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INDUSTRIAL IMPORTANCE OF LIPASES Lipases are a class of enzymes which catalyze the hydrolysis of long chain triglycerides and constitute the most important group of biocatalysts for biotechnological applications. Lipolytic enzymes are currently attracting an enormous attention because of their biotechnological potential. They constitute the most important group of biocatalysts for biotechnological applications further more, novel biotechnological applications have been successfully established using lipases for the synthesis of biopolymers and biodiesel, the production of enantiopure pharmaceuticals, agro-chemicals, and flavour compounds. The chemo- regio- and enantio-specific behavior of these enzymes caused tremendous interest among scientists and industrialists.The knowledge of lipolytic enzymes in industrial applications is increasing at a rapid and exciting rate. Lipases in organic synthesis For synthetic chemists, the application of lipases as catalysts in organic synthesis is of much advantage. These enzymes can show stereoand regiospecificity, and tolerate organic solvents in the incubation mixture. Both the activity of the enzyme and the identity of the product depend upon the solvents used, which may vary from aqueous buffer systems through biphasic emulsions and microemulsions, to organic solvents. For synthetic purposes, crude enzyme preparations are often convenient. Because the catalyst is always an expensive factor in a chemical reaction, strategies for enzyme recycling are being developed. Synthetic strategies involving microbial lipases can be used to prepare molecules of high positional and configurational purity. Lipases can be used to create biologically active
Lipase catalyses the hydrolysis of triglycerides at the waterlipid interface. Under given experimental conditions, the amount of water in the reaction mixture will determine the direction of the lipase-catalysed reac-tion. While in absence of water or its presence in trace quantities esterification and transesterification are favoured; in presence of excess water hydrolysis occurs.
Medical and pharmaceutical application Enantioselective interesterification and transesterification have great significance in pharmaceutical for selective acylation and deacylation (Stinson, 1995). Lipases are important in application in pharmaceuticals in transesterificatrion and hydrolysis reaction. They play a prime role in production of specialty lipids and digestive aids (Vulfson, 1994). The alteration of temperature during the esterification reaction drastically changes the
Domestic applications The most commercially important field of application for hydrolytic lipases is their addition to detergents, which are used mainly in household and industrial laundry and in household dishwashers. Godfrey and West (1996) reported that about 1000 t of lipases are sold every year in the area of detergents. The use of cold active lipase in the formulation of detergents would be of great advantage for cold washing that would reduce the energy consumption and wear and tear of textile fibers (Feller and Gerday, 2003). The industrial dehairing of hides and skin at low temperature using psychrophilic protease or keratinase would not only save energy but also reduce the impacts of toxic chemicals used in dehairing. Enzymes can reduce the environmental load of detergent products since they save energy by enabling a lower wash temperature to be used; allow the content of other often less desirable chemicals in detergents. Addition of cold active lipsase in detergent become biodegradable, leaving no harmful residues, have no negative impact on sewage treatment processes and do not present a risk to aquatic life. Commercial preparations used for the desizing of denim and other cotton fabrics contain both alpha amylase and lipase enzymes. Lipases are stable in detergents containing protease and activated bleach systems.
Lipases in dairy industry Lipases are used extensively in the dairy industry for the hydrolysis of milk fat. Current applications include flavour enhancement of cheese, acceleration of cheese ripening, manufacture of cheese-like products,
Lipases in detergents The usage of enzymes in washing powders still remains the single biggest market for industrial enzymes. The world-wide trend towards lower laundering temperatures has led to much higher demand for household detergent formulations. Recent intensive screening programmes, followed by genetic manipulations, have resulted in the introduction of several suitable preparations, for example, Novo Nordisks Lipolase (Humicola lipase expressed in Aspergillus oryzae).
