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Dna Based Characterization Of Triacyl Glycerol Lipase Gene From Geobacillus Sp. Sbs-4s

By: Maheen Aslam (2012-VA-803) | Dr. Muhammed Tayyab.
Contributor(s): Ms. Asma Waris | Dr. Sehrish Firyal.
Material type: materialTypeLabelBookPublisher: 2014Description: 56p.Subject(s): Department of Biochemistry and Biotechnology (IBBT)DDC classification: 2234-T 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 Introduction 3 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 Introduction 4 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 Introduction 5 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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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
Introduction
3
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
Introduction
4
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
Introduction
5
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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