Production Of Polyhydroxybutyrate From Azotobacter Vinelandii Using Molasses And Whey As Substrates
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Publisher: 2014 Dissertation note: Polyhydroxybutyrate (PHB) is biodegradable polyester produced in nature by microbial fermentation and it is used as thermoplastic. Azotobacter vinelandii is a bacterium that accumulates PHB as intracellular granules in response to physiological stress such as excess of carbohydrate sources and limitation of nutrients e.g. nitrogen, oxygen and phosphorus etc. PHB produced in this work have great potential be used in various industries like pharmaceutics and food industry for packaging purposes and medical field. Recent research work was conducted to produce PHB form cheap agro industrial wastes like Molasses and Whey by fermentation. Different parameters such as substrate water ratio, incubation period, volume of inoculums and pH were optimized for maximum yield of PHB.
In this study fermentation media containing whey and molasses as substrates was used to check the production of PHB from the Azotobacter vinelandii. 0.5ml of inoculum media was taken in fermentation media and then kept for incubation for 24-72 hours. After incubation, culture media was centrifuged and then sediment was used for extraction, determination and identification of PHB.
It was found that Azotobacter vinelandii in molasses contained medium gives maximum yield of PHB (mg/100mL) at 4% substrate water ratio after 48 hours of incubation period (140 mg/100mL), at 2.5 mL of volume of inoculum (204 mg/100mL), at pH 8.0 (220 mg/100mL), at 0.2% of peptone (252 mg/100mL) and 0.25% (234 mg/100mL) of yeast extract. While 4% of substrate water ratio after 60 hours of incubation (128 mg/100mL), 2.0 mL of volume of inoculum (176 mg/100mL), pH 7.0 (192 mg/100mL), 0.25% of peptone (248 mg/100mL) and 0.25% of yeast extract (240 mg/100mL) were observed to be optimum parameters for maximum production of PHB from Azotobacter vinelandii in whey based medium. Data was analyzed by means of linear regression analysis to determine R (regression coefficient), which was used to find significant differences (P?0.05) in each experiment.
Conclusion: The results of present study show that molasses and whey are economically good substrates for production of polyhydroxybutyrate (biodegradable polymer) from Azotobacter vinelandii. The results also suggest that Azotobacter vinelandii is a good potential strain for production of PHB under optimized conditions.
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Production Purification And Characterization Of Alkaline Proteasefrom Aspergillus Flavus Using Agricultural
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Publisher: 2014 Dissertation note: Abstract
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Effect Of Orally Adminisrered B-Gulcan From Different Sourves On Lipid Profile Of Hypercholestrolemic Rata
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Publisher: 2014 Dissertation note: Abstract
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Optimization For The Production Of Amylase By Geobacillus Sbs-4S
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Publisher: 2014 Dissertation note: Abstract
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Effect of Ginger and Turmeric Against Cadmium Induced Hepato-Renal Toxicity in Albino Rats
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Publisher: 2014 Dissertation note: Metal compounds and metal is natural elements of all ecosystems, moving between biosphere, hydrosphere, atmosphere and lithosphere. Metal complexes are increasingly introduced in the environment and could finally accumulate in a/biotic systems (Florea et al. 2005).
Contact to heavy metals is potentially damaging particularly for those metal compounds, which do not contain any physiological function in the metabolism of cells. A heavy metal is a part of an ill-defined subset of constituents that show metallic properties, which would mostly include the some metalliods, actinides, lanthanides and transition metals. Heavy metals have a high density and atomic weight much greater at least 5 times than water. Anthropogenic basis of heavy metals, i.e. contamination, have been introduced to the ecosystem waste-derived fuels are particularly prone to have heavy metals. More than 20 heavy metals, but inorganic arsenic, lead and cadmium are of particular concern (Gornal 1949).
Although, carcinogenic and toxic effects of metals have been observed in animals and humans, and that these metals form a key part in the normal functioning of biological cells. Some necessary transition metals like manganese, iron, zinc and copper contribute in controlling a variety of signaling and metabolic pathways. On the other hand their redox properties and coordination chemistry gave them an additional advantage that these metals might escape from the control mechanism such that homeostasis, partitioning, transport and binding to the designated cell elements and they interrelate with protein sites other than those which are tailor- made for them by displacing other metals from their natural binding sites. While, this process does not take place regularly, but the toxicity of metals can lead to impairment and dysfunctioning of cells (Leonard et al. 2004).
Oxidative stress is one of the main mechanisms of heavy metal toxicity. These metals are able to interact with DNA causing oxidative worsening of biological macromolecules and nuclear protein (Chen et al. 2001).
Metals like mercury, iron, cadmium, lead, copper and nickel, have the capability to produce reactive radicals, leading to cell damage like damage to lipid bilayer, depletion of enzyme activities and DNA (Stohs 1995). Moreover, these reactive radical species comprise a broad diversity of sulfur-, oxygen, nitrogen- and carbon radicals, initiating not only from lipid peroxides, hydrogen peroxide and superoxide radical but also in chelates of proteins complex peptide and amino acid, with the toxic metals. Metals produce reactive species, which in turn can cause nephrotoxicity, hepatotoxicity and neurotoxicity in humans and animals (Chen and Sthos 1995).
Cadmium is a natural metal located in the Periodic Table of the elements between mercury and zinc and the chemical behavior of cadmium is like a Zn. There is usually a divalent cation, complexd through other constituents (e.g CdCl2). Cadmium in the soil crust around 0.1ppm (Hans 1995) frequently being found as a contaminant in Pb or Zn deposits. In Zn or Pb smelting cadmium produced as a by product. Commercially, Cd is used in batteries, galvanizing steel, lasers, ink color, television screens, cosmetics and was used as an obstacle in nuclear fission and zinc to weld seals in water pipes made of lead before 1960. In the United States, approximately 600 metric tons are produced annually and about 150 tons are imported (US 2012).
Contact of Cd in human occurs mainly through ingestion or inhalation. Absorption through the skin contact is negligible. Intestinal absorption of cadmium is greater in individuals with zinc, calcium or iron deficiency (Nordberg et al. 2007).
The main source of cadmium exposure in human is considered to be the cigarette smoking (Friberg et al. 1983). Cd levels in blood and kidney are consistently elevated in smokers than nonsmokers. Inhalation exposure due to industry can be major occupational settings for example, soldering or welding and can cause a severe chemical pneumonitis (Nordberg et al 2007).
Exposure to cadmium from getting unhygienic food (eg, shellfish, leafy vegetables, rice regions of Japan and China and organ meats,) or water (either the old tap closed Zn / CD or a long-term industrial pollution) and can produce long-term effects on health (Abernethy et al. 2010).
After absorption, Cd is transported all over the body, often linked to a sulfhydryl group of protein such as metallothionein and about 30% deposits in the kidneys and 30% in the livers, and the rest scattered throughout the body (Argonne et al 2001). Half life of cadmium in the blood was estimated 75 to 128 days. (Jarup et al 1983). As a result urine, blood and hair Cd levels are poor substitutes for body burden and primarily reflect current contact; it is also true with the other heavy metals. Urine provocation test will require the estimation of cadmium in the body (Bernhoft et al. 2012).
The toxicity of cadmium has been shown in parts of body, cadmium induces tissue damage by creation of oxidative stress (Matovic et al. 2011; Patra et al. 2011; Cuypers et al. 2010) epigenetic changes in DNA expression (Wang et al. 2012; Martinez et al. 2011; Luparello 2012) mainly in the proximal segment of the renal tubule S1 (Vesay et al. 2010) inhibition or up regulation of transport routes (Therenod et al. 2012; Wan et al. 2012; Vankerkhove 2012).
Other pathologic mechanisms comprise competition disruption of the physiologic effects of Mg or Zn (Abdulla et al. 1989; Moulis et al. 2010; Shukla et al. 1984), destruction of mitochondrial function and inhibition of heme synthesis (Schauder et al. 2010), and potentially inducing apoptosis (Cannino et al. 2009). Glutathione reduction is observed, as structural deformation of proteins due sulfhydryl groups bind to the cadmium (Valko et al. 2005). Moreover, these effects are amplified by contact with other toxic metals such as As and Pb (Whittaker et al. 2011) and may be ameliorated by Se or Zn and by factors increasing levels of Nrf2 (Wang et al. 2012; Kcwill 2012).
Medicinal plants are plants having inherent active components used to treat disease or relieve pain (Okigboet et al. 2008). In most developing countries traditional medicines and medicinal plants are used as healing agents for the maintenance of good physical condition (UNESCO 1996) and in developing countries 80% of the peoples relies on traditional medicines, usually herbal remedies, for their prime health care needs (Schmincke et al. 2003). Plants extracts and their products are used in medicines as herbal remedies and they are being used to cure diverse infections (Arekemase et al. 2011). Moreover, there has been an increased concern in the beneficial potential of medicinal plants or plant products containing antioxidant properties in plummeting free radical induced tissue injury (Gupta & Flora 2005). Plants make a vital contribution to health care. The medicinal properties of plants could be based on the antimicrobial, antipyretic, antioxidant, effects of the phytochemicals in them (Cowman 1999; Adesokan et al. 2008).
Natural antioxidants also in the form of crude extracts or their chemical ingredient are very efficient in retarding the devastating processes create by oxidative stress (Zengin et al. 2011) and the toxicity analysis of the majority of the medicinal plants are not yet fully appreciated it is usually accepted that drugs which are derivative of plant products are safer than their imitative counterparts (Oluyemi 2007).
Ginger (Zingiber officinale), is a part of the Zingiberaceae family, is a eminent spice used in your daily diet (Demin et al. 2010) and also utilized for the traditional treatment of several infirmities (Afzal et al 2001). Major components of ginger like shogaol, gingerol, diarylheptanoids and volatile oil, work as antioxidant, anti-diabetic, analgesic, antipyretic, anti-inflammatory, anti-lipid and anti-tumor (Penna et al. 2003; Kadnur et al. 2005; Islamr et al. 2008; Shim et al. 2011; Kim 2008; Wangw et al. 2009). Latest scientific research has exposed that ginger has many therapeutic such as anti-oxidant effects, a capability to restrain the formation of inflammatory complexs and direct anti-inflammatory effects (Thomson et al. 2002). Ginger extract have antioxidative features, since it can scavenge hydroxyl radicals and superoxide anion. Z. officinale was found to slow down the activity of peroxidation and lipoxygenase (Topic et al. 2002).
Another, frequently used spice of Zingiberaceae: ‘curcuma longa’ (turmeric) has shown its strong intrinsic activity as a healing agent for several ailments. The active ingerdient of turmeric is the Curcumin that (Curcuma langalinn) shows antioxidant property. It is a yellow coloured phenolic pigment yield from the turmeric rhizomes (family Zingiberaceae).The most significant characteristic of curcumin is that it has no side consequences, regardless of the therapeutic agent in a number of useful purposes. It acts as a scavenger of free radicals (Khanna et al. 1999). Curcumin is considered to be an efficient antioxidant against oxidative tissue damage. It can considerably restrain the generation of reactive oxygen species (Joe et al. 1994) Moreover, curcumin is considered to be a powerful inhibitor tumour cells proliferation (Joe et al. 2004) a powerful cancer chemopreventive agent (Duvoix et al. 2005; Aggarwal et al. 2005) an dexhibits anti carcinogenic, anti-infective and anti viral properties (Araujo et al. 2001).
