Articles

Optimization of PCR Conditions for Adding XhoI Restriction Sites to the Glucose Oxidase Gene of Aspergillus niger IPBCC 08.610

Optimization of PCR Conditions for Adding XhoI Restriction Sites to the Glucose Oxidase Gene of Aspergillus niger IPBCC 08.610

An efficient CRISPR/Cas9 genome editing system based on a multiple sgRNA processing platform in Trichoderma reesei for strain improvement and enzyme production.

The CRISPR/Cas9 technology is being employed as a convenient tool for genetic engineering of the industrially important filamentous fungus Trichoderma reesei. However, multiplex gene editing is still constrained by the sgRNA processing capability, hindering strain improvement of T. reesei for the production of lignocellulose-degrading enzymes and recombinant proteins. Here, a CRISPR/Cas9 system based on a multiple sgRNA processing platform was established for genome editing in T. reesei. The platform contains the arrayed tRNA-sgRNA architecture directed by a 5S rRNA promoter to generate multiple sgRNAs from a single transcript by the endogenous tRNA processing system. With this system, two sgRNAs targeting cre1 (encoding the carbon catabolite repressor 1) were designed and the precise deletion of cre1 was obtained, demonstrating the efficiency of sgRNAs processing in the tRNA-sgRNA architecture. Moreover, overexpression of xyr1-A824V (encoding a key activator for cellulase/xylanase expression) at the ace1 (encoding a repressor for cellulase/xylanase expression) locus was achieved by designing two sgRNAs targeting ace1 in the system, resulting in the significantly enhanced production of cellulase (up to 1- and 18-fold on the Avicel and glucose, respectively) and xylanase (up to 11- and 41-fold on the Avicel and glucose, respectively). Furthermore, heterologous expression of the glucose oxidase gene from Aspergillus niger ATCC 9029 at the cbh1 locus with the simultaneous deletion of cbh1 and cbh2 (two cellobiohydrolase coding genes) by designing four sgRNAs targeting cbh1 and cbh2 in the system was acquired, and the glucose oxidase produced by T. reesei reached 43.77U/mL. Besides, it was found the ER-associated protein degradation (ERAD) level was decreased in the glucose oxidase-producing strain, which was likely due to the reduction of secretion pressure by deletion of the major endogenous cellulase-encoding genes. The tRNA-gRNA array-based CRISPR-Cas9 editing system was successfully developed in T. reesei. This system would accelerate engineering of T. reesei for high-level production of enzymes including lignocellulose-degrading enzymes and other recombinant enzymes. Furthermore, it would expand the CRISPR toolbox for fungal genome editing and synthetic biology.

Combining manipulation of integration loci and secretory pathway on expression of an Aspergillus niger glucose oxidase gene in Trichoderma reesei

Trichoderma reesei (T. reesei) is well-known for its excellent ability to secret a large quantity of cellulase. However, unlike the endogenous proteins, little is known about the molecular mechanisms governing heterologous protein production. Herein, we focused on the integration loci and the secretory pathway, and investigated their combinatorial effects on heterologous gene expression in T. reesei using a glucose oxidase from Aspergillus niger as a model protein. Integration in the cel3c locus was more efficient than the cbh1 locus in expressing the AnGOx by increasing the transcription of AnGOx in the early stage. In addition, we discovered that interruption of the cel3c locus has an additional effect by increasing the expression of the secretory pathway component genes. Accordingly, overexpressing three secretory pathway component genes, that were snc1, sso2, and rho3, increased AnGOx expression in the cbh1 transformant but not in the cel3c transformant.

Molecular Cloning, Expression, and Enzyme Activity of Glucose Oxidase Gene from Soil Thermophilic Streptomyces.

