Purification Of Glucose Oxidase Research Articles

Evaluation of Antimicrobial Activity of Glucose Oxidase from Aspergillus niger EBL-A and Penicillium notatum

This work aimed to study the production and purification of glucose oxidase by Aspergillus niger and Penicillium notatum using corn steep liquor as the substrate and evaluate its antimicrobial activity for use in pharmaceutical and food industries. The enzyme was purified by ammonium sulfate precipitation (60-85%), DEAE-cellulose ion exchange and Sephadex G-200 size exclusion chromatography. The crude enzyme extracts of A. niger and P. notatum showed 2.32 and 5.53 U mg-1 specific activities, respectively, which after desalting was 15.52 and 12.05 U mg-1, and after ion exchange and gel filtration chromatography was 29.09 - 62 and 25.72 - 59.37 U mg-1 for A. niger and P. notatum, respectively. The antimicrobial activity was determined by disc diffusion method against selected microbial strains where glucose oxidase from A. niger showed anti-bacterial activity, while no fungicidal effects were shown by both A. niger and P. notatum glucose oxidases.

Purification of Glucose Oxidase from Aspergillus niger by Liquid-Liquid Cationic Reversed Micelles Extraction

The aim of this work was to select the operating conditions for the extraction and recovery of glucose oxidase (GOX) by reversed micelles from mixtures of commercial enzyme and Aspergillus niger homogenates. For this purpose, the influence of the main operating parameters (pH, surfactant concentration, and presence of cell debris or not) on GOX extraction was investigated at 25 degrees C. Without cell debris, the highest yield of GOX activity recovery (90.8%) was obtained performing (a) the forward extraction in isooctane as solvent and hexanol and butanol as cosolvents at 76/6/18 ratio, pH 7.0, 0.2 M cetyl trimethylammonium bromide as cationic surfactant, and electric conductivity of 5.0 mS cm(-1) and (b) the backward extraction at pH 5.5. Forward and backward extractions furnished comparable results when using raw homogenate, which demonstrates a negligible impact of the presence of cell debris on the process. The highest extraction yield (94%) was obtained under the same forward and backward conditions adopted without cell debris. The promising results of this work suggest that the proposed methodology could be profitably exploited at an industrial level.

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.

Purification of Glucose Oxidase and β‐Galactosidase by Partitioning in a PEG‐Salt Aqueous Two‐Phase System in the Presence of PEG‐Derivatives

Purification of glucose oxidase from Aspergillus niger and that of β‐galactosidase from Kluyveromyces lactis have been attempted using poly(ethylene glycol) (PEG)‐sodium sulfate aqueous two phase system (ATPS) in the presence of PEG‐derivatives, i.e. PEG‐Coomassie brilliant blue G‐250 and PEG‐benzoate, PEG‐palmitate and PEG‐TMA, respectively. The enzymes showed poor partitioning towards the PEG phase in comparison with other proteins in ATPS containing no ligands. Selective partitioning of other proteins was observed towards the PEG phase in the presence of PEG‐benzoate and PEG‐palmitate enriching β‐galactosidase in the salt phase whereas in the case of glucose oxidase, PEG‐Coomassie brilliant blue G‐250 derivative worked as a better affinity ligand for other proteins. A 19‐fold purification was obtained with the PEG dye derivative after 5 stage cross extractions with 80% recovery of glucose oxidase and an enrichment factor upto ∼7 for β‐galactosidase with the PEG‐TMA derivative. The interaction of PEG‐benzoate and PEG‐TMA ligands with the active site of β‐galactosidase has been evaluated by molecular modeling. The effect of the molecular weight of glucose oxidase on its partitioning was confirmed as the molecular simulation shows strong affinity interaction of PEG‐glucoside with the enzyme.

Purification of Glucose Oxidase and Catalase Produced by the Apple Blue Mold, Penicillium expansum O-385-10, and Their Characteristics Including the Browning of Apple Fruit

Penicillium expansumO-385-10 produces glucose oxidase and catalase but showed poorgrowth in medium containing glucose as a sole carbon source. In this culture, gluconate wasaccumulated, the pH of the culture medium decreased during incubation, and growth wasremarkably inhibited. Glucose oxidase and catalase were purified as electrophoreticallyhomogeneous proteins from the culture in which the pH was adjusted using calcium carbonate.The molecular weights of glucose oxidase and catalase were estimated at about 130 kDa (homodimer) and 300 kDa (homotetramer). Glucose oxidase and catalase showed about 80%and 50% activity at pH 3, respectively. The catalase was remarkably activated by 1mM Ca2+and 1mM Ba2+. When glucose oxidase and catalase were simultaneously incubated withsmall pieces of apple, the fruit tissue browned remarkably. No browning reaction occurredwhen each enzyme was incubated independently.

8] Spectrophotometric method for glucose-6-phosphate phosphatase

Publisher Summary This chapter describes the assay method and procedure used for the determination of glucose-6-phosphate phosphatase. Glucose-6-phosphatase is located in the endoplasmic reticulum and is commonly used as a marker enzyme for this organelle. The procedure described in the chapter is based on a coupled enzymic reaction that can be followed spectrophotometrically. The assay method is used for the determination of glucose and cholesterol. Glucose released by the enzyme is oxidized by glucose oxidase and peroxidase. Commercially available glucose oxidase is contaminated by a phosphatase that hydrolyzes glucose 6-phosphate at low pH values. The purification of glucose oxidase is done by isoelectric focusing. The focusing of glucose oxidase in the pH gradient and its elution from the focusing column is followed easily owing to the yellow color of the enzyme. The coupled enzyme reaction is slightly inhibited by inorganic phosphate. Thus, the specific activity decreases less than 10% in 5 m M and approximately 30% in 20 m M sodium phosphate buffer, pH 6.5. The glucose is removed by gel filtration or dialysis.