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Biochemical Evaluation Of Armoracia Rusticana And Raphanus Sativus On Alloxan Induced Diabetic Rats
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Publisher: 2014 Dissertation note: Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia in which the body does not produce or properly utilize insulin. It the reason of interruption in protein, carbohydrate and lipid metabolism and caused the complications such as nephropathy, microangiopathy and retinopathy. It is the most widespread endocrine disorder, affects more than 176 million people worldwide (WHO 2004).
Diabetes mellitus is generally classified into three types; type I, type II diabetics and gestational diabetes (Velho and Foguel, 2002). Type I diabetes mellitus is commonly occur among young people, it is also known as juvenile-onset diabetes or insulin dependent diabetes mellitus. Type I is the result of absolute deficiency of insulin that is commonly caused by the chronic autoimmune disease that results from complex interaction of both genetic and environmental factors (Pietropolo 2001). Type II diabetes mellitus is mostly occur in adults aged 40 years or above, it is commonly known as non-insulin diabetes mellitus characterized by too much hepatic glucose production, reduced insulin secretion from beta cells of pancreas, and peripheral tissues such as muscle adipose and liver become resistant to insulin (Ahmad 2006).
Association of hyperglycemia with long term damage, dysfunction as well as ultimate organs failure, mainly the heart, blood vessels, eyes, kidney and nerves has previously been recognized (Hung et al. 2005).
Dyslipidemia is another main reason of mortality and morbidity that results in development of cardiovascular complications (Reasner 2008). It is a main risk factor of diabetes and mostly result from prolonged hyperglycemia and insulin resistance in both (type I and type II) diabetic patients is called ‘diabetic dyslipidemia’ (Mooradian 2009). Hyperlipidemia and an increase in blood cholesterol and triglyceride are results from decrease in lipolysis which is caused by deficiency of insulin, eventually increases the risk of heart attack and atherosclerosis (Avramoglu et al. 2006). The risk of heart disease, stroke, kidney disease, retinopathy, neuropathy, ulceration and gangrene of extremities is increased with association of diabetes mellitus (Rotshteyn and Zito, 2004).
According to current statistics, diabetes mellitus is worse or greater in developing countries than the developed countries worldwide (Oputa 2002). So there is a great need to discover, design and test new drugs having dual therapeutic properties to control and cure both closely related critical diseases, diabetes and dyslipidemia and their mutually linked chronic complications (Bhandari et al. 2002).
In order to design and develop the drugs for the treatment, one of the best strategies is experimental animal models to understand pathophysiology of any disease (Rees and Alcolado, 2008; Chatzigeorgiou et al. 2009). For studying and testing anti-hyperglycemic agent, several animal models have been developed for the past few decades (Srinivasan and Ramarao, 2007). Chemical induction of experimental diabetes by alloxan is one of the most effective methods (Etuk 2010).
Alloxan is a widely used diabetogenic agent that induced the type I diabetes in animals but it also represent the end stage type II diabetes milletus: as there is severe deficiency of insulin in plasma, the end stage type II diabetes mellitus also adopts the characteristics of T1DM (Viana et al. 2004). Alloxan exerts its action by generating reactive oxygen species (ROS) along with cytosolic calcium raised in islet B of pancreas, when administered parenterally (Szkudelski 2001). Diabetic dyslipidemia is also acquired by the untreated alloxan induced diabetic animals (Alnoory et al. 2013).
Currently herbal remedies are in great demand due to side effects associated with therapeutic synthetic drugs (Mahmood et al. 2011). There are large numbers of plants that have shown effective hypoglycemic activity after laboratory testing, more than 1200 plants species are used in the treatment of diabetes mellitus worldwide (Eddouks et al. 2005).
It is believed that antioxidants present in the diet help to reduce certain diseases, vegetables are rich in these compounds (Astley 2003; Bazzano et al. 2002). There are large number of herbs, spices and other plant materials that have shown hypoglycemic and antioxidant properties, and are less harmful than synthetic drugs (Eidi et al. 2006). For the development of new pharmaceutical lead along with dietary supplement to already existing therapies, medicinal plants provide a valuable source of oral hypoglycemic compounds (Bailey and Day, 1989).
Raphanus sativus (radish) belong to the family Brassicaceae and it is an edible root vegetable (Lewis-Jones et al. 1982). Radishes contain high quantity of calcium, magnesium potassium, copper, ascorbic acid, folic acid, vitamin B6, and riboflavin and low amount of saturated fat and are very low Cholesterol (Nunes et al. 2011). Roots, seeds and leaves are the different parts of radishes (Raphanus sativus) that are used for medicinal purposes (Nadkarni et al. 1976). Radish roots are beneficial to protect the cell membranes against lipid peroxidation and also inhibit the changes in membrane caused by fat rich diet (Sipos et al. 2002).
Radishes (Raphanus sativus) have good hypoglycemic potential coupled with antidiabetic efficiency (Shukla et al. 2011). Due to hyperlipidemia the probabilities of cardiovascular disease increases in diabetic patient. Raphanus sativus (radish) is a traditional plant which is used to lower plasma lipid. It has the capability to lower the plasma triglyceride, cholesterol, and phospholipids in normal rats (Taniguchi et al. 2006).
Radishes are recommended as an alternative treatment for various diseases including hyperlipidemia, coronary heart diseases and cancer due to its high medicinal and nutritional value (Cetin et al. 2010). Phosphatase, catalase, sucrase, amylase, alcohol dehydrogenase and pyruvic carboxylase are the main enzymes that found in the radish roots (Singh et al. 2013). It is beneficially used in curing poor digestion and liver dysfunction (Lugasi et al. 2005), antioxidant activities (Wang et al. 2010), anti tumorigenic (Kim et al. 2011), anti-diabetic (Shukla et al. 2010). The leaves of radish are good source of protein (Singh and Singh, 2013).
Armoracia rusticana (Horseradish) belongs to the Brassicaceae family; it is a hardy perennial plant, mustard and cabbage are also including in this family. The roots of horseradish are rich in vitamin C and B1, iron, potassium, calcium and magnesium, phytoncide and essential oils; Allyl isothiocyanate a (volatile aglycone) which is released by a glycoside is identical with the essence of mustard plant (Istudor 1998). Root of horseradish smells pungent due to the allyl sulfide, a substance present in garlic and onion.
Armoracia rusticana is a source of many compounds that have been broadly studied for various health benefits (Lin et al. 2000). It contains several substances that have beneficial effects on peripheral blood flow. Its utilization normalizes the blood pressure and prevents the risk of thrombosis and sulfurous substances also improve the elasticity of cerebral and coronary blood vessels (Cirimbei et al. 2013). It has antibacterial properties due to allyl isothiocyanate present in volatile oils, especially mustard oil (Rosemary 1976).
The main component of the horseradish and the other vegetables from Brasicaceae family is sinigrin, degraded by the myrosinase enzyme complex to the allyl isothiocyanate (Wang et al. 2010). The enzyme horseradish peroxidase, is a heme-containing enzyme found in the plant that utilizes hydrogen peroxide to oxidise a extensive variety of organic and inorganic compounds, widely used in molecular biology and biochemistry (Bladha and Olssonb, 2011).
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Production Of Laccase By Immobilized White Rot Fungi And Its Application For The Decolorization Of Textile Effluent Dyes
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Publisher: 2014 Dissertation note: Textile wastewater effluent contains several types of dyes that are toxic, carcinogenic, and dangerous for environment (Nyanhongo et al. 2002). More than 10,000 different kinds of dyes and pigments are used in dyeing and textile industries. Approximately 8, 00, 000 tons colorant is produced annually and 10% of used dyes are enters the environment in the form of wastes. There are different types of textile dyes such as direct dyes, disperse dyes, reactive dyes, acid dyes, and basic dyes. Wastewater effluents discharge from textile industries contain more than 10-15% of these dyes (Kunamneni et al. 2007). Such wastewater effluents are being discharged into water stream without or after only partial treatments, causing water pollution and negatively affecting the aquatic life. The treatment of textile wastewater effluents are of major environment concerns (Nyanhongo et al. 2002).
White rot fungi (WRF) is a wide class of fungi and it is mostly comprised of basidiomycetes, ascomycetes and lignin-decomposing fungi (Wesenberg et al. 2003). WRF are the most abundant wood degraders, and are so named because they leave a bleached appearance of the wood fibers following their attack. WRF has the ability to degrade contaminants by virtue of the nonspecific nature of its extracellular ligninolytic enzyme system (Nyanhongo et al. 2002)
The white rot fungus is also known as lignin degraders because it degrades lignin effectively due to some enzymes present in it. The important enzymes involves in degradation of lignin are following: (i) lignin peroxidase: It oxidizes both phenolics and non pheolics compounds, (ii) manganese-dependent peroxidase, (iii) laccase: It oxidises phenolic compounds and produce phenoxy radicals and quinones; (iv) glucose oxidase and glyoxal oxidase used for H2O2 production, and (v) celloulobiose quinone oxidoreductase for quinone reduction (Kunamneni et al. 2007).
Laccase (oxidoreductase, EC 188.8.131.52) belongs to polyphenol oxidases group of enzymes. Copper atoms are present in the catalytic center of enzyme so it is also known as multicopper oxidases (Baldrain et al. 2006). The molecular mass of laccase is 50–100 kDa (Couto and Toca 2006). According to the mechanism of laccase, it carries out the reduction of oxygen to water along with the oxidation of its substrate. Laccases oxidize wide range of compounds such as polyphenols, methoxy substituted phenols, aromatic diamines, and other compounds (Baldrain et al. 2006).
The substrate specificity of laccase is very wide and broad. In ortho and para substituted mono and polyphenolics substrate, it carries out reduction by removing hydrogen atom from hydroxyl group. In aromatic amines, it removes one electron and produces free radicals. These radical are able of many other reactions such as depolymerization, repolymerization, demethylation, or quinone formation. During lignin degradation, oxidation of methoxyhydroquinones followed by auto-oxidation of the methoxysemiquinones. Furthermore, formation of superoxide anion radicals undergoes more chemical reactions. The activity of laccase may be increased by using different kind of activators, such as ABTS (2, 2-azinobis (3-ethylbenzthiazoline- sulfonic acid), 1-hydroxybenzotriazole, or compounds secreted by fungi (Abadulla et al. 2000). In the presence of ABTS, the decolorization efficiency increases up to 45% (Tong et al. 2007).