Background: Glucose oxidase is an oxidoreductase that depletes oxygen in food processing and is used in biosensors, glucose diagnostic kits, food processing, cosmetics, and chemical industries. This enzyme is often isolated from fungi, such as Penicillium and Aspergillus Niger.Objectives: The objective of this study was to clone and express a full-length GOX gene from soil thermophilic streptomyces for bioinformatic and enzyme activity evaluations.Materials and Methods: After collecting samples from the Gandom Beryan area of Kerman province, Iran, Streptomyces strains were identified with specific biochemical and molecular tests. Streptomyces strains with glucose oxidase gene were detected by PCR, and the amplified gene fragment was cloned into Escherichia coli host bacterium using TA cloning technique. The expression of the cloned GOX gene in the host bacterium was measured using real-time PCR, and the recombinant plasmids were sequenced. The enzymatic activity was measured in the extracts of E. coli cells carrying the plasmids.Results: After screening the samples, 12 strains of Streptomyces were identified, 4 of which carried the GOX gene. The GOX open reading frame, obtained by PCR, was cloned into a vector and transformed into Escherichia coli origami to generate GOX-producing bacteria. Enzyme activity was confirmed and a phylogenetic tree showed the degree of kinship between Streptomyces species and other species, including Streptomyces SP MI02-7b. The expression levels of GOX genes mRNA were found to be approximately 4-fold higher in transformed E. coli than in soil thermophilic Streptomyces (P <0.001).Conclusion: This study showed that natural thermostable streptomyces producing glucose oxidase enzyme could be found in Iran. The enzyme gene was successfully transformed into Escherichia coli generating a recombinant host with high yield capability that can be a major step towards the production of this enzyme from indigenous strains. It should be emphasized that the GOX enzyme produced by these strains is profitable due to high production levels correlated to the optimum condition in cheap culture media, short fermentation cycles, high expression capability, and ease of growth.

Expression of Glucose Oxidase Gene from Aspergillus niger in Pichia pastoris and Optimization of Enzyme Production Conditions

Expression of Glucose Oxidase Gene from Aspergillus niger in Pichia pastoris and Optimization of Enzyme Production Conditions

High-level production of glucose oxidase in Pichia pastoris: Effects of Hac1p overexpression on cell physiology and enzyme expression

Secretion is a common bottleneck in the production of industrial proteins. Although overexpression of the unfolded protein response regulator Hac1p has been widely used to enhance protein secretion, its effects on the physiology of host cells were often overlooked, which would attenuate and even neutralize its beneficial effects on overall protein production.In order to achieve high-level glucose oxidase (GOX) production in Pichia pastoris, we used a set of Hac1p homologues from Saccharomyces cerevisiae (ScHac1p), P. pastoris (PpHac1p), Trichoderma reesei (TrHac1p) and Homo sapiens (HsXbp1), to evaluate the effects of Hac1p overexpression on the secretion capacity, cell physiology and overall enzyme production in P. pastoris strains containing different copies of the GOX gene. Results showed that overexpression of Hac1ps was able to remarkably alleviate the secretion bottleneck in the 3-copy strain, to improve its GOX secretion capacity by 21.2–140.2 % and its overall enzyme production by 23.7–69.2 %. However, overexpression of ScHac1p, PpHac1p and TrHac1p led to reduced cell growth in GS-3GOX, possibly due to increased oxidative stress. Overexpression of ScHac1p and PpHac1p in the 6-copy strain (resulting in GS-6GOX-Sc and GS-6GOX-Pp, respectively) further increased the overall GOX production levels by 88.9–103.3 %, and GS-6GOX-Pp exhibited the highest overall GOX production and GOX secretion capacity among all constructed strains. Nevertheless, in addition of significantly reduced growth, loss of GOX gene was also observed for GS-6GOX-Pp and GS-6GOX-Sc during the fermentation process. With higher induction cell density and co-feeding of yeast extract, the GOX titer of GS-6GOX-Pp reached 2125.3 U/mL on 1-liter fermentor.This study not only achieves a record high GOX production level but also provides new insights into secretion pathway engineering using Hac1p overexpression strategy.

