1.
Production Of Laccase By Immobilized White Rot Fungi And Its Application For The Decolorization Of Textile Effluent Dyes
by Iqra Ghulam Rasool (2012-VA-579) | Ms. Faiza Masood | Dr. Muhammad Tayyab | Prof. Dr. Tahir Yaqub.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation 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 1.10.3.2) 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.
Availability: Items available for loan: UVAS Library [Call number: 2208,T] (1).
2.
DNA Based Characterization Of Protease Gene From Geobacillussp.Sbs-4s
by Anam Shabbir (2012-VA-608) | Dr. Muhammad Tayyab | Ms. Huma Mujahid | Prof. Dr. Tahir Yaqub.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation 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).
Availability: Items available for loan: UVAS Library [Call number: 2242-T] (1).