Laccases have been produced from different kind of sources such as some species of fungus like white rot fungi, different kinds of bacteria, and some insects (Heinzkill et al. 1998; Diamantidis et al. 2000; Dittmer and Kanost 2010). This enzyme is widely distributed in Ascomycetes, Deuteromycetes, and Basidiomycetes, WRF is the major source for the production of laccase enzyme because this fungi is involved in metabolism of lignin (Bourbonnais et al. 1995).
There are many applications of fugal laccases such as effluent decolorization discharged from industries, degradation of pulp released from paper and pulp industries, removal of phenolics compounds from alcohols, synthesis of organic compounds, biosensors, pharmaceutical sector (Yaver et al. 2001). This enzyme can also improve animal performance, increase nutrient digestibility when added to animal feed (Sharma et al. 2013). Fungal laccases have higher redox potential of +800mV as compared to plants or bacterial laccases that’s why there are several applications of laccase in biotechnology field especially in the decolorization of dyes. Enzymes can be produce in larger amount so that laccase based decolorization techniques are advantageous to bioremediation technologies (Devi et al. 2012).
Pleurotus is a species of WRF and few laccases have been isolated, purified and cloned from Pleurotus species. However, the physiological signiﬁcance of laccase produced by the white rot fungi is not known. Literature reports that mycelia culture of Pleurotus ﬂorida produces at least two laccases (L1 and L2), one of which appears to be linked with the mycelia growth of the fungus (Das et al. 1997). The L1 isoenzyme is dominantly involved in the dye decolorization process.
Submerged fermentation (SmF) is a type of fermentation in which microorganism is grow in liquid broth and enzymes and other compounds are released in the broth. This technique used free liquid substrates such as nutrients etc. The substrates are utilized quite rapidly and constantly supplemented with nutrients. In fermentation broth, microorganisms are provided with appropriate nutrients and conditions such as high oxygen concentration for the production of microorganism in order to get desired products. In this technique, mycelium formation is takes place. Mycelium formation can lead to pellet formation which hinders the diffusion of oxygen and nutrients in the medium.
In recent times, wide variety of secondary metabolites has been produced commercially by fungal fermentation. Fungi are complex microorganism that is different morphologically and structurally at different phases of their life cycles form others. It is also differ in form between surface and submerged growth in fermentation media. Nature of liquid media also effect on the growth of fungi. Different culture conditions such as temperature, pH and mechanical forces are important for fungi growth but these parameters are different for different fungi (Kossen et al. 2000).
Enzymes act like catalyst and they speed up any chemical reaction without being used up in the reaction. The uses of enzymes are advantageous due to its several characteristics and features as compared to conventional chemical catalyst. However, there are some problems that can reduce the operational life time of any enzymes. These problems includes; non-reusability of enzyme, the instability of their structure, high cost of isolation, purification and characterization and their sensitivity to harsh condition of reaction.
These objectionable limitations of enzymes may be reduced by the use of immobilized enzymes. There are mainly four procedures present for immobilization of any cell (Kunamneni et al. 2007). These procedures are following: adsorption, gels entrapment or polymer entrapment, covalent coupling, and cross-linking to insoluble matrices (Brouers et al. 1989). For immobilization different kinds of matrices, such as agar, calcium alginate beads, polyacrylamide gel, etc have been used. In order to select suitable matrix and immobilization procedure, type of the cell, type of the substrate, medium conditions and products are major factors (Prasad et al. 2005).
During immobilization, enzyme is fixed to or within solid matrix in order to get heterogeneous immobilized enzyme system. Naturally enzymes are bounded to cellular membrane in living cells for most cases so in order to get the natural form of enzyme, immobilization of the cell is done. This immobilized system stabilized the structure and activity of the enzyme for longer period of time. When enzymes are immobilized, they are stronger and more challenging to harsh environment changes. Immobilization also allows easy recovery of enzyme and also it’s multiple re-use in processes. The Michaelis constant of immobilized enzymes increased and their activity usually lowered when compared to free enzyme. When immobilization procedure applied, different structural changes introduced to an enzyme which leads to these alterations. Immobilization helps to maintain the structure, stability and activity of enzyme for longer time without being de-activated (Kunamneni et al. 2007).
Immobilization represents an attractive option to obtain enzymatic catalyst for dyes treatment. This technique provides different advantages: (i) it prevents enzyme leakage even under harsh conditions; (ii) it facilitates enzyme use in different types of reactors like packed bed, stirred tank and continuous bed; (iii) it causes stabilization of the enzyme tertiary structure, usually as a consequence of multipoint attachment of the enzyme to the support, providing enzyme rigidity. The stabilization provided by covalent bonding is usually counter balanced by partial enzyme deactivation. This negative effect can be mitigated by carefully optimizing the immobilization conditions in order to maximize the ratio between immobilized enzyme activity and activity of the primary enzyme solution (Pezzella et al. 2014).
Immobilization of laccase was extensively investigated with broad range of different techniques and substrates. Inactivation of enzyme occurs when oxidized products are absorbed on the surface of the immobilization matrix support (Kunamneni et al. 2007).
Textile industries discharged wastewater effluents comprised of toxic dyes are dangerous for aquatic life and have harmful impacts on the environment. There are different methods including physical and chemical methods which are use previously to decolorized dyes. These physical and chemical methods are quite costly, prolonged, ineffective and insecure (Shang and Chi 1996; Mechichi et al. 2006). In comparison to these methods, biological processes are quite suitable and helpful. Biological processes are less expensive, safe and take less time and effective. Biological processes used microorganisms to decolorize dyes. Laccase as an extracellular oxidative enzyme produced by white rot fungi are eco-friendly and cheap. In order to decolorize dye, three day old fermentation media is used and dyes is added in the broth. To get 70-75% decolorization in fungal culture, more than 48 hours are required. Pleurotus Species produced laccase efficiently and this laccase could decolorize malachite green dye upto 70% within 24 hours (Yan et al. 2009).
Laccases can degrade several dye structures such as phenol, polyphenols and diamines (Abadulla. et al. 2000) to degrade harmful compounds into less toxic compounds and may be helpful to reduce environmental pollution (Gianfreda et al. 1999). The specific features and mechanism of laccase helps to make it a versatile biocatalyst. Due to its versatility, it is suitable for several applications such as biopulping, biobleaching, and industrial wastewater treatment. Due to the severe environment legislation, the textile industry is trying to introduce new innovative technologies for the treatment of wastewater effluents discharged from textile industries. Laccase has potential to degrade dyes of various chemical structures so that development of techniques based on laccase seems an attractive and suitable solution in decolorizing dyes (Madhavi and Lele 2009). The decolorization and detoxification of the wastewater effluent would help to use it again and again in dying process in textile wet processing.
The major purpose of this research is to decolorize the textile effluents dyes discharged by industries after partial treatment and cause water pollution and have negative effect on aquatic life and ecosystem. It is necessary to established most effective and efficient method to produce sufficient amount of laccase enzyme through immobilized white rot fungus and then utilized it in the process of bioremediation.
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Comparative Evaluation Of Anti-Hyperglycemic Effect Of Herbal Medicinal Plants Extracts On Alloxan Induced Diabetic Albino Rats
Material type: Book ; Literary form:
Publisher: 2014 Dissertation note: Diabetes mellitus is a clinical syndrome described as inappropriate hyperglycemia triggered by a relative or absolute deficiency of insulin or by a resistance to the action of insulin at the cellular level. It is the most shared endocrine disorder, upsetting 16 million individuals in the United States and as many as 200 million worldwide (Debra, 1991).
The word diabetes was devised by the Greek physician Aeretaeus in the first century A.D. In the 17th century, Willis detected that the urine of diabetics as ideally sweet as if infused with honey or sugar. The existence of sugar in the urine of diabetics was established by Dobson in 1755 (Straton et al. 2000).
Diabetes mellitus is a global health crisis, which has been obstinately disturbing the humanity, regardless of the socioeconomic profile and geographic location of the population. According to an estimate, one person is identified with diabetes every 5s somewhere in the world, while someone dies of it every 10s. Diabetes mellitus has achieved a pandemic form. Hence, it is very vital to control diabetes and its difficulties to lessen the human suffering (Wild et al. 2004).
Alloxan a glucose equivalent and is toxic by selectively abolishing insulin-producing cells in the pancreas (that is beta cells) of many animal species. This produces an insulin-dependent diabetes mellitus (called "Alloxan Diabetes") in these animals, with features similar to type 1 diabetes in humans. Alloxan is selectively toxic to insulin-producing pancreatic beta cells because it preferentially amasses in beta cells through uptake via the GLUT2 glucose transporter. Alloxan, in the presence of intracellular thiols, produces reactive oxygen species which start toxicity by its redox reaction (Lenzen et al. 1998).
There are diverse methods to the management of diabetes, like insulin treatment in type 1 diabetes: Sulphonylureas, which discharge insulin from pancreas by blocking the ATP-sensitive potassium channels; Biguanides, which reduce the insulin resistance; Thizaolidinediones, which upsurge the insulin sensitivity; alpha-glucodase inhibitors like acarbose, which lessen glucose absorption from intestine, thus reducing postprandial hyperglycemia; metiglinides like repaglimide and nateglamide, which are insulin secretogogues (Aslam, 1988).
In spite of the statistic that synthetic drugs such as insulin, investigators have been building efforts to find insulin-like substances from plant sources for the treatment of diabetes. More than 1200 plant species have been suggested for the managementof diabetes (Radha et al. 2011) Natural resources such for example plants are cherished source of bioactive compounds. A large number of drugs have been recognized in medicinal practice from natural products (Philipeon et al. 2010).
Recent scientific research and clinical studies have established the usefulness of some medicinal plants and herbal preparations in the development of standard glucose homeostasis. Herbal treatment have been used in patients with insulin-dependent and non-insulin dependent diabetes, diabetic retinopathy, diabetic peripheral neuropathy and other penalties of this metabolic disease (Ahmed et al. 2006). The herbal drugs are recommended extensively because of their effectiveness, less side effects and comparatively low cost (Lezney et al. 2004).
Ethno pharmacological reviews show that more than 1200 plants are used in customary medical systems for their suspected hypoglycemic activity (Marles and Farnsworth, 1995, Dey et al. 2002, Grover et al. 2002). The hypoglycemic activity of a huge number of these plants/plant products has been appraised and inveterated in animal models (Gupta et al. 2005, Kesari et al. 2006) as well as in human beings (Herrera et al. 2004, Jayawardena et al. 2005). In some circumstances the bioactive principles have also been secluded and identified. However, the mechanism of action whereby most of these plants and yields lesser the blood glucose level rests hypothetical.
This study reveals the comparative effect of different herbal plants effect on alloxan induced diabetic rats. Six different herbal plants have been used in this study to investigate the hypoglycemic activity. These plants areAllium sativum (Garlic), Aloe vera(Kanwargandal), Gymnemasylvestre (Gurmar), Momordicacharantia (karela), Trigonellafoenum-graecum (Methidana), and Syzigiumcumini(Jamun).