Characteristics of Glucose Oxidase Gene (GGOx) from Aspergillus niger IPBCC 08.610

Glucose oxidase is used in various industries for the development of enzymatic fuel cell. Based on prior studies, this compound is sourced from the local isolates of Aspergillus niger IPBCC 08.610, although investigations on the encoding gene have not been conducted. The purpose of this research, therefore, is to identify and characterized the gene responsible for encoding glucose oxidase, in the aspect of sequence, length, and restriction patterns. This experiment involved the amplification of genomic DNA using specific primers for gene recognition, which was followed by the restriction technique with EcoRI and PstI endonucleases. Furthermore, the gene is inserted into vector pGEM®T-Easy and transformed into competent E. coli DH5α cells, in an attempt to perform sequencing. The glucose oxidase gene from A. niger IPBCC 08.610 was confirmed to possess a size of 1848 bp, and a GC content of 57.8%, with a possibility of restriction into two fragments of size 908 bp and 980 bp, using the EcoRI restriction.

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.

Oxidative Stress, Endoparasite Prevalence and Social Immunity in Bee Colonies Kept Traditionally vs. Those Kept for Commercial Purposes.

Commercially and traditionally managed bees were compared for oxidative stress (superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and malondialdehyde (MDA)), the prevalence of parasites (Lotmaria passim, Crithidia mellificae and Nosema ceranae/apis) and social immunity (glucose oxidase gene expression). The research was conducted on Pester plateau (Serbia—the Balkan Peninsula), on seemingly healthy colonies. Significant differences in CAT, GST and SOD activities (p < 0.01), and MDA concentrations (p < 0.002) were detected between commercial and traditional colonies. In the former, the prevalence of both L. passim and N. ceranae was significantly (p < 0.05 and p < 0.01, respectively) higher. For the first time, L. passim was detected in honey bee brood. In commercial colonies, the prevalence of L. passim was significantly (p < 0.01) lower in brood than in adult bees, whilst in traditionally kept colonies the prevalence in adult bees and brood did not differ significantly. In commercially kept colonies, the GOX gene expression level was significantly (p < 0.01) higher, which probably results from their increased need to strengthen their social immunity. Commercially kept colonies were under higher oxidative stress, had higher parasite burdens and higher GOX gene transcript levels. It may be assumed that anthropogenic influence contributed to these differences, but further investigations are necessary to confirm that.

Characterization, stability improvement, and bread baking applications of a novel cold-adapted glucose oxidase from Cladosporium neopsychrotolerans SL16

Glucose oxidases are widely used in various industrial processes, including bread baking. In this study, a novel glucose oxidase gene, CngoxA, from Cladosporium neopsychrotolerans SL16, was cloned and expressed in Pichia pastoris. Recombinant CnGOXA exhibited maximal activity at 20 °C and pH 7.0, and was stable at 30 °C and pH 6.0–9.0 for 1 h, with a half-life of 15 min at 40 °C. Compared with CnGOXA, the half-life of its mutant CnGOXA-M1 (Y169C-A211C), at 40 °C increased approximately 48-fold, and was stable at 30 °C and pH 3.0–12.0 for 1 h. The kcat and catalytic efficiency of CnGOXA-M1 were enhanced 0.7- and 1.6-fold, respectively. Both enzymes were cold-adapted and highly resistant to SDS. Furthermore, CnGOXA-M1 had a more significant effect on bread volume than that of GOX from Aspergillus niger. These favorable enzymatic properties of CnGOXA-M1 make it a potentially useful enzyme for many industrial applications.

Genome sequencing of Aureobasidium pullulans P25 and overexpression of a glucose oxidase gene for hyper-production of Ca2+-gluconic acid.

Gluconic acid (GA) has many applications such as in the food and pharmaceutical industry. Aureobasidium pullulans P25 strain is able to produce high levels of Ca2+-GA. The genome length, GC content and the gene number of this yeast were found to be 30.97Mb, 50.28% and 10,922, respectively. The pathways for gluconic acid biosynthesis were annotated. Glucose oxidase (Gox) sequences from different strains of A. pullulans were highly similar but were distinct from those of other fungi. The glucose oxidase had two FAD binding sites and a signal sequence. Deletion of the GOX gene resulted in a strain that showed no Gox activity and that was unable to produce Ca2+-GA. Overexpression of the GOX gene in strain P25 generated strain GA-6 that produced 200.2 ± 2.3 Ca2+-GAg/l and 2480U/mg of Gox activity. The productivity of Ca2+-GA was 2.78g/l/h and the yield was 1.1g/g.

