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
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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
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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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Detoxification Of Aflatoxins Using Different Organic Acids
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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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