Glucose Oxidase (GOD) is an important industrial enzyme in the food industry. System name of glucose oxidase is β-D-glucose oxidoreductase (EC1.1.3.4), which is widely used in wine, beer, fruit juice, and milk powder deoxidation, as well as flour improvement, browning prevention, food rapid detection and biosensors. Glucose oxidase is widely distributed in animals, plants and microorganisms. Microbial growth is the main source of glucose oxidase production because of the fast reproduction and wide source of microorganisms. The main production strains of glucose oxidase production are Aspergillus niger and Penicillium.
Structure
Glucose oxidase is a homodimeric molecule containing two binding sites of flavin adenine dinucleotide (FAD). Each monomer contains two distinct regions: one non-covalently but tightly bound to a portion of the FAD, predominantly β-sheet; the other is bound to the substrate β-D-glucose and supported by four α-helices with an antiparallel β-sheet.
Properties
High purity glucose oxidase is light yellow crystals, which is soluble in water and insoluble in organic solvents such as ether, chloroform, butanol, pyridine, glycerol, and ethylene glycol. The relative molecular mass of glucose oxidase is about 1.5 × 105. The pH range is 3.5 to 6.5, the optimum pH is about 5.0. The temperature range is generally 30 to 60℃, the optimum temperature is 50 to 55℃. Glucose oxidase binds to β-D-glucopyranose with a high degree of specificity. The hydroxyl group on glucose C1 is necessary for the enzyme to catalyze, and the activity of glucose oxidase is about 160 times higher when hydroxyl group at the β-site than at the α-site. Glucose oxidase activity is greatly reduced when changes occur in the molecular structure of the substrate C1, C2, C3, C4, C5, C6 sites. Glucose oxidase is completely inactive against L-glucose and 2-O-methyl-D-glucose.
Catalytic mechanism
Glucose oxidase can consume molecular oxygen or atomic oxygen to oxidize glucose, protecting easily oxidizable components in foods. According to the reaction conditions, there are two forms of glucose oxidase catalytic reaction.
(1) In the absence of catalase: Glucose oxidase catalyzes the oxidation of β-D-Glucose to D-glucono-δ-lactone with the concurrent release of hydrogen peroxide.
(2) In the presence of catalase: First, glucose oxidase catalyzes the oxidation of β-D-Glucose to D-glucono-δ-lactone and hydrogen peroxide. Catalase then catalyzes hydrogen peroxide to produce water and oxygen. Finally, water combines with D-glucono-δ-lactone to produce gluconic acid.
(3) In the presence of catalase and ethanol: First, glucose oxidase catalyzes the oxidation of β-D-Glucose to D-glucono-δ-lactone and hydrogen peroxide. Catalase then catalyze hydrogen peroxide and ethanol to produce water and acetaldehyde. Finally, water combines with D-glucono-δ-lactone to produce gluconic acid.
Production
At present, industrialized production of glucose oxidase is produced by microbial fermentation. The microorganisms that can produce glucose oxidase are mainly bacteria and mold. UV mutagenesis and chemical mutagenesis of production strains are commonly used in the glucose oxidase industry. A single mutagenesis method or combination of several methods can be used in to screen positive mutant strains to improve the enzyme producing capacity. Aspergillus niger or Penicillium fermentation is commonly used in industrial production to obtain glucose oxidase. However, during the fermentation of Aspergillus niger and Penicillium, other hybrid proteins such as catalase are usually produced, which makes it difficult to separate and purify glucose oxidase. Therefore, construction of a better strain using genetic engineering has long been the focus of scientists in various countries. A large amount of glucose oxidase protein can be obtained by inducing glucose oxidase gene expression. Because the small amount of endogenous protein secreted by the genetically engineered, there are mainly the exogenous protein secreted in the matrix which can simplify the later protein purification operation.
Applications
In the food industry, glucose oxidase is mainly used in the following five areas: deoxidation, improved flour, glucose, glucose content, and sterilization. Besides, glucose oxidase is a new type of enzyme feed additive, which can improve animal intestinal environment, regulate diet digestion and promote animal growth. A mixed feed additive containing glucose oxidase, lactic acid peroxide and lactoferrin can be used to prevent gastrointestinal infections and diarrhea of livestock. Since glucose oxidase can catalyze the production of gluconic acid and hydrogen peroxide in the intestinal glucose, the growth and reproduction of Escherichia coli, Salmonella, Pasteurella, Staphylococcus and Vibrio can be directly inhibited when hydrogen peroxide accumulates to a certain concentration.
Enzyme preparations, such as glucose oxidase with lactoperoxidase (LPO), amyloglucosidase, glucanase, lysozyme, etc., can remove or alleviate dental plaque, tartar and dental caries formation. The stability of glucose oxidase-containing drugs increased 3 times than others. Preparations containing glucose oxidase, lactoperoxidase and iodine compounds can be used for oral hygiene to stop bad breath. Double-enzyme chewing gum with glucose oxidase and lactoperoxidase has an antibacterial efficiency of 96% to 99% when chewed. Glucose oxidase can be used for the targeted treatment of hydrogen peroxide-sensitive lymphomas. Enzyme electrode can be used for in vitro glucose quantitative analysis in serum (pulp), urine and cerebrospinal fluid.