Table 1: Plants used in present study
Plant and family Plant part used Active ingredient Mechanism of action Reference
Allium sativum, Alliaceae Garlic gloves S-methyl cysteine sulphoxide-precursor of allicin and garlic oil Arouse in vitro insulin discharge,Hinder glucose making by the liver Sheela et al. 1992, Augusti and Shella 1996.
Aloe vera, Aspholedeceae Leaf pulp Phytosterols Excite production or discharge of insulin Modify action of carbohydrate processing enzymes Rajasekaran et al. 2004,
Tanaka et al. 2006.
Asclepiadaceae Leaves Gymnemosides and gymnemic acid (from the saponin fraction)
Triterpene glycosides Kindle exudation of insulin from rat islets.
Declines the activity of gluconeogenicenzymes,Induce beta cell regeneration. Shanmugasundaram, 1990,
Cucurbitaceae Fruit pulp Charantin (a peptide),Insulin like polypeptide P ("vegetable insulin") Encourage insulin secretion,
Quash the activities of gluconeogenic enzymes
Rises the quantity of beta cells in diabetic rats Rao et al. 1981,
Day et al. 1992,
Sarkar et al. 1996.
nicotinic acid, and coumarin
Galactomannan depress digestion and absorption of carbohydrates
Upsurge glucose induced insulin release Khosla et al. 1995,
Hannan et al. 2007.
Syzigiumcumini Seeds Mycaminose Kindle kinases intricate in peripheral utilization of glucose Achrekar et al. 1992.
Kumar et al. 2008
1.1: Allium sativum(Garlic) is a common zesty flavoring agent used since prehistoric times. Garlic has been cultured in all over world for its distinctive flavor, foodstuff, and medicinal properties. It has mostly been ascribed to its hypoglycemic, anticoagulant, antibiotic, hypo-cholesterolaemic, antihepatotoxic, anticancer, immune system modulatory and antioxidant possessions (Bakri and Douglas, 2005).
Figure 1: structure of allicin
1.2: Aloe vera(Kanwargandal)is one of the therapeutic plants which are conventionally well accredited plant in the controlling of diabetes. It fits to family Liliaceae (sub-family of the Asphodelaceae). Many studies titles that the high innards of phenolic compounds, glycosides (aloins), 1,8-dihydroxyanthraquinone derivatives,β -1,4 acetylated mannan, mannose-phosphate and alprogenglucoprotein in the A. vera is vital for its biotic action. Through past two years, Aloe vera used as helpful beneficial agent which defensively act as a free radical scavenging and other antioxidant characteristics on diabetic patients, by monitoring raised anions in an alloxan or STZ-induced diabetic animal models (Nakamura, et al. 2011).
Figure 2: structure of phytosterole
1.3: Gymnemasylvestre (Gurmar) is a plant used in Asia as a usual cure for diabetes or “sweet urine.” The hypoglycemic action of Gymnema leaves was first recognized in the late 1920s. Gymnema is testified to upsurge glucose uptake and utilization. It also mends the utility of pancreatic β-cells and may also decline glucose captivation in the gastrointestinal tract. Phytochemically the plant has been described to comprehend gymnemagenin- the sapogenin. Gymnemic acid was sequestered in pure states from the hot water extract of leaves of G. sylvestre (Puratchimani and Jha, 2004).
Figure 1: structure of gymnemic acid
1.4: Momordicacharantia (Bitter Melon) also known as karela, is one of the plants normally used for its glucose-lowering properties (Ahmed et al., 1998). The slices of the plant usually used contain the entire plant, its fruit or seeds, all of which are bitter due to the manifestation of the chemical momordicin. The anti-diabetic constituents in bitter melon comprise charantin, vicine, polypeptide-p, alkaloids and other non-specific bioactive components such as anti-oxidants (Beloin, et al. 2005).
Figure 4: structure of momordicin
1.5: Trigonellafoenum-graecum (Fenugreek L. Leguminosae) is one of the ancient therapeutic plants, originating in India and Northern Africa.The leaves and seeds, which ripe in long pods, are used to formulate extracts or powders for medicinal use.The hypoglycemic properties of fenugreek have been recognized to numerous mechanisms.The amino acid 4-hydroxyisoleucine in fenugreek seeds amplified glucose-induced insulin release in human and rat pancreatic islet cells. Fenugreek seeds apply hypoglycemic effects by exciting glucose-dependent insulin discharge from pancreatic beta cells, as well as by impeding the actions of alpha-amylase and sucrase, two duodenal enzymes involved in carbohydrate breakdown (Gupta, et al. 2001).
Figure 5: structure of 4-hydroxyisolucine
1.6: Syzigiumcumini(Jamun) tree belongs to the Myrtaceae family. This is also called as Jamun, Jambul and Jambol in Pakistan, India and Malaya. The barks, leaves and seeds extracts of SC have been testified to have anti-hyperglycemic, anti-inflammatory, antibacterial and anti-diarraheal effects. A complex mycaminose is extracted from its seeds which display anti-diabetic characteristic (WL Li, et al. 2004).
Figure 6: structure of mycaminose
There are numerous potential mechanisms through which these herbs can perform to regulate the blood glucose level (Tanira, 1994). The mechanisms of action can be associated, commonly to the capability of the plant in question (or its active principle) to lesser plasma glucose level by meddling with one or more of the procedures involved in glucose homeostasis. The described mechanisms whereby herbal antidiabetic remedies decrease blood glucose levels are more or less alike to those of the artificial oral hypoglycemic drugs and are abridged as follows (Acharya et al. 2008, Bastaki, 2005, Bnouham et al. 2006).
i) Stimulation of insulin production and/or discharging from pancreatic beta-cells
ii) Revival of impaired pancreatic beta cells
iii) Development of insulin sensitivity
iv) Imitating the action of insulin
v) Modification of the action of some enzymes that are tangled in glucose metabolism reducing the absorption of carbohydrates from the gut.
The effectiveness of herbal drugs is substantial and they have insignificant side effects than the synthetic antidiabetic drugs. There is growing demand by patients to use the natural products with antidiabetic activity. In recent times there has been improved concern in the plant remedies. Plants grasp certain potentials in the organization of Diabetes mellitus. Isolation and documentation of active ingredients from these plants, preparation of unvarying dose and dosage schedule can play a noteworthy role in improving the hypoglycemic action (Jung et al. 2006).
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Production, Purification And Characterization Of Laccase From White Rot Fungus
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Publisher: 2014 Dissertation note: Laccase (oxidoreductase, EC 184.108.40.206) are blue copper dependent oxidases and the mainligninolytic enzyme produced by white rot fungus. Laccase catalyze the oxidation of large snumbers of phenolic compounds (Kunamneni et al. 2007; Poonkuzhali et al. 2011). These enzymes have a molecular weight 60-90 kDa and consist of 15–30% carbohydrate. Laccases are the earliest and maximum investigated enzymatic systems. Laccase was initially found by Yoshilda in 1883 in the sap of Japanese laquer tree named as Rhusvernicifera. After a while in 1896, Bertrand and Laborde determined that laccase is a fungal enzyme.(Shraddha et al. 2007; Giardina et al. 2010).
Laccases are present extensively in nature, originating from plants, bacteria and fungi (Poonkuzhali and Palvannan 2011). In fungi, laccases are widely distributed in ascomycetes, deuteromycetes and basidiomycetes. The laccase producing fungus include Trametes versicolor, Pleurotus ostreatus, Polyporus, Trametespubescens, Cerrenaunicolour,PhanerochaetechrysosporiumandFunaliatrogiietc (Dwivediet al. 2011). Laccases occur morein fungi, than in the higher plants. Laccases are also present in few bacteria such as S.lavendulae, S.cyaneus, and M.mediterranea(Viswanath et al. 2008; Arias et al. 2003). In vegetables laccases have been recognized in turnips, apples, pears, cabbages, potatoes, beets, asparagus and various other vegetables (Jhadav et al. 2007).
Enzymes are produced by every living organism, however enzyme produced by microbes have various benefits over the enzyme originated from plants and animal origins.Laccases by nature
are important because of its huge diversity of catalytic activities, economical in production and comparatively more stable than other enzymes.The field of biotechnology proposes expanding possibilities for the production of several enzymes from microorganisms. New methods and techniques have been advanced by using enzyme as biocatalysts to produce big added value products like growing food requirements,good quality chemicals and medicines. Moreover enzymes are also utilized for environmental actions and for diagnostic and analytical motives. (Buchholz et al. 2005). Microbial enzymes are used as cost effective and environmentally sensitive substitutes for chemical processing in several industries and bioremediation. Therefore the commercial demand for microbial enzymes is increasing (Radhika et al. 2013).
Fungal laccases have boundless biotechnological functions across the globe like the decolouration and detoxification of industrial effluent, bleaching of pulp, phenolicselimination from wines, in preparation of biosensors in detergents blockindye transfer- functions (Yaver et al. 2001).It is also used in the formation of anticancer drugs, and included in few cosmetics to lessen their toxicity (Couto and Herrera 2006).In recent years, laccase have been skillfully practiced to the field of nanobiotechnology due to its capacity to mobilize electron transfer reactions without further addition of cofactor(Shraddha et al. 2007).
Laccase is ample in several white- rot fungi that are involved in lignin metabolism (Bourbonnais et al. 1997, Leontievskyet al. 1997). Fungal laccases have immense redox potential (up to +800 mV) than bacteria or plant laccases. The action of these laccases seems to be appropriate in nature and also has significant applications in the field biotechnology. These laccases are associated with the deterioration of lignin and also in the elimination of conceivably lethal phenols appear during the breakdown of lignin (Thurston et al. 1994).
The white rot fungus is corporeal in preference to morphological and composes of those fungi that are adequate of degrading lignin, which is a heterogenous polyphenolic compound in huge amount within the lignocellulose wastes(Eaton and Hale. 1993).Theircapability to deteriorate cellulose, hemicellulose, these are the polysaccharides forming the essential part of lingo cellulose is the basic metabolic processbetween the fungi and happen under the span of environmental conditions.The degeneration of lignindoesn’tprovide net energy so it is degraded during the secondary metabolism in order to gain polysaccharides present in lignin and carbohydrate complexes, supplying energy to which the organisms don’t have access(Jeffrics. 1990).The white rot fungi varyingly secrete one or more three extracellular enzyme namely manganese peroxidase, lignin peroxidase and laccase that are fundamental for degradation of lignin, ant they are generally mentioned as lignin modifying enzymes LMEs (Pickard et al. 1999).
Laccase is the subjects of demanding research in the recent years, because of their several properties like extensive substrate relevance, doesn’t required the inclusion of cofactors because they use oxygen as cofactor which is frequently present in the environment (Eugenio et al. 2009). Maximum number of laccases produced by various organisms is excreted as extracellular enzymes and this makes the purification process quite accessible. Laccase commonly display appreciable extent of stability. Due to these properties laccases are ideally applicable in diverse biological processes such as the treatment of industrial effluent, biopulping and biobleaching (Eggert et al. 2006).