Cloning, deletion, and overexpression of a glucose oxidase gene in Aureobasidium sp. P6 for Ca2+-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.

Expression, characterization and one step purification of heterologous glucose oxidase gene from Aspergillus niger ATCC 9029 in Pichia pastoris

Glucose Oxidase (GOD), is a common flavoprotein from Aspergillus niger ATCC 9029 with a broad application in biotechnology, food and medical industries. In this study, GOD gene was cloned into the expression vector, pPIC9 and screened by the alcohol oxidase promoter. The enzyme production increased at 28 °C. GOD activity induced by 1.0% methanol and the highest level of GOD production was the result of shaking rate at 225 rpm. The highest enzyme activity obtained at a pH value ranged from 5 to 7 at 50 °C. The enzyme was stable at a broad pH range and temperature.

Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase

Glucose oxidase (GOx) mutants with higher activity or stability have important role in industry and in the development of biosensors and biofuel cells. Discovering these mutants can be time-consuming if appropriate high-throughput screening (HTS) systems are not available. GOx gene libraries were successfully screened and sorted using a HTS system based on GOx activity dependent fluorescent labeling of yeast cells with tyramids and quantification of the amount of expressed enzyme by yeast enhanced green fluorescent protein (yGFP) tagging and flow cytometry. For this purpose, we expressed wild type and a mutant GOx as a chimera with the yGFP to confirm differences in catalytic activity between wild-type and mutant GOx. Fluorescence of yGFP is preserved during expression of chimera, and also after the oxidative enzymatic reaction. We have obtained a 2.5-fold enrichment in population of cells expressing active enzyme, and percentage of enzyme variants with enzymatic mean activity higher than wild type activity was increased to 44% after a single round of GOx gene library sorting. We have found two mutants with 1.3 and 2.3-fold increase in Vmax values compared to the wtGOx. By simultaneous detection of protein expression level and enzyme activity we have increased the likelihood of finding GOx variants with increased activity in a single round of flow cytometry sorting.

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.

Extremely low frequency magnetic field enhances glucose oxidase expression in Pichia pastoris GS115.

In this study attempts were made to investigate the effect of extremely low frequency magnetic field (ELF-MF) on expression of glucose oxidase gene from Aspergillus niger Z-25 in Pichia pastoris GS115 at optimal environmental conditions. GOX encoding gene from A. niger was expressed in P. pastoris GS115 employing methanol inducible AOX1 promoter. From resistant transformants to high zeocin concentrations, maximum GOX expressing transformant was isolated and used throughout the study. Optimal environmental conditions for achieving maximum glucose oxidase (GOX) production was determined by varying temperature (20-30°C) and final methanol concentration from 0.25-1% (v/v). Maximum GOX production was obtained at 20°C and 0.25% (v/v) final methanol concentration. Exposure carried out at extremely low frequencies ranging from 1 to 5Hz and the field strength of 30-100mT unceasingly. Biomass, GOX, protease productions and cell viability were examined in exposed and non-exposed groups every 12h up to 96h. Among different exposure conditions, the frequency of 2.8Hz significantly increased GOX production and cell viability, decreased non-specific protease activity and improved biomass production regardless of changes in field intensity. Overall, supported by results, P. pastoris exposure to ELF-MF in certain circumstances could effectively further increase maximum production of GOX as achieved by optimized preliminary environmental parameters.

