Bioconversion Of Molasses To Glucose Oxidase Through Solid State Fermentation With Aspergillus Niger
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Publisher: 2014 Dissertation note: Enzymes can be defined as soluble colloidal organic catalysts which are produced by living cells and are capableof acting independently of the cells. Glucose oxidase belongs to oxidoreductaseand is also called as glucose dehydrogenase. The glucose oxidase enzyme (GOX) oxidizes glucose to gluconic acid. In cells, it aids in breaking the sugar down into its metabolites. Glucose oxidasehas found several commercial applications including glucose removal from dried egg; improvement of color, flavor, and shelf life of food materials; oxygen removal from fruit juices, canned beverages. It has also been used in an automatic glucose assay kit in conjunction with catalase and chiefly in biosensorsfor the detection and estimation of glucose in industrial solutions and in body fluids such as blood and urine. It is often extracted from Aspergillusniger. GOX is a dimeric protein. The active site where glucose binds is in a deep pocket. This enzyme acts outside of cells, is covered with carbohydrate chains (Raba and Horacio 1995).
Aspergillus niger is the potential source for the production of glucose oxidase and is preferreddue to its high production ration of extracellular enzyme. The ability of Aspergillus niger toutilize a wide range of waste products as nutrition source makes it more economical source of the enzyme (Rajesh et al .2002).
The glucose oxidase fromA. nigerisalso an intracellularenzyme present in the mycelium of the organism. Aspergillus nigeris a filamentous fungus belonging to phylum Ascomycota. It Produces microscopic conidia on conidiophores that are produced asexually. Hyphae possess septa and are hyaline. They are supported at their base by foot cells from which conidiophores originate. It possesses long, double-walled, smooth and colorless to brown conidiophores.It is commonly foundin mesophilic environments such as soil, plants and enclosed air environments. It is capable of surviving in various environments, it is not only a xerophilic fungus, but is also a thermo tolerant organism. It is because of this property that it exhibits a high tolerance to freezing temperature(Schuster 2002).
Glucose oxidase was first isolated from mycelia ofA. nigerandPenicilliumglaucumby Müller.
A large number of microbes including bacteria and filamentous fungi have been used for the production of glucose oxidase. Glucose oxidase is produced at large scale using A. nigerand P. amagasakiense. Many bacteria are also involved in the production of this enzyme; some of these are Zymomonasmobilis, Micrococcus and Enterobacte(Yogananth et al. 2012).
Glucose oxidase (GOX) from Aspergillus niger is a well-characterised glycoprotein consisting of two identical 80-kDa subunits with two FAD co-enzymes bound. Both the DNA sequence and protein structure at 1.9 A have been determined that these identical subunits size vary from 70 to 80 KDa. It catalyzes the oxidation of D-glucose (C6H12O6) to D -gluconolactone (C6H10O6) and hydrogen peroxide. It is produced naturally in some fungi and insects where its catalytic product, hydrogen peroxide, acts as an anti-bacterial and anti-fungal agent (Ikram et al . 2014).
Glucose oxidase has a molecular weight of 160,000 a.m.u. (Tsugeet al .1975) and consists of two identical polypeptide chain subunits having nearly equal molecular weights linked by disulphide bonds (O'Malley and Weaver 1972) and it is highly specific for β-D-glucose (Bentley 1963). Each subunit of the glucose oxidase contains one mole of Fe and one mole of FAD (Flavin adenine dinucleotides) and it contains 74% protein, 16% natural sugar and 2% amino sugars (Tsugeet al. 1975). The Glucose oxidase enzyme in its purest form is pale-yellow powder. The molecular weight of GOX ranges from approximately 130 kDa to 175 KDa (Kalisz et al. 1997).
Gluconic acid, the oxidation product of glucose, is a mild neithercaustic nor corrosive, non-toxic and readily biodegradable organic acid of great interest for many applications. As a multifunctional carbonic acid belonging to the bulk chemicals and due to its physiological and chemical characteristics, gluconic acid itself, its salts (e.g. alkali metal salts, in especially sodium gluconate) and the gluconolactone form have found extensively versatile uses in the chemical, pharmaceutical, food, construction and other industries (Anastassiadis and Morgunov 2007 ).
This enzyme is present in all aerobic organisms and normally functions in conjunction with catalase (Coxon and Schaffer 1971). This enzyme is also used as an antioxidant (Berg et al. 1992). It is mainly available from microbial sources and is normally produced by aerobic fermentation of Aspergillus nigerand Penicillium species (Fiedurak1996; Lu et al. 1996; Plush et al .1996; Rando et al. 1997). It has high specificity for D-glucose (Kuly and Cenas 1983).
This enzymeis also widely used to produce gluconicacidthatGOXtogether with Horse Reddish peroxidase has a range of applications in the food industry for glucose determination. GOX is being used in the textile industry producing hydrogen peroxide for bleaching process. This enzyme is also used to determine capillary glucose in screening of gestational diabetes (Mesiggi et al. 1988). This enzyme is utilized to extend the shelf life of fish(Field et al.1986) andproduction of calcium gluconate, gluconic acid and its derivatives (Khurshid2009).
Solid state fermentation [SSF] has been recently considered as the most cheapest and more environmentally friendly relative to submergedliquid fermentation [SLF] in the production of value added industrial based products such as enzymes, bio fuels.Advantages of Solid State Fermentation over Submerged Fermentation isHigher volumetric productivity, usually simpler with lower energy requirements, Might be easier to meet aeration requirements, Resembles the natural habitat of some fungi and bacteria and Easier downstream processing (Mienda et al. 2011).
Molasses is a dark brown, almost black, moist granular sugar. Its distinctive molasses taste is due to its high content of minerals. Nutritively, it has high iron content (Draycott and Philip 2008).
Aspergillus niger is a fungus, one of the most common species of the genus Aspergillus.The genus Aspergillus isimportant economically, ecologically and medically (Nizamuddinet al.2008).
Glucose oxidase enzymewas producedthrough the microbial fermentation. For that purpose solid state fermentation wasdevelopedwithA.niger. Solid state fermentation was applied to utilize agricultural residue such as Molasses as substrate.
The current research work was focused on production ofextracellular Glucose Oxidase (GOX) fromAspergillusniger using industrial waste such as molasses as substrate by Solid state ( static ) fermentation.
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