The huge potential of laccase requires advancement in its production and, with huge activities and low cost (Herrera et al. 2007). The use of lignocellulosic agricultural waste as substrates is a tradition for the production of enzyme like laccase because it is ligninolytic in nature (Niladeviet al.2011). It is highly crucial to optimize the fermentation parameters for the adequate production of laccase (Revankaret al. 2007). .
The advantages of agro-industrial leftovers for cultivation media is of immense concern as agriculture waste cut down the expenditure of enzyme production and enhance the understanding on energy protection and recycling (Mansuret al. 2003).These agriculture wastes are comparatively economical and also contain ample nutrients such as lignin, cellulose andhemicellulose. These nutrients serve as inducer to energize the production of enzyme (Vassil et al. 2000).Due to these properties these agricultural waste can be used as substrate for the production of ligninolytic enzymes during the process of fermentation.
Laccase can be produced at varying rates by using a wide range of organisms grown on different substrates and by using several methods of fermentation, such as solid state, semisolid state, and submerged (Rodriguez et al. 1999; Boran et al. 2011). However, for effective laccase production, it is very important to use efficient laccase-producing organisms, suitable fermentation methods, and cheap and widespread sources. Accordingly, one of the most suitable approaches for the production of this enzyme is to use the most efficient agricultural wastes for increasing the production of the ligninolytic enzymes (Elisashviliet al. 2008).
Pakistan is an agricultural country and each year manufactures tons of agricultural by products. These agricultural wastes are accessible in markets at a very reasonable price and can be utilized as substrates in fermentation technique (Minussi et al. 2007). Agricultural waste products like rice husk, wheat bran, corn cob, millet husk and cereal huskhave been utilized by various scientists for laccase production (Osma et al. 2011; jhadav et al. 2009).The chemical properties of these agricultural wastes make them important and economical fermentation medium for biotechnological purposes(Giardina et al. 2010).The cellulose and hemicellulose constituents of lignocellulose wastes are widely used by several organisms but lignin, which is the maximum contrary material to microbial degradation, is transformed conveniently by only few organism of thw white rot fungus (Dwivedi et al. 2011).Lignin serves as a barrier that protects cellulose and hemicellulose from enzymatic attack, however, white rot fungi can attack this barrier in order to obtain energy from cellulose. These fungi produce different extracellular ligninolytic enzymes such as laccase, manganese peroxidase, and lignin peroxidase (Couto et al. 2006).
Fermentation is a biological approach that is used for the transformation of complicated substrates into basic composites by different microorganisms like bacteria and fungi. In the procedure of this metabolic breakdown the microorganisms also release various added compounds like carbon dioxide and alcohol asidefrom the conventional products of fermentation. These added compounds are known as secondary metabolites (Pandey et al. 1999). These Secondary metabolites span from enzymes, antibiotics, peptides and growth factors (Balakrishnan and Pandey. 1996; Machado et al. 2004; Robinson et al. 2001). They are also known as bioactive compounds becausethey carry biological activity(Demain et al. 1999).
Submerged fermentation is a type of fermentation in which components are present in a liquid media like broths and syrup. The co-active composites are poured into the fermentation broth. In this media the substrates are employed quiet immediately, due to this reason the nutrients in the media are either fortified or regained continuously. This type of fermentation approach is optimum for microorganisms such as bacteria, fungi because they depend upon on immense moisture content. The increased benefit of this approach is that the purification of the desired products or enzymes is quiet effortless. Submerged fermentation is especially used in the abstraction of secondary metabolites that are utilized in liquid form (Subramaniyam et al. 2012).
Furthermore 75% of the commercial enzymes are made by using submerged fermentation, it also supports the usage of genetically modified organisms to a large expanse then solid state fermentation. Submerged fermentation is also used on large extent because it doesn’t require equipment concerning solid state. On the contrary solid state fermentation is a mechanism operated in absence of free flowing water by utilizing solid support in form of natural substance ( Poonkuzhali et al . 2011). .
The major purpose of conducting this research is to design optimized fermentation process which produces effective amount of enzyme by using agricultural wastes. The use of agricultural wastes as substrates is economical and increase awareness on energy conservation .The enzyme can be used further for bioremediation because it not substrate specific and can act on broad range of substrates.
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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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Delignification Of Rice Husk By Organic Solvent Treatment To Increase It’s In Vitro Digestibility
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Publisher: 2014 Dissertation note: The major constituent of plant cell wall is lignocellulose. Plant biomass mostly consist of cellulose, hemicellulose and lignin alongside little measures of pectin, protein, extractives (dissolvable nonstructural materials, for example, sugars, nitrogenous material, chlorophyll, waxes) and ash. Lignocellulosic biomass is the most abundant organic material in nature. There is an expected yearly overall production of 10–50 billion dry tons representing about 50% of the worldwide biomass yield (Parveen et al. 2009).
Numerous physicochemical, structural and compositional variables decrease the digestibility of cellulose present in lignocellulosic material. So a treatment is required to increase the digestibility of lignocellulose biomass by exposing the cellulose present in plant fibers. Different techniques have been utilized for treatment, including chemical treatment, ammonia fiber explosion, biological treatment and steam explosion to modify the cellulosic structure to increase the availability of cellulose for digestion (Haoran et al. 2013). At that point, acids, bases and enzymes might be utilized to break down the cellulose into its respective sugars. Cellulolytic enzymesare broadly used to break down cellulose into its constituent sugars.
Among various agricultural wastes a broadly available waste is Rice husk (RH) which is rich in lignocellulosic material. Internationally, roughly 600 million tons of rice paddy is delivered every year. By and large 20% of the rice paddy is husk, giving a yearly aggregate generation of 120 million tons (Abbas et al. 2010). Pakistan is a rice producing country a great part of the husk produced from processing of rice is either blazed or dumped as waste. Rice husk yield in Pakistan is more than 1780 thousand tons every year (Asif et al. 2013).
Rice husk produced during rice refining, makes disposal issue because of less business interest. Additionally, handling and transportation of RH is hazardous because of its low density. Rice husk ash (RHA) is an incredible environmental risk bringing about harm to land and encompassing range here it is dumped. Thus, business utilization of rice husk and its ash is the option answer for disposal problem (Dilip et al. 2014).
RH are essentially made up of lignocellulose (60wt. %) and silica (11wt. %). The greater part of past investigations concentrated on the preparation of silica or other silicon based materials from RH, while the lignocellulose in RH was mostly glazed and then wasted. Thus, a methodology for comprehensive usage of RH has been produced to expand its digestibility by the breakdown of lignocellulosic mass. (Ajay et al. 2012)
Numerous techniques have been adopted for treating lignocellulosic feedstocks. However just a few of them appear to be encouraging. These treatment techniques include dilute acid treatment, steam blast (CO2 blast), pH controlled water treatment, ammonia fiber expension, ammonia recycle percolation (ARP) and lime treatment. Some survey articles have been appeared for microbial biomass treatment. But the present study gave presentations on organosolv treatment process. Despite the fact that organosolv treatment is more expensive at present than the leading treatment forms, it can give some significant side products. It appears that organosolv treatment is more practical for biorefinery of lignocellulosic biomass which considers the usage of every bit of biomass parts. An essential streamlining and usage of side products may lead the organosolv treatment to be a guaranteeing one for bio refining lignocellulosic feedstock in future. Organosolv treatment yields three different parts: dry lignin, a watery hemicellulose stream and a moderately pure cellulose division (Xuebing et al. 2009).
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DNA Based Characterization Of Protease Gene From Geobacillussp.Sbs-4s
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Publisher: 2014 Dissertation note: Proteases are hydrolytic enzymes responsible for the hydrolysis of proteins(Qadar et al.2004).These enzymes contribute major role in textile and leather industry,accounting 60% of the world wide enzyme market(Nascimento et al.2004).These enzymes are also being used in food ,pharmaceutical ,detergent, brewage sweet industry and as digestive additives in human and animal feed (Wilson, 2012).
Proteases are produced by microbes,animal and plants but microbial proteases are preferred due to ease in production and cheaper cost (Ningthoujam et al.2010).Microbes produce a variety of proteases according to their requirement that are specific in their function (Neurath 1999).Microbes might be involved in the production of intra or extracellular proteases.Extracellular proteases help the organism to absorb and utilize hydrolytic products from proteinious substrates in order to get energy by catabolism or to synthesize the biomolecules through anabolism reactions(Ningthoujamet al.2010).
Proteases can be classified in different ways.On the basis of cutting preferences these can be divided in to two groups:endopeptidases and exopeptidases (Barret and Mcdonald 1985).Exopeptidases are involved in hydrolysis of the peptide bond near N or C terminal whereas endopeptidases are responsible for the hydrolysis of peptide bond, with the chain, distant from the peptide ends(Motyan et al .2013).On the basis of catalytic residues in active site the proteases can be divided into six groups including glutamate,serine, therionine cysteine,aspartate and metalloproteases(Li et al.2013).
Microorganisms occupy all possible environments including habitats that provides appropriate conditions for growth(Sharma et al.2009).Thermophiles have ability to grow at highertemperature whereas other microbes fail to survive.There has been increasing interest in thermophilic bacteria because of their thermostable enzyme(Obeidat et al.2012).Hyperthermophiles can survive in extremely hot environment. Hyperthermophiles occupy the most basal positions of the phylogenetic tree of life(Bouzas et al. 2006). About 70 species of hyperthermophilic bacteria and archea has been isolated from different terrestrial, marine and thermal areas in the world.Hyperthermophiles are very divergent in their phylogeny and physiological properties.Proteolytic enzymes from hyperthermophiles are catalytically active at high temperature and they can alsoretain their catalytic activity in the presence of detergent and other denaturing substances (Stetter et al.1993).
Geobacillusis widely distributed thermophiles isolated from geothermal areas (Chalopagorn et al.2014).On the basis of16SrRNA gene sequences, Geobacillus belongs to Bacillus genetic group 5. It is phenotypically and phylogeneticallyconsistent group of thermophilicbacilli (Rahman et al. 2007).Bacillus and Geobacillus species are the dominant workhorses in industrial biotechnology. These bacteria produce a variety of extracellular enzymes, such as amylases, xylanases, proteases, phytases, carbonic anhydrases, catalases, pectinases. Bacillus and Geobacillus species hasability to grow at acidic, alkaline, neutral pH and at elevated temperature has positioned them among the most important industrial enzyme producers(Satyanarayana et al. 2012).
