Glucose oxidase from Aspergillus niger. Cloning, gene sequence, secretion from Saccharomyces cerevisiae and kinetic analysis of a yeast-derived enzyme.

Frederick, K.; Tung, J. S.; Emerick, R S; Masiarz, Frank R.; Chamberlain, Scott; Vasavada, Amit; Rosenberg, Steven; Chakraborty, Sumita; Schopfer, Lawrence M.; Schopter, L M

doi:10.1016/s0021-9258(19)39664-4

Cited by 212 publications

(57 citation statements)

References 64 publications

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“…The binding interaction between the negatively charged GOx and AuNP surfaces can be related to many collective week forces such as van Deer Waals forces, hydrogen bonds, hydrophobic interactions, but covalent thiol bonds at one of the cysteine rich side of GOx has also shown to contribute by strong binding affinity to gold. [29][30][31][32] For creating GOx-AuNP conjugates with varying enzyme-to-AuNP molar ratios, different size aliquots from a 2 mg/ml GOx stock solution in 10 mM NaHCO3 buffer, pH 8.2 was added to a fix volume of 1 nM AuNP solution in 2 ml Maxymum Recovery® centrifuge tubes (Maxyclear Boil Proof, VWR, USA). The final volume of the conjugate samples was adjusted with NaHCO3 buffer solution and was incubated in the dark, at room temperature for 1.5 hours.…”

Section: Number Of Gox Per Aunp= a E A Aunpmentioning

confidence: 99%

“…4,7,9 According to our flocculation experimental results suggesting a minimum ratio of 50:1 of GOx:AuNP is necessary in solution during conjugation to eliminate aggregates forming, while also considering the amplitude of the AuNP plasmonic peak suggest that a 100:1 or higher ratio is needed to completely coat and producing a monolayer of GOx on the surface of the AuNPs in the sample solution. GOx has been shown to contain a cysteine rich patch containing thiol groups that may create a strong binding affinity for the Au surface, [29][30][31][32] which may direct the binding of GOx to AuNP, but other binding strategies might influence enzyme immobilization. Previous work on examining the interactions between the enzymes horse radish peroxidase and glutamate oxidase, respectively, with the surface of AuNP have found that the enzyme:AuNP ratio necessary to prevent flocculation can vary greatly from theoretical predictions.…”

Section: Figure 3 (A) Schematic Of a Flocculation Assaymentioning

confidence: 99%

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Molecular Crowding and a Minimal Footprint at a Gold Nanoparticle Support Stabilize Glucose Oxidase and Boost its Activity

Wang¹,

Jonkute²,

Lindmark³

et al. 2019

Preprint

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Enzymes conjugated to nanomaterials are used in the design of various biotechnologies. In development of biosensors, surface modifications with the enzyme glucose oxidase (GOx) serve to aid the detection of blood glucose. In order to optimize sensor effectiveness, the enzyme tertiary structure needs to be preserved upon immobilization to retain the enzyme´s catalytic activity. Due to the nature of GOx, it suffers from tendency to denature when immobilized at a solid surface, methods to optimize enzyme stability are of great importance. Here, we introduce the study of the interaction of GOx to the highly curved surface of 20 nm gold nanoparticles (AuNP) that shows how placing a monolayer of enzyme where the enzyme spreads thin at the AuNP surface still provides stable catalytic performance up to14 days compared to enzymes free in solution. Moreover, by increasing enzyme density and creating a molecularly crowded environment at the highly curved nanoparticle surface, which limits the size of the enzyme footprint for attachment, the activity per enzyme can be enhanced up to 300%. This is of great importance for implementing stable and sensitive sensor technologies that are constructed by enzyme-based nanoparticle scaffolds. Here, we show by using the conditions that maintain GOx structure and function when limiting the enzyme coating to an ultra-thin layer, the design and construction of ultrafast responding diagnostic sensor technology for glucose can be achieved, which is crucial for monitoring rapid fluctuations of for instance, glucose in the brain.

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Section: Number Of Gox Per Aunp= a E A Aunpmentioning

confidence: 99%

Section: Figure 3 (A) Schematic Of a Flocculation Assaymentioning

confidence: 99%

Molecular Crowding and a Minimal Footprint at a Gold Nanoparticle Support Stabilize Glucose Oxidase and Boost its Activity

Wang¹,

Jonkute²,

Lindmark³

et al. 2019

Preprint

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“…The standard curve was made using fresh A. niger GOD (Sigma, St. Louis, MO, USA) as described in. [15,16] The GOD activity was detected based on the standard curve. Assays were performed in a volume of 3.2 mL in 2 mL glycerol, 1 mL glucose solution (2 mg/mL, which was formulated with 0.1 mol/L sodium citrate buffer, pH 5.6), 100 mL o-dianisidine solution (2 mg/mL, formulated with sterilized water) and 100 mL of horseradish peroxidase solution (100 U/mL, which formulated with 0.1 mol/L sodium citrate buffer, pH 5.6).…”

Section: Detection Of God Enzymatic Activitymentioning

confidence: 99%

Expression ofAspergillus nigerglucose oxidase in yeastPichia pastorisSMD1168

Qiu

Guo

Bao

et al. 2016

Biotechnology & Biotechnological Equipment

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The 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 pGAPZaA plasmid carrying a pGAP promoter and was expressed in yeast Pichia pastoris. The expression vector, pGAPZaA-GOD, was validated by colony polymerase chain reaction (PCR), agarose gel electrophoresis, restriction enzyme analysis and sequencing methods. The pGAPZaA-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, pGAPZaA-GOD, was successfully constructed to express A. niger accc30161 GOD protein. After transformation, the pGAPZaA-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.

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“…The primary structure has been described from the c-DNA sequence corresponding to 583 aminoacids, 3 cysteine residues and 8 potential sites for N-linked glycosilation 21 . A high negative charge at neutral pH can be predicted from this information.…”

Section: Glucose Oxidasementioning

confidence: 99%