Lipases in synthesis of triglycerides The commercial value of fats depends on the fatty acid composition within their structure. A typical example of a high-value asymmetric triglyceride mixture is cocoa butter. The potential of 1,3regiospecific lipases for the manufacture of cocoa-butter substitutes was clearly recognized by Unilever and Fuji Oil. Comprehensive reviews on this technology, including the analysis of the product composition, are available. In principle, the same approach is applicable to the synthesis of many other structured triglycerides possessing valuable dietic or nutritional properties, for example, human milk fat. This triglyceride and functionally similar fats are readily obtained by acidolysis of palm oil fractions which are rich in 2-palmitoyl glyceride with unsaturated fatty acid(s). Acidolysis, catalysed by 1,3-specific
Lipases in synthesis of surfactants Polyglycerol and carbohydrate fatty acid esters are widely used as industrial detergents and as emulsifiers in a great variety of food formulations (low-fat spreads, sauces, ice-creams, mayonnaises). Enzymic synthesis of functionally similar surfactants has been carried out at moderate temperature (6080C) with excellent regioselectivity. Adelhorst etal. have carried solvent-free esterification of simple alkyl-glycosides using molten fatty acids and immobilized Candida antarctica lipase. Fregapane etal. obtained mono- and diesters of monosaccharides in high yields, using sugar acetals as starting materials. Lipase from A. terreus synthesizes a biosurfactant by transesterification between natural oils and sugar alcohols. Lipases may also replace phospholipases lipase has in the production used for of the lysophospholipids. Mucor miehei been
transesterification of phospholipid in a range of primary- and secondary alcohols. Lipases may also be useful in the synthesis of a whole range of amphoteric bio-degradable surfactants, namely amino acid-based esters and amides.
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Lipases in pharmaceuticals and agrochemicals The utility of lipases in the preparation of chiral synthons is well recognized and documented. Several processes have recently been commercialized which have been described by Sainz-Diaz etal. and Davis etal.. The resolution of 2-halopropionic acids, the starting materials for the synthesis of phenoxypropionate herbicides, is a process based on the selective esterification of (S)-isomers with butanol, which is catalysed by porcine pancreatic lipase in anhydrous hexane. Another impressive example of the commercial application of lipases in the resolution of racemic mixtures is the hydrolysis of epoxyester alcohols. The reaction products, (R)-glycidyl esters and (R)-glycidol are readily converted to (R)- and (S)-glycidyltosylates which are attractive intermediates for the preparation of optically active blockers and a wide range of other products. A similar technology has been commercialized to produce 2(R),3(S)-methylmethoxyphenyl glycidate, the key intermediate in the manufacture of the optically pure cardiovascular drug Diltiazem. Lipases have applications as industrial catalysts for the resolution of racemic alcohols in the preparation of some prostaglandins, steroids, and carbocyclic nucleoside analogues. Regioselective modification of polyfunctional organic compounds is yet another rapidly expanding area of lipase application, particularly in the field of AIDS treatment. Lipases from A. carneus and A. terreus show chemo- and regiospecificity in the hydrolysis of
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Bioconversions in aqueous media A typical lipase-catalysed reaction in aqueous media is ester hydrolysis. This enzymic conversion can be used for the synthesis of triglycerides as shown for the preparation of platelet-activating factor. Another application of the hydrolytic specificity of lipases is the partial hydrolysis of triglycerides to di- and monoglycerides in the food industry, where di- and monoglycerides serve as biocompatible emulsifiers and food additives. These and other applications of lipases in industry and research have been discussed in the review by Iwai and Tsujisaka.
Bioconversions in organic media The synthetic potential of lipases in organic solvents has been widely recognized and is well documented, particularly on the basis of activity of lipases in organic solvents containing low water content. The main application of lipases in organic chemistry is the resolution of enantiomeric compounds, making use of the enantioselectivity of these enzymes. The use of organic solvents for lipase-catalysed resolutions has four main advantages in comparison with water as the solvent: (i) racemic mixtures of alcohols or acids need not be esterified before resolution into enantiomers, (ii) these enzymes are more stable in organic solvents than in water, (iii) lipases used need not be immobilized for recovery, owing to their insolubility in organic solvents; they can be collected by filtration in their active state, and (iv) substrates and products may be unstable in aqueous solution. In this case, reaction in organic solvents is essential for formation and isolation of the
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Applications
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Lipase on various environmental pollutants There are number of uses of the cold active enzymes, presently it is conceivable that they could be used for environmental bioremediation
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Isolation and molecular confirmation of lipase producing organisms from oil spills The aim of this study is to characterize the microbes in genetic level. Lipases are the very commercially important enzymes which have the wide application in degradation studies. Once if the microorganism confirmed in molecular level, based on modifying the cultural characteristics we can increase the production of lipases enzyme expression. Its very important for knowing the cultural characteristics of organisms, because the cultural characteristics and expression of desired products varies from organism to organisms. Here we aimed to isolate and molecular characterize the microbes which have the lipids degrading activity. By using the PCR based amplification of 16s rDNA gene we can classify the microbes in species and strain level. The species and strain level information provides the much information about the organisms and its lipase production. After getting the much information about the organism we used to assay it both biological and enzyme kinetic assays for lipase production.