Geobacillus are gram-positive, rod-shaped, aerobic,endospore-forming obligate thermophiles.The growth temperature for various Geobacillus species ranges from 37 to 75 °C and pH range of 6.0 to 8.5.The members of Geobacillusare homologus to each other and share homology 99% among them(Tayyab et al.2011). The genus Geobacillusthermophilicstrains, produce a variety of thermostable hydrolytic extracellular enzymes, such as proteases, amylases, and lipases used in various industrial applications (Wiegand et al. 2013)
GeobacillusSBS-4S was isolated from a hot spring located in Gilgit, Northern areas of Pakistan.Geobacillus SBS-4S strain is Gram positive, rod-shaped bacteria and occurs in chains. That could grow at a wide range of temperature (45 to 75˚C) and pH ranging 5.5 to 9.5.Geobacillus SBS-4S produced several extracellular enzymes including amylase, protease and lipase.The comparison of the strain SBS-4S with the already reported species of genus Geobacillus showed that SBS-4S is resistant to antibiotics such as streptomycine, spectinomycin and rifampicin(Tayyab et al.2011).
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DNA Based Characterization of Xylanase Gene From Hyperthermophilic Archeon
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Publisher: 2014 Dissertation note: Blank CD
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Dna Based Characterization Of Triacyl Glycerol Lipase Gene From Geobacillus Sp. Sbs-4s
Material type: Book ; Literary form:
Publisher: 2014 Dissertation note: Lipases are hydrolases responsible for the liberation of fatty acids from triglycerides (Akoh et al. 2004). With the exception of hydrolysis, lipolytic enzymes can also catalyze transesterification, esterification and interesterification in low aqueous conditions (Goldberg et al. 2005). Under micro-aqueous conditions, lipases have exceptional ability to catalyze the reverse reactions that leads to acidolysis, alcoholysis and esterification (Jaegar and Reetz 1998). Previously production of lipases has been reported from various sources like microorganisms, animals and plants (Lee et al. 2006). Lipases extracted from different sources have broad spectrum properties depending on their sources regarding pH optima, positional specificity, thermostability, fatty acid specificity, etc (Gupta et al. 2004). Thermostable lipases are important for many industries due to their distinct feature (Demirjian et al. 2001). Psychrophilic lipases have high activity at low optimum temperature so they are fascinated for the production of relatively frail compounds and their use has been increased in the organic synthesis of chiral intermediates (Joseph et al. 2008). Alkali stable lipases have ability to work optimally at alkaline pH and are highly suitable to be used in detergents (Sarethy et al. 2011). Lipases are the component of additives in biotransformations, environmental bioremediations, molecular biology applications, food and detergent industry and heterologous gene expression in psychrophilic hosts to prevent formation of inclusion bodies (Houde et al. 2004). Lipases occur in almost all organisms from bacteria to complex organisms. In complex eukaryotes, pig and human pancreas are the main source for lipase production. In eukaryotes, lipases carry out lipoproteins metabolism, fat digestion, reconstitution and adsorption. Lipases have also been extracted from plants. They are found in higher plants and energy reserve tissues.
(Treichel et al. 2010). However, microorganisms are preferred for the production of enzymes over plants and animals because of their shortest generation time, the high yields, great flexibility in environmental conditions, ease of cultivation conditions, variety in catalytic activities, regular supply due to absence of seasonal fluctuations, simplicity in genetic manipulation and quick growth of microorganisms on economical media (Gurung et al. 2013). The production of microbial enzymes is safer and more expedient and they have more stability than their corresponding animal and plant enzymes (Messaoudi et al. 2010). Lipases share a common architecture of α/β-hydrolase fold and a highly conserved pentapeptide catalytic triad G-X1-S-X2-G, where G for glycine, S for serine, X1 for histidine and X2 for glutamic or aspartic acid (Widmann et al. 2010). In the highly conserved catalytic triad there is a nucleophilic residue comprising serine and a catalytic residue containing aspartic or glutamic acid and histidine (Anobom et al. 2014). Lipases have alkyl groups on the surface of their structure due to which they are strongly hydrophobic. Broad substrate specificity is another remarkable characteristic of lipases. Also they catalyze the hydrolysis of alcohols with various chain lengths and esters of fatty acids. The long chain fatty acids of varying chain lengths hydrolysis form triglycerides correspondingly (Patil et al. 2011). Lipases are biotechnologically important enzymes and they have vast applications in leather, food, textile, pharmaceutical, detergent, paper, cosmetic industries and in biodiesel formation (Gupta et al. 2004). Lipases are used in processing of food by the esterification and transesterication of oils and fats. These enzymes are involved in the enhancement of flavor, prolong shelf life and improves aroma of bakery goods, beverages, dairy products, fruits and vegetables. In food
industry egg yolk is treated with phospholipase to hydrolyze egg lecithin and isolecithin which improves its heating stability and emulsification capacity. This treated egg yolk is then used for the processing of mayonnaise, baby foods, custards, salad or food dressings and sauces. Lipases are also used to remove fats from meat and fish (Aravindan et al. 2006). In textile industry lipases are used in processing of fabrics, thus improving its quality and absorbing ability by removing size lubricants. Polyethylene terephthalate is an important synthetic fiber in the textile industry (Araujo et al. 2008). Lipases action on that fiber improves its hydrophilicity and anti-static ability (Contesini et al. 2010). Lipases in therapeutics are involved in the synthesis of macrolide products. Macrolide products have potential antitumor activity against a broad spectrum of human tumor lines including multidrug resistant cell lines. In pharmaceutical industries, lipases are used for esterification, transesterication and asymmetric hydrolysis of racemic alcohols and carboxylic acids to produce their enantiomeric forms. Many β-blockers, nonsteroidal anti-inflammatory and anti-asthamic drugs are pharmacologically active in their one enantiomeric form while toxic in other form like “profens and ibuprofen” are pharmacologically active in their (S)-enantiomeric form whereas (S)-thalidomide has severe side-effects (Jegannathan and Nielsen 2014). Leather manufacturing industries use lipases for degreasing which is the process of removing fats and grease from skins and hides of cattle. Organic solvents and surfactants are also used to process leather but these methods are not eco-friendly and results in the emission of volatile organic compounds. Besides fat dispersion lipases also improve the quality of leather by making it water proof and low fogging (Horchani et al. 2012). Lipase is used as a catalyst in the tranesterification of vegetable oil or alcohols to form emollient esters like myristyl myristate. Emollient esters due to their moisturizing properties are
used in beauty creams. Lipases have also been used in anti-obese creams and they are added as texturing agents to improve the consistency of creams and lotions (Sharma and kanwar 2014). Laundry detergents have surfactants as their primary constituent which remove stains. But they require a considerable amount of energy and also they are toxic to our environment, released in water even they are harmful to aquatic life. The detergent industries are developing trends to use such agents that are eco-friendly and require less energy. Nowadays enzymes are being used in the detergents to remove tough stains and give softness, resiliency to fabrics, antistaticness, dispersible in water and mild to eyes and skin. Lipases are used specially to remove oil and grease stains (Ghuncheva and Zhiryacova 2011). The demand of industries for lipases has grown in the past decade for their environment friendly nature, biodegradability, high specificity and high catalytic efficiency. The commercial applications of lipases are a billion-dollar business that comprises their use in a broad spectrum of industries. Many techniques are being used nowadays to improve the features of lipases e.g., stability, activity, specificity and selectivity, reduction of inhibition (Rebeiro et al. 2011). The main advantage of using immobilized lipases is that it is possible to reuse them, since they can be easily recovered, thus making the process economically feasible, not interacting chemically with the polymer, thus avoiding its denaturation in detergent industry and ester formation (Sharma and Kanwar 2014). Genetic engineering has been used to modify the industrial enzymes to enhance its properties (Adrio and Demain 2014). For lipases as potential candidates of detergent industry, these have to be thermostable, alkali stable, stable against proteolysis, action of oxidative compounds and other chemicals used in detergents. In food and pharmaceutical industry usage
lipases should be more stable in organic solvents and they must show high stereospecificity (Verma et al. 2012). Geobacillus sp. SBS-4S is a thermophillic microorganism that was isolated from Gilgit bultistan, Northern areas of Pakistan. It was found to be gram positive, rod shaped aerobic endospore-forming bacterium. It grows optimally on pH 7 and temperature 55 °C. It produces several industrially important extracellular enzymes including amylases, proteases and lipases (Tayyab et al. 2011). The present study deals with the characterization of triacylglycerol lipase gene responsible for the hydrolysis of triglycerides.
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Sequence Analysis Of Mitochondrial Atpase 8/6 Gene Variants In Equine
Material type: Book ; Literary form:
Publisher: 2014 Dissertation note: Human has been using horses for doing different jobs like transportation, hunts, carrying loads, warfare and sports (Zhang et al. 2012). In Pakistan, horses and donkeys are mostly used for transportation whilehorses are also used for racing and playing games like polo.There are two main types of horses:Equuscaballusare domesticated horses and Equusferus are the wild horses. There are more than 300 breeds of horses in the world today (Barbara and Dafydd, 2007). The horse population is estimated as 0.32 million and has been decreasing over the years in Pakistan. Main breeds of horses that are found all over the Pakistan are Kajlan, Kakka, Balochi, Morna, Shien, Anmol, Makra, Pak-thoroughbred,Heerzaiand Waziri (Khan, 2004).
Seventy percent of the population earns living from the land. Agriculture contributes nearly 21% to gross domestic product and generates 43% of all jobs. Over 30 million people in rural areas derive their livelihood from livestock production. The number of impoverished communities moving from the country to find work in Pakistan’s towns and cities is rising. Many of these people rely on working equine animals to earn a living.
Nuclear and mitochondrial genomes are frequently used in animal genetic research. Nuclear genomeis generally a huge and complicated molecule and is not well studied in many species. However mitochondrial DNA being small sized and having high mutation rate is used frequently for the purpose of genetic research (Stanley et al. 1994). Characteristic of having fast evolution rate as compared to nuclear DNA makes mitochondrial genes a good tool for genetic studies (Avise, 1994).
Several studies have investigated the genetic relationship among horse and donkey breeds using mitochondrial sequences as a marker for breed characterization and phylogenetic. Each mitochondrion contains its own circular DNA, replication, transcription and translation machinery and serves as semi-autonomous organelle. Mitochondria perform so many important functions in our body like metabolism(oxidative phosphorylation), apoptosis and aging(Weinberg, 2007).
The advent ofpolymerase chain reaction and direct sequencing techniques with the use of mtDNA as a phylogenetic marker has been extended to much greater levels of phylogenetic inclusiveness (Zardoya and Meyer,1996). The special features of mtDNAi-e,lack of introns, maternal inheritance, absence of recombination events and haploidy have made it the most common type of sequence information used to estimate phylogenies among both closely and distantly related texa(Meyer, 1993).