Cloning &amp; expression of glucose oxidase fromAspergillusniger

Our aims is to clone the Glucose oxidase (GOx) gene which has been isolated from Aspergillus niger, in turn isolated from the fertile garden soil. Detection of the expression of gox gene in prokaryotic host. Aspergillus niger has been isolated from soil samples taken from Navsari region. Enzyme assay has been done to check enzyme. DNA extraction & PCR amplification was done using Banglore GeNei TM kits. 90% success has been achieved by obtaining the GOx protein from Escherichia coli BL21 in inclusion bodies. The protein has been purified onto Ni-NTA column. gox gene was successfully isolated and expressed in prokaryotic host, which would be purified by simple procedure. –We conclude that the by-product of gluconic acid fermentation has a great industrial importance. So, for greater production of GOx, the gene can be transferred from donor to the recipient. The gox gene of Aspergillus niger has been isolated and studied for the identification & its expression for improvement of enzyme production by establishing a process based on heterologous expression.

Amelioration of insulin resistance by Rk1 + Rg5 complex under endoplasmic reticulum stress conditions.

Background:Diabetes mellitus is a metabolic syndrome exaggerated by stress conditions. Endoplasmic reticulum stress (ERS) impairs the insulin signaling pathway making the diabetic conditions worsen. Pharmacological agents are supplied externally to overcome this malfunction. Ginsenosides from Panax ginseng C.A Meyer possesses many pharmacological properties and are used for the treatment of diabetes.Objective:To investigate the effects of the Rk1 +Rg5 complex on the amelioration of insulin resistance in 3T3-L1 cells under endoplasmic reticulum stress conditions.Materials and Methods:Heat-processed ginseng extracts are found to contain many pharmacologically active ginsenosides. Among them Rk1 +Rg5 is found to be present in higher concentrations than the other minor ginsenosides. The Rk1 +Rg5 complex was tested for its effect in the 3T3-L1 insulin-resistant model and subjected to the MTT assay, glucose oxidase assay and gene expression studies using RT-PCR and real-time PCR under endoplasmic reticulum stress conditions.Results:Rk1 +Rg5 treatment is found to increase the glucose uptake into the cells when compared to that of a positive control (tunicamycin treatment group, TM). Further we have analyzed the role at gene expression level. The Rk1 +Rg5 complex was found to show an effect on the IGF 2R receptor, CHOP-10, and C/EBP gene at a particular treated concentration (50 μM). Moreover, stress condition (about 50% decreases) was overcome by the ginsenoside treatments at 50 μM.Conclusion:The present results showed that under endoplasmic reticulum stress conditions Rk1 +Rg5 complex exhibits a potential protective role in insulin-resistant 3T3-L1 cells.

Disease Resistance Conferred by Constitutive Expression of a Fungal Glucose Oxidase Gene in Transgenic Tobacco Plants

Disease Resistance Conferred by Constitutive Expression of a Fungal Glucose Oxidase Gene in Transgenic Tobacco Plants

Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions

A Vanadium bromoPeroxidase-coupled fluorescent assay (ViPer) for ultrahigh-throughput screening of glucose oxidase (GOx) gene libraries employing double emulsions and flow cytometry was developed. The assay is based on detection of the product of a GOx reaction, hydrogen peroxide, that is first converted to a hypobromide by vanadium bromoperoxidase in the presence of sodium bromide. The hypobromide is afterwards detected in a reaction with a fluorogenic probe, 3-carboxy-7-(4'-aminophenoxy)-coumarine, where fluorescent 3-carboxy-coumarine is released. The ViPer screening system is three times more sensitive than a horseradish peroxidase coupled detection system and more resistant to bleaching of fluorescence in excess of peroxide. Using the ViPer screening system a high epPCR gene library containing 100,000 different GOx variants was screened for active clones in less than 1 h by flow cytometry. A library containing 0.15% of yeast cells expressing active enzyme variants and with an average GOx activity in the liquid culture of 0.47 U/mL, after one round of sorting, had 28.12% of the yeast cells expressing the active GOx (an enrichment factor of 200) and 26.8 U/mL of the GOx activity in the liquid culture (an enrichment factor of 57). The developed screening system could be adapted and used in a directed evolution of GOx and other hydrogen peroxide-producing enzymes (oxidases) and glycosidases if coupled with a carbohydrate oxidase.