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Bacterial lipases A relatively smaller number of bacterial lipases have been well studied compared to plant and fungal lipases. Bacterial lipases are glycoproteins, but some extracellular bacterial lipases are lipoproteins. Winkler et al. reported that enzyme production in most of the bacteria is affected by certain polysaccharides. Most of the bacterial lipases reported so
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Pseudomonas
Staphylococcus
Chromobacterium sp. have been exploited for the production of lipases. Staphylococcal lipases are lipoprotein in nature. Lipases purified from S.aureus and S. hyicus show molecular weights ranging between 3446 kDa. They are stimulated by Ca++ and inhibited by EDTA. The optimum pH varies between 7.5 and 9.0. The lipase genes from S. hyicus and S. aureus have been cloned, sequenced, and compared with other lipases. This revealed two conserved domains separated by 100 amino acids which are likely to form active site. Putative active site residues around His 269 and Ser 369 of the S. hyicus lipase are highly conserved in the two S. aureus lipases and in several eukaryotic lipases. Lipases from different species of Psuedonomas were purified by acidification of the culture supernatant, ammonium sulphate precipitation, sepharose CL-6B chromatography, and isoelectric focussing using CHAPS 47. The purified lipase of P. fragi, P. fluorescens, and P. aeruginosa were monomeric with molecular weight of 33 kDa, 45 kDa, and 29 kDa, respectively. The lipase was inhibited by Zn++, Fe++, and Al+++ and activa-ted by Ca++ (ref. 48). The lipase gene of P. fragi has been cloned and sequenced.
Fungal lipases Fungal lipases have been studied (Lawrence etal., in 1950) have presented comprehensive reviews. These lipases are being exploited due to their low cost of extraction, thermal and pH stability, substrate specificity, and activity in organic solvents. The chief producers of commercial lipases are Aspergillus niger, Candida cylindracea, Humicola lanuginosa, Mucor miehei, Rhizopus arrhizus, R. delemar, R. japonicus, R. niveus and R. oryzae.
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Optimization parameters Varying the following parameters one at a time with optimization of fermentation media. The parameters varied were i. incubation period, ii. temperature, iii. pH and iv. lipid substrates. The influence of growth period on biomass and lipase production of Bacillus sps (B1 B5) was assessed by culturing it on production media for different time duration (24, 48 and 72 h). The effect of medium pH on lipase production was assessed at different pH ranged from 4 to 9 by culturing the isolated Bacillus sps (B1 B5) on medium with different temperatures (27, 37 and 47C) was also assessed.
Optimization of growth parameters A number of reports exist on influences of various environmental factors such as temperature, pH, nitrogen, carbon and lipid sources, agitation, and dissolved oxygen concentration on lipase production. Lipase production is generally stimulated by lipids. The lipase activity steadily increases to a peak and declines. Lipase production is usually coordinated with, and dependant on the availability of triglycerides. Besides this, free fatty acids, hydrolysable esters, bile salts, and glycerol also stimulate lipase production. High production of lipase in case of P. fragi occurs in peptone-supplemented medium, although different peptones vary in their effective-ness. Though Pseudomonas sp. grow in a basal medium with ammonium sulphate, glucose, citrate or pyruvate, it requires an organic nitrogen source for lipase production. A mixture of arginine, lysine and glutamic acid in medium was observed to be effective for lipase production. A strain of Penicillium
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Lipase purification Lipases have been purified from animal, plant, fungal and bacterial sources by different methods involving ammonium sulphate precipitation, gel filtration, and ion exchange chromatography. In recent years, affinity chromatographic techniques have come into use as this technique decreases the number of steps necessary for lipase purification as well as increases specificity. Currently, reversed-micellar and two-phase systems, membrane processes, and immunopurification are being used for purification of lipases. Lipase assay
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Temperature optima and thermal stability The pancreatic lipases loose activity on storage at temperatures above 40C, but some microbial lipases are more resistant to heat inactivation. While lipases of A. niger, R. japonicus, and C. viscosum are stable at 50C, lipases of thermotolerant H. lanuginosa and P. sp. nitroreducens are stable at 60C and 70C (ref. 141), respectively. C. gigantea lipase had half life for inactivation of 35.7, 46.4 and 22.9 min. at 45C, 50C and 55C (ref. 12), similar to lipases of R. japonicus. In our laboratory, we observed that purified lipase from A. terreus retained 100% of its activity at 60C after 24 h. But, the maximum activities of C. gigantea and other lipases from mesophiles were at 3035C (ref. 144). Thermophilic bacterial lipases obtained from Icelandic hot spring showed higher lipase activity at 40 to 60C (ref. 145).