Four of the five mitochondrial respiratory chain complexes, namely C1, C3, C4 and C5 (ATP synthase) contain subunits encoded by mitochondrial DNA (Kadenbach, 2012). ATP synthase (Complex5) functions to make ATP that is used by the cell (Von et al. 2009). ATP synthasecomprisesan integral membrane cylindrical, the F0 particle and a peripheral matrix-facing F1 particle, the catalytic ATP synthase domain (Boyer, 1997). All aerobically respiring organisms possess ATP synthase enzymes and are located inthe cell membrane in prokaryotes, the mitochondrial inner membrane in eukaryotes and the chloroplast thylakoid membrane (Ackerman and Tzagoloff, 2005). This enzyme is responsible for the final step of oxidative phosphorylation. The protons move down their concentration gradient from inter membrane space to matrix through F0 particle while F1particleuses the energy provided by influx of these protons and converts ADP molecule into ATP. ATPase 6 and ATPase 8 proteins are components of F0 particle where they play direct role in maintaining the structure and function of ATP synthase (complex 5). All five subunits of F1 and most of the F0 subunits are nuclear encoded(Collinson et al. 1996). Only two proteins i-e, ATPase 6 and ATPase 8 are encoded by mtDNA (Boyer, 1993).
The present study is designed to investigate the diversity and phylogenetic analysis of Thoroughbred Pakistani horse and donkey breeds on the basis of ATPase 6 and ATPase 8 genes.
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Production, Purification And Characterization Of Exoglucanase By Arachniotus Rubber
Material type: Book ; Literary form:
Publisher: 2015 Dissertation note: Cellulose is well thought-out as the major renewable biological resource which is constantly replenished by the photosynthesis (Ragauskas et al. 2006). It is the most plentiful carbohydrate and is considered world’s abundant organic substrate. Cellulose, lignin and hemicellulose are the main components in plant cell walls with cellulose being the mainly abundant part (Saleem et al. 2008). Enzymatic conversion of cellulose is inexpensive. The cellulose hydrolysis is brought about by mixture of β-glucosidase, endoglucanase and exoglucanase. The entire hydrolysis of cellulose is carried out by these enzymes (Mathew et al. 2008).
Biosynthesis of cellulose via land plants as well as marine algae occurs at a rate of 0.85×1011 tons per year (Niranjane et al. 2007). By using it in proper way this vast quantity of cellulose can be used for different purposes with some enzymatic conversions. Cellulose can be transformed into simple sugars that can be used for the production of ethanol or other energy products and for the food purposes.
Lignocelluloses are agro-industrial wastes worldwide. These can be used for the production of different important products that may include renewable sources to accomplish the energy demand by making biofuels and to cover up the high food demand of present century. Cycling and recycling of these materials may also decrease pollutants in the environment (Doran et al. 1984).
Cellulases which are resistant to proteases are preferably used in detergent and soap industries and are also used in detergents for depilling, care agents of colour, washing of stone, biopolishing and smoothing of surface in cotton fabric (Godfrey and West 1996). Cellulase is being used in improving digestibility of animal feed (Lewis et al. 1996).
When cellulases are added in detergents it brightens the color of cotton textiles and smoothens the fabric (Niehaus et al. 1999). Several applications of cellulases include development of the nutritional rate of cellulosic materials and forage crops, improvement of pulp class, and enhanced digestibility of organic matter via elevated fiber content (Garcia et al. 2002).
Cellulases are also used in paper, lumber and textile industries, in making of food and feed supplements for cattle and poultry feed stocks, preparations of baking, brewing, pharmaceuticals, malting, removal of fruit juices, dealing out of vegetables and processing of starch (Petre et al. 1999). The main significant application is in the production of single cell protein, alcohol, beer, biofuels, chemical feedstock, ethanol, and high fructose syrup (Solomon et al. 1999).
Development of an inexpensive method for food production through enzymatic hydrolysis is slowed down by sky-craping rate of cellulase making, low enzymatic behavior and low conc. of sugar syrup obtained on hydrolysis of such materials. However research have been conducted on pure cellulose and cost of isolation of cellulose from lignocellulosic is added up in the overall production cost (Chahal et al. 1985)
Production of Cellulase can be improved by studying media composition and optimizing fermentation parameters, microbial strain and some other factors that control production and growth (Han and Chen 2010). Different lignocellulosic materials are used for economic enzymes production like, corn cobs, bagasse, wheat straw, rice straw, and wheat bran (Hussain et al. 1999). The hydrolysis catalyzed by cellulases has found like a practicable method to make reducing sugar or glucose from cellulose for making biofuels and some other value added goods by means of microbial fermentation (Zhang et al. 2006)
Two important classes of enzymes, Cellulases and hemicellulases are produced by filamentous fungi and secreted into the cultivation medium (Sadia et al. 2008). Cellulose can be degraded by numerous microorganisms like bacteria, fungi and plant cell wall fibers. Degradation of cellulosic biomass is carried out by cellulases in nature. Production of industrial enzymes has been carried out by filamentous fungi for more than 50 years (Saleem et al. 2008). A range of microorganisms have the ability to secrete cellulases including fungi and bacteria (Jiang et al. 2011).
Various fungal strains secrete large quantity of cellulases as compared to bacterial ones. Due to high production rates microorganisms are compatible for the production of cellulases through fermentation of cheap and non conventional sources like cellulosic agro industrial wastes and byproducts (Ghosh et al. 1984).
Most of the cellulases exploited for industrial applications are from soft rot and white rot fungi such as Trichoderma, Penicillium, Phanerochaete (Dashtban et al. 2009). There are different microorganisms which can produce cellulose effectively belonging to genus Cellulomonas, Clostridium, Ruminococcus, Bacillus, Bacteriodes, Microbispora, Streptomyces and Arachniotus (Saratale et al. 2008). Arachinotus sp. is a white rot fungus and had used for economic consumption of many waste products. It act as antagonist to other microbes and prevents contamination (Alexopoulos and Mims 1985).
The earlier period have shown significant progress in separation of microorganisms that produce cellulases, civilizing the yield of cellulases via mutation, purifying also characterizing the cellulase components (Wood TM and McCrae SL. 1977).
Viable production of cellulases had tried by means of solid or submerged culture with batch, fed batch and continous run processes. Production of cellulase on profitable scale is increased via growing the fungus on top of solid cellulose (Persson 1991 et al). Production of these enzymes by culturing Aracniotus sp. on a fibrous substrate like wheat bran would not only reduce the pollutants but will also serve as potential source of energy.
The hydrolysis of cellulose is brought about by mixture of endoglucanase, exoglucanase and β- glucosidase. These enzymes act synergistically to accomplish the entire hydrolysis of cellulose. Endoglucanase works internally on cellulose chain by cleaving 1,4-β associated bonds. The exoglucanase acts processively starting from reducing and non-reducing ends eliminating cellobiose in an order (Mathew GM et al. 2008). β-glucosidase completes the hydrolysis by means of changing cellobiose and also small oligosaccharides in the glucose units (Kumar R et al. 2008).
Use of industrial wastes for making cellulases increases the financial effectiveness of the chief production method. A lot of cellulosic residues including corn stover, corn stalks, bagasse, rice straw, wheat straw, cotton stalks etc. accumulated up to 50 million tones only in Pakistan (Azad 1986) are not wasted properly, it could provide as an cost-effective resource of Cellulase.
The incorporation of inexpensive sources, such as sugar cane bagasse and wheat bran in the media for the manufacturing of lignocellulose enzymes can help in decreasing the production costs of enzyme complexes which can hydrolyse lignocellulosic residues that can be used for the formation of fermented syrups therefore contributing to the cost-effective production of bioethanol. (Camassola and AJP Dillon 2007).
Fungal biomass can also be formed by Solid substrate fermentation (SSF) and submerged fermentation (SMF). Along with a variety of groups of microorganisms used in SSF, the filamentous fungi are mainly exploited as they have ability to grow up on absolute solid substrate. Submerged fermentation is the development of microorganisms in fluid nutrient broth. Industrial enzymes can also be formed with this process. In this type of fermentation the substrate is solublized or suspended as excellent particles in a huge volume of water. In submerged fermentation, substrate concentration from 0.5 to 6% are used which depends upon the concentration of the substrate (Chahal et al. 1982).
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Detoxification Of Aflatoxins Using Different Organic Acids
Material type: Book ; Literary form:
Publisher: 2015 Dissertation note: From global prospective of food safety and food security, mycotoxin contamination of foods has gained much attention as potential health hazards for humans and animals. Cereals and other crops are exposed to fungal attack in the field or during storage and this attack may result in mycotoxin contamination of crops. Animal feed is basic necessity for all the live stock, poultry and other animals. AF is the most important for human and animal health perspective and in developing countries such as Pakistan where climate conditions favor the formation of these toxic metabolites. Governments and private organizations of international level have established maximum residue levels (MRIs) which usually guide to control AF in feed. Therefore, the current study was planned to detoxify AF by using different organic acid treatments in animal feed collected from different dairy farms of Punjab.
The samples of cotton seed cake, maize oil cake and animal feed were collected and checked the presence of AFB1 qualitatively by TLC and quantitatively by HPLC. The samples which gave positive results were treated with different acidic treatments applied on it. Firstly checked the results of citric acid, acetic acid and lactic acid on feed sample qualitatively by TLC. TLC plates were checked under UV box and the samples which showed the detoxification of AF were quantitatively analyzed by HPLC in Toxicology Laboratory, QOL, UVAS, Lahore, Pakistan.
The average concentration of AFB1 found in the cotton seed cake, maize oil cake and mixed feed were 279.8 ppb, 34.2 ppb and 25.5 ppb, respectively much greater than permissible levels proposed by European Union. Treatments of varying concentration of citric acid, acetic acid and lactic acid were applied on positive samples (≥20 ppb) and checked their effect on rate of detoxification.
All the above mention treatments applied on the feed samples in order to obtained in vitro detoxification of AFB1. Sprayed different concentration of acetic acid, citric acid and lactic on positive samples by varying volumes and placed them over night then extracted and analyzed.
It has been observed that 1N concentration of citric acid, acetic acid and lactic acid showed complete detoxification. However, when these samples were treated with 0.5N solution of organic acids then variation was seen in rate of detoxification.
Statistically these results were analyzed by ANOVA which showed that effect of these treatments on rate of detoxification was highly significant (P<0.05). In vitro detoxification of AF by these organic acids was proved beneficial in order to reduce the animal and human health risks. However, in vivo detoxification of aflatoxin by using these organic acids should be studied in future.
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Production Of Single Cell Protein By Using Banana Peels As Substrate And Its Biological Evaluation In Broiler Chicks
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Publisher: 2015 Dissertation note: The term single cell protein (SCP) refers to dead, dry microbial cells or total proteins extracted from pure microbial cell culture and is produced using a number of different microorganisms including bacterium, fungus and algae. It can also be called biomass, bioprotein or microbial protein.
Besides high protein content (about 60-82% of dry cell weight), SCP also contains fats, carbohydrates, nucleic acids, vitamins and minerals.
Fermentation media containing grinded banana peel as substrate was used to check the production of single cell protein for the selected Arachniotus sp. Different parameters were optimized for higher production of SCP e.g: Incubation period, pH, volume of inoculum, carbohydrate source, concentration of corn steep liquor and ionic salts concentration.
