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Production, Purification And Characterization Of Exoglucanase By Arachniotus Rubber

By: Saira Bano (2012-VA-602) | Ms. Shagufta Saeed.
Contributor(s): Ms. Faiza Masood | Dr. Tahir Yaqoob.
Material type: materialTypeLabelBookPublisher: 2015Description: 65p.Subject(s): Department of BiochemistryDDC classification: 2257-T 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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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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