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Substrate specificity Specificity of lipases is controlled by the molecular properties of the enzyme, structure of the substrate, and factors affecting binding of the enzyme to the substrate. Substrate specificity of lipases is often crucial to their application for analytical and industrial purposes. Specificity is shown both with respect to either fatty acyl or alcohol parts of their substrates. Many microbes produce two or more extracellular lipases with different fatty acid specificities. Tributyrin is hydrolysed slowly by some microbial lipases. In contrast, M. miehei lipase preferentially releases butyric acid from milk fat especially at low pH (ref 150). Geotrichum candidum produces a lipase, which shows pronounced specificity for the hydrolysis of esters of a particular type of long-chain fatty acid. Substrate specificity of this lipase has been summarized by Jensen, Jensen and Pitas, and Macrae. Lipases show both regio- and stereospecificity with respect to the alcohol moeity of their substrates. Lipases can be divided into two groups on the basis of the regiospecificity exhibited acylglycerol substrates. Lipases in the first group catalyse the complete breakdown of triacylglycerol to glycerol and free fatty acids together with diacylglycerols and monoacylglycerol as intermediates in the reaction. These intermediates do not accumulate since they are hydrolysed more rapidly than the triacylglycerol. Examples of the first group of lipases include lipase from C. cylindracea. The second group of lipases release fatty acids regiospecifically from the outer 1 and 3 positions of acylglycerols. These lipases hydrolyse triacylglycerol to give free fatty acids, 1,2-diacylglycerols, and 2monoacylglycerol. Many extracellular microbial lipases, such as those from A. niger and R. arrhizus, show 1,3-(regio)-specificity. Lipases excreted by R. japonicus, M. miehei, H. lanuginosa, C. viscosum, and P. fluorescens are also 1,3-(regio)-specific. Till date, there are no authentic reports of lipases which
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Rhizopus arrhizus lipase of Novo Nordisk converts selectively geraniol into its acetate ester, when a mixture of geraniol and nerol is subjected to esterification in hexane. Thus, a reaction between 0.1 M each of geraniol and nerol mixed with acetic acid in hexane for 2 days produced 80% of the total esters as geranyl acetate, and the remaining 20% as nerol acetate. Laboratory-scale studies on an enzyme dose of 1% w/v for the synthesis of geranyl butyrate gave a conversion of almost 100% after 1.5 days at 30C when 0.1 M geraniol and 0.1 M butyric acid were used as reactants in hexane.
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Lipase degradation current research going for lipase Industrial significance lipases have been widely studied with main focus on enzyme action, kinetics, sequencing and cloning of lipase genes and structural characterization but the studies on using RSM for validating and optimizing nutritional parameters for R. oryzae have been left relatively unexplored. Development of novel bioprocess is based on the
advancements in optimization of process which is a tedious and time taking process because effects of multivariable process parameters. For such purposes screening nutritional factors of importance initially carried out and selected factors are then optimized on priority basis by different available techniques. Response surface methodology (RSM) is an advanced tool now a days commonly applied involves three factorial designs giving number of input (independent) factors and their corresponding relationship between one or more measured dependent responses. RSM is advantageous over conventional methods available and it includes less experiment numbers, its suitability for multiple factor experiments and search for common relationship between various factors towards finding the most suitable production conditions for the bioprocess and forecast response . In this, linear or quadratic effects of experimental variables construct contour plots and a model equation fitting the experimental data. This facilitates the determination of optimum value of factors under investigation and prediction of response
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Current status of lipase research in India Researches on microbial lipases in India date back to late seventies when a few reports on screening and production of lipase from a few fungi and bacteria appeared. The initial emphasis on screening exercises was followed by process optimization for maximum lipase production. Scientists at the National Diary Research Institute, Karnal113,115,117 investigated physicochemical conditions of lipases produced by M. racemosus, A. wentii, and P. chrysogenum. In 1981, one group highlighted the lipolytic activity of thermophilic fungi of paddy straw compost295. Systematic screening strategies were employed by Bhaduria296. This study reported A. niger, A. flavus, A. fumigatus and Penicillium glaucum as the potential lipase producers isolated from the kernels of chironji and walnut.