The biomass yield was estimated for total protein content by Lowrymethod. Biomass produced from fermentation was used for biological evaluation in feed trials of broiler chicks.
It is found that Arachniotus sp gave maximum single cell protein 7.49 g/L using 10 g banana peels at 72 hours incubation period. And protein concentration increased 7.58 g/L by optimizing volume of inoculum 2ml. It is observed in present study carbohydrate source also increases the protein concentration 8.41 g/L when carbohydrate source was optimized (glucose 3%).
Later on it was found that nitrogen source also enhance the protein production upto 12.61 g/L by using 2% corn steep liquor. Results also revealed that ionic salt concentration also play important role in the production of biomass protein, addition of 0.075% CaCl2.H2O produced 14.45 g/L single cell protein using above mentioned optimized conditions. 0.050 %
K2HPO4 produced 15.06 g/L. Addition of 0.050% MgSO4.7H2O produced maximum protein 15.86 g/L.
Biological evaluation in broiler chicks of this biomass protein shown there is no deleterious effects on weight gain, feed conversion ratio, protein efficiency ratio and net protein utilization. Maximum weight gain observed 215.6 grams in the group (C) in which 50% sunflower meal was replaced with biomass protein.
Feed conversion ratio in group (C) was 2.64 in which 50% sunflower meal was replaced by biomass protein and in group (B) was 2.51 in which 25% sunflower meal was replaced. And in control group (A) feed conversion ratio was 2.41.
Protein efficiency ratio was observed with non-significant value. And same results were shown by Chaves et al (1988) who reported non-significant differences among the standard and test diet when Chaetominumcellulolyticum biomass was fed to chicks. Net protein utilization observed in present study gave significant P value among the groups.
So it is concluded that single cell protein produced by this method is cheap and can be used in the food industry as food supplements and can also be included in poultry feed. The study findings suggested that microbial biomass produced by Arachniotus sp using banana peels as substrate can be replaced upto 50% of the protein supply by sunflower meal without any deleterious effects on growing broiler chicks. Moreover, it will also help in the reduction of pollution by using waste i.e. banana peel for useful purpose.
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Production Of Single Cell Protein By Arachniotus Ruber Using Remnants Of Carrot As Substrate And Its Biological Evaluation In Broiler Chicks
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Publisher: 2015 Dissertation note: CD Error. Summary could not opened.
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Production Oflaccase From White Rot Fungususing Rice Bran As A Substrate By Solidstate Fermentation
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Publisher: 2015 Dissertation note: Laccase are copper oxidases and are found in large quantities in several white rot fungi that are involved in lignin metabolism. Fungal laccases have boundless biotechnological functions across the globe like the decolouration and detoxification of industrial effluent, bleaching of pulp, phenolic elimination from wines, in preparation of biosensors in detergents blocking dye transfer- functions. Laccase showed vast variety of substrates due to this ability they can enhance different types of industrial mechanism such as methylation, demethylation, polymerization, mineralization of pollutants like hydrocarbons. White rot fungus is efficient for the production of laccase using agro-waste as substrate.
In this research white rot fungus was isolated from stock cultures of Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan and the organism was maintained on Tien& Kirk media slants and petri plates. Solid state fermentation technique was used using basal fermentation medium and agro waste rice bran was used as substrate for the production of laccase.Proximate analysis was performed of the substrate rice bran to analyse crude protein, fat, ash, moisture and fibre content. The fermentation was performed at room temperature and flasks were placed on orbital shaker at 100 rpm and 30˚C.
Enzyme activity was checked using (ABTS) as substrate at 420nm, for every 24 hour to observe the maximum enzyme production. Fermentation parameters like substrate concentration, incubation period, pH, temperature and nitrogen source (corn steep liquor and ammonium sulphate) were optimized. The concentration of substrate optimized for rice bran was 7.5g/100ml and optimum production of 6.11 IU/ml of enzyme was observed. The optimum day for the production of enzyme was day 7 and the amount of enzyme produced was 6.91 IU/ml. The optimum pH and temperature were 4 and 40˚C respectively, and the amounts of enzyme produced were 7.48 IU/ml and 7.96 IU/ml respectively. Two nitrogen sources optimized were maize steep liquor 1ml and ammonium sulphate 0.2 g, and the enzyme produced was recorded 7.67 IU/ml and 9.41 IU/ml respectively. Large scale fermentation batch of one litter was carried out under the optimized conditions and the enzyme produced was 9730 IU/L. Triplicates of each parameter were prepared.
The enzyme was purified using the purification techniques like ammonium sulphate precipitation, then by dialysis excess salt was removed, and then gel filtration was performed to collect different fractions on the basis of size of molecules and molecular mass of the laccase was analysed by SDS-PAGE. The size of the protein was found to be 70kDa. Characterization of laccase was performed in terms of optimum pH, temperature and in response to inducers and inhibitors. The optimum pH and temperature of the purified enzyme was 6 and 40˚C respectively. The inducer copper sulphate enhanced the activity of enzyme up to 9.7 U/ml and then inhibitors EDTA and 2-merceptoethanol reduced the activity level up to 4.17IU/ml and 3.98 IU/ml respectively. To study and analyse the effects of optimization parameters Pearson correlation, descriptive statistics and one way Anova were used.
The optimum production of laccase was achieved using agro waste rice bran. The enzyme produced was economical and it can provide effective solutions for bioremediation of hazardous compounds and pollutants.
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Bioconversion Of Agricultural Waste To Alginate By Azotobacter Vinelandii Using Fermentation
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Publisher: 2015 Dissertation note: Alginate is an exopolysaccharide composed of varying ratios of β-D mannuronic acid and its C5 epimer α-L-guluronic acid linked together by β-1,4 - glycosidic bond. It has wide range of industrial applications particularly in food sector as a viscosifier, stabilizer, thickener, emulsifier, gelling and water binding agent. Commercial alginate is extracted from brown algae but due to variation in composition of biopolymer isolated from species of different locations, there is growing interest in bacterial alginate.
At present two strains of bacteria are reported to produce alginate, Pseudomonas and Azotobacter. Hence present study was designed to produce alginate by Azotobacter vinelandii utilizing cheap substrates to save the foreign exchange. To achieve the goal, different physio-chemical parameters were optimized to have hyper-production of alginate through submerged fermentation. Different agricultural wastes like wheat bran, rice polishing and molasses were utilized as substrates through fermentation with Azotobacter vinelandii.On fermentation of 7.5% (w/v) wheat bran by A.vinelandii, maximum alginate production (5.21 g/L) was observed at 48 hours of incubation time with 6% (v/v) inoculum size, pH 7.0, 300C and agitation speed of 200 rpm. Addition of different optimum levels of ionic salts i.e. 1.5% CaCl2 and 2% MgSO4. 7H2O in the growth medium gave significantly (P< 0.05) higher quantity of alginate (6.08 g/L) where as addition of KH2PO4 and NaCl reduced the yield of alginate. Among different nitrogen sources tested, 2% corn steep liquor resulted significantly (P<0.05) higher yield of alginate (7.46 g/L).
The bacterial strain was improved by exposure to physical (UV irradiation) and chemical mutagens (Nitrous acid and ethidium bromide) to obtain more than 90% killing. The survivors were screened for hyper-production of alginate against the wild strain of A.vinelandii using pre-optimized conditions. The highest alginate production (13.8 g/L) was obtained by the ethidium bromide treated strain (EtBr-02). The mutant strain was used for optimization of fermentation parameters. The maximum concentration of alginate (15.61 g/L) was obtained by utilizing 10% (w/v) wheat bran, 8% (v/v) inoculum at 48 hours of incubation, pH 7.0, 300C and an agitation speed of 200 rpm. Inclusion of 2.5% cornsteep liquor raised the alginate concentration to 15.8 g/L.
Batch fermenter studies were carried out in 2 L fermenter with working volume of 1.5 L using the mutant strain A.vinelandii, EtBr-02. Optimization of process parameters like agitation, aeration and pH in the fermenter showed that maximum alginate (16.8 g/L) was achieved at 300 rpm, 2.5 vvm aeration and controlled pH condition at 32 hours of incubation time.
The alginate produced was identified by FTIR spectrum after precipitation. The purity of alginate was estimated by HPLC against the standard alginic acid from Sigma-Aldrich and was found to be 98% pure. The alginate produced was used at 3% concentration for immobilization of yeast cells. Immobilized and free cells were compared for ethanol production using 10% sucrose as the carbon source in fermentation medium. The maximum amount of ethanol obtained was from free cells i.e. 38 g/L whereas immobilized cells produced 32.5 g/L ethanol. The advantage of immobilization is that beads can be reused in eight sequential fermentation cycles of 10 h each. Thus a cheap and practical bioprocess of alginate production was developed, that can be exploited commercially to save foreign exchange.
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Physical, Chemical and Biological Treatment of Rice Husk to Improve Its Nutrative Value
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Publisher: 2015 Dissertation note: Thesis submitted without CD.
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Bio-Conversion of Molasses to Phytase Through Solid State Fermentation With Aspergillus Niger
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Publisher: 2015 Dissertation note: CD Corrupt.
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Biochemical And Homology Analysis Of Jak2 Gene In Canines And Hominidae
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Publisher: 2016 Dissertation note: Cancers are considered to be the most lethal of all diseases known out of which myeloproliferative neoplasms comprise of a very little percentage.The frequency of these disorders is known in human beings and a lot of work has been done on humans. But there is a lot of scope for research on this area in canines. As dogs were found to have strong homology with human beings, we compared canine cJAK2 exon 13 sequence with the humanhJAK2 exon 13 and found 96 % homology. Mutations in JAK2 gene are well known to cause three types of disorders i.e. polycythemia vera caused by a well-known point mutation in exon 14 causing substitution of valine for phenylalanine in JH2 domain of the protein.Essential thrombocythemia and idiopathic myelofibrosis may also be caused by this mutation but similar clinical conditions arise without the presence of this mutation. Studies have revealed that other point mutations such as deletion, addition or substitution are also responsible for these disorders.
JAK2 is an intracellular protein which performs phosphorylation of STAT molecules upon their activation. Although the whole protein in its good state is important for its function but the two domains JH1 and JH2 are vital. JH1 domain acts as a tyrosine kinase enzyme and its activity is controlled by JH2 domain also known as pseudo tyrosine kinase domain. Any mutation in these domains leads to protein conformation defect and thus prevents its performance. Besides V617F mutation, other mutations are being discovered in this part of gene. Researchers have found mutations in exon 12, 13 and 15 that have been found to be involved in development of myeloproliferative neoplasms in different cases of patients.
Blood picture do not reveal any direct clue except for increased erythrocytes alone or along with other cells like increased platelets. Therefore blood indices are not reliable parameter to indicate the type of mutation involved in these disorders. Also LDH and EPO levels are not correlated with the disorder. Although EPO test must be done to exclude the possibility of secondary PV and erythropoiesis.
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