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Future outlook In spite of the importance of cold active lipases, studies on the mechanisms of production of microbial lipases and the role of lipidic substances used as inducers in lipase production are scanty. Cold active lipases represent an extremely versatile group of bacterial extracellular
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In nature, the lipases available from various sources have considerable variation in their reaction specificities: this property is generally referred as enzyme specificity. Thus, from the fatty acid side, some lipases have affinity for short-chain fatty acids (acetic, butyric, capric, caproic, caprylic, etc.), some have preference for unsaturated fatty acids (oleic, linoleic, linolenic, etc.), while many others are nonspecific and randomly split the fatty acids from the triglycerides. From the glycerol side of the triglycerides, the lipases often show positional specificity and attack the fatty acids at 1 or 3 carbon position of glycerol or at both the positions but not the fatty acid at the 2 position of the glycerol molecule. However, through random acyl migration, the 2-fatty acid monoglyceride undergoes rearrangement pushing the fatty acid to the 1 or 3 position of the glycerol molecule; as acyl migration is a slow process and as the available lipases do not act on glycerol 2-mono fatty acid esters, the hydrolysis slows down and awaits the acyl migration to complete for enabling the lipase to attack the glyceride at the 1 and/or the 3 position. Interestingly, lipases function at the oilwater interface (Figure 2). The amount of oil available at the interface determines the activity of the lipases46. This interface area can be increased substantially to its
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Enzyme assay and enzyme activity. The lipase triacylglycerol acylhydrolases, EC 3.1.1.3) assay was carried out by using modified lipase assay media (Peptone: 15g, NaCl: 5g, CaCl2: 1g, Tween 20: 10mL, Agar: 15g, Water: 1000 mL; pH: 7.0). The lipase activity was detected due to occurrence of a zone of clearance around the colony and subsequent formation of white precipitate of calcium monolaurate around the colony [25-26].
Enzyme activity was measured by titrimetric method. The oilwater emulsion and enzyme extract in a ratio 0.1 mL : 9.9 mL : 1 mL was titrated at constant temperature against 0.1 N NaOH using Phenolphthalein as indicator. A blank (9.9mL water, 0.1mL Tween20, 1mL sterilized broth media) was previously run to find the standard deduction in titer value. The principle behind this method is that Lipase release free fatty acid from the Tween 20 and cause acidic pH, which is neutralized by titrating with NaOH in constantly stirring vessel. Each reading was taken in triplicate. The activity is measured as amount of enzyme required liberating one micromole equivalent fatty acid per mL min-1.
Lipid optimization by substrates By using different substrates sources such as olive oil, coconut oil and sunflower oil, their effect on lipase production by the selected Bacillus
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Enzyme assay Isolated Bacillus sps (B1 B5) were assayed for extracellular lipase production using titrimetric method (Sadasivam and Manickam, 1996).
Lipase activity One unit of lipase activity was defined as the amount of enzyme releasing one mole of free fatty acid in one minute under standard assay condition. Volume of alkali consumed x Normality of NaOH Lipase activity = ( g/ml/min) Time of incubation x Volume of enzyme solution Mohan et al. 2729
Table 1. Biochemical identification of lipase-producing Bacillus strains from oil mill waste. Test Results Catalase + V-P +
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1a. At pH 4.0 1b. At pH 5.0 1c. At pH 6.0 1d. At pH 7.0 1e. At pH 8.0 1f. At pH 9.0 Figure 1. Effect of coconut oil on lipase activity of Bacillus sp. (B1 - B5) cultured for different time intervals (24 72 h) at different medium pH 4 9. mum medium pH (7.0), (Table 2) (Figure 2) Two-way analysis of variance for the data on lipase production indicated that the influence of different Bacillus strains was statistically more significant (P < 0.01) than the independent influence (P < 0.05) of medium pH. The result of the effect of medium pH on the tested olive oil indicated that the lipase production was maximum (0.026 g/ml/min) at pH 7. In low 4, 5, and also at high 8 and 9 medium pH the lipase activity was less for all the tested bacillus strains similar to that recorded in the first experiment has also the bacillus strain B5 showed maximum lipase production at the optimum me.
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GastroZyme Supports healing and repair of the gastrointestinal tract Helps control heartburn, ulcers, diarrhea, colitis, gastritis, and Crohn's Disease. LypoZyme Helps control cholesterol Aids in proper digestion of fats and supports overall cardiovascular health. Also beneficial for those with gall bladder problems or who have had their gall bladder removed.
MasterZyme Supports the endocrine system and controls hormonal imbalances Can help regulate or balance blood sugar levels, PMS, and thyroid disorder. MasterZyme contains animal glandulars and may not be appropriate for vegetarians. ReleaseZyme The natural way to regularity Formulated to help reduce the discomfort of constipation and assist with elimination. RepairZyme Supports muscle, skeletal, and tissue health Blend of enzymes, herbs, and minerals provides excellent post-workout supplement.
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Super CellZyme All-natural whole food supplement Contains enzymes along with all 45 vitamins and minerals necessary for overall cell health
Mechanism of lipid degradation enzymes Lipases are not involved in any anabolic processes. Since this enzyme acts at the oilwater interface, it can be used as a catalyst for the preparation of industrially important compounds. Lipases catalyse the hydrolysis of triglycerides into diglycerides, monoglycerides, glycerol and fatty acids, and under certain conditions the reverse reaction leads to esterification and formation of glycerides from glycerol and fatty acids. As lipases act on ester bonds, they have been used in fat splitting, interesterification (transesterification), development of different flavours in cheese, improving pallatability of beef fat for making dog food, etc. A current application involves using lipases in water-deficient organic solvents for synthesizing different value-added esters from organic acids and alcohols. Lipases which are stable and work at alkaline pH, say 8 to 11, which are usually the suitable wash conditions for enzymated-detergent powders and liquids, have also been found, and these hold good potential for use in the detergent industry.
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Figure 2. Lipolytic reaction at the oilwater interface. Fungal lipases which degrade lipids from palm oil were investigated by Turner (cited by Lazer and Schroder, and listed by Sztajer and Zboinska). Among Mucorales, the lipolytic enzymes of the moulds Mucor hiemalis, M. miehei, M. lipolyticus, M. pusillus, Rhizopus japonicus, R. arrhizus, R. delemar. R. nigricans, R. nodosus, R. microsporus, and R. chinesis have been studied in great detail50. The thermophilic M. pusillus is well known as a producer of thermostable extracellular lipase. From a lipaseproducing strain of M. miehei, two isoenzymes with slightly different isoelectric points but a high degree of antigenic identity could be isolated92. A lipase of M. miehei, immobilized on a resin (Lipozyme TM) has been commercialized by Novo Industries. Due to the 1,3-(regio)-specificity of Rhizopus, lipases that are especially suited for the conversion of triglycerides to their corresponding monoglycerides, and interesterification reactions of fats and oils, have food and pharmaceutical applications. R. japonicus lipase has been used to produce hard butter suitable for chocolate manufacture by interesterification of palm oil with methyl stearate54. The lipases (40 to 45 kDa) of various Rhizopus species show maximum activity towards medium-chain fatty acids (C8C10). In case of R. delemar, extracellular94 and intracellular95 lipase isoenzymes have been isolated.
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centropunctatum, Arthrobacter, Bacillus hexacarbovorum, Candida lipolytica, Candida tropicalis, Candida utilis, Candida parasilosis, Candida guilliermondii, Candida rugosa, Trichosporan cutaneum, Aureobasidium pullulans, The preferred carrier materials when used include clays such as kaolin, zeolites and other microporous silicaalumina materials, silica gels, vermiculities and perlites, and particularly these in hydrophillic forms. The operable materials, however, include microporous materials of the class into which microorganisms and nutrients or microorganisms alone can be absorbed and freeze-dried, and which will subsequently adsorb oil so as to bring this oil into a close relationship with the microorganisms for digestion. A particularly preferred material is vermiculite and ideally an exfoliated vermiculite. Vermiculite as used herein refers to the group of rock-forming mineral species characterized by a layer of latticized structure 10A which the silicatelayer units have a thickness of approximately 10a (Angstrom units). The main elements present in the layer are magnesium, aluminum, silica, iron and oxygen with the latter being separated by one or two sheets of water molecules associated with cations, such as magnesium, calcium, sodium and hydrogen. The layers have considerably lateral extent relative to the thickness of the basic 10 Angstrom-unit layer. Further, vermiculite belongs to the phyllosilicate group, which are characterized by the presence of Si--O sheets formed by the linkage of three corners of each SiO The type formula is Am(B X )
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tetrahedron to
neighbors so that each tetrahedron has three shared and one free oxygen.
2 5 n
plates and a hardness of one. The term "vermiculite" as used herein therefore
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The microorganisms act on the film of oil and on the oil so as to break it up into smaller globules in a period of over four hours or less, the globule particle size in diameter decreases from 200 microns to approximately 20 to 40 microns. After the globules reach the size of 6 to 20 microns, the oxidation products are realized in that the hydroxides, alcohols, aldehydes, and acids are formed by the microorganisms, then die. When the assimiable nutrients are exhausted, there is in fact, no contamination of the sea water by
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It has also been found that when sand, rocks, concrete, metals, plastics and so forth are treated with a water slurry of the above-described microorganisms such materials are not readily contaminated by oil. It was found that plastic in contact with oil acquires a rather tenacious oil film in the absence of treatment with the described microorganisms. However, after the plastic is treated with a water slurry of the microorganisms by dipping the plastic into the slurry followed by draining, oil no longer stick to the plastic. Similar tests were conducted on metals, gears, rocks and sand.
The concentration of the viable cells per mole of slurry ranges from 10 5 to 10 8 . These microorganisms also can be employed for recovering petroleum oil from oil-bearing earth formations and can be useful in the secondary and tertiary recovery of petroleum oil from such formations. The process of this invention of preparing freeze-dried cultures of selected bacterial species of hydrocarbon consuming microorganisms involves growing the mixed population in a suitable aqueous medium containing high inorganic phosphate concentration and a suitable easily assimiable inorganic nitrogen source, trace metals and a hydrocarbon containing C 8 to C
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carbons as the
only carbon source. Generally the mixed culture is produced in gram quantities in 20 to 50 liter quantities under vigorously aerated and agitated conditions. After the cell density has reached the desirable concentration, it is then preferably centrifuged to collect the cells and lyophilized or directly freeze-dried or adsorbed on to a suitable carrier selected from the inert supports described in this invention. The latter procedure is preferred since the desired amounts of nutrients are already present in this spent broth. In use, the preparation is generally reconstituted in suitable aqueous medium
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Table VII sets forth the viability of the bacterial pool during storage at -18C prior to lyophylization and also the viability of lyophilized preparations after a week of storage at ambient temperatures. In addition, Table VII also sets forth the ability of the lyophylized preparations to disperse No. 4H.O heating oil in laboratory TABLE ____________________________________________________________ ______________ VIABILITY OF LYOPHYLIZED BACTERIAL POOL EM-30 AND INDIVIDUAL ISOLATES DAYS* VIABILITY % ** TIME FOR 100% DISPERSION LOT No. TYPE STORED CELLS/GRAM VIABILITY OF No. 4H.O HOURS ____________________________________________________________ ______________ 1. EM-30 5 1 10 10 1 1 Bacterial pool 2. EM-30 12 3 10 10 3 1 3. EM-30 20 2 10
8 10
tests. VII
0.1 1 6. Arthrobacter 3 4 10
10 9 4.0 0.5 ATCC No. 21909 8. Achromobacter 5 5 10 9 0.5 0.75 ATCC. No. ____________________________________________________________ ______________ * Wet cell paste stored at -18C prior to lyophylization. ** Viability tested after a week of storage at ambient temperatures. By employing the microorganisms in the manner shown and described hereinabove they produce a solution to the problems of not getting a large enough quantity into an oil slick in order to effectively accomplish its dispersion and degradation. The above procedure is inexpensive to prepare,
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Mechanism of Microbial Oil Dispersion In order to determine the mechanism(s) adopted by these hydrocarbonophilic microorganisms a series of experiments were conducted. The affinity of the bacterial pool for oil and the initial changes in the physical nature of the oil were determined by examining a sample of oil from control and reaction flasks at 0, 10 and 20 minutes under phase contrast microscope. The results recorded are that initially the single oil globule is surrounded by a thin film of water and the oil-water interface is coated by bacteria. After the 20 minutes of exposure to the microorganisms the oil droplets are further reduced in size and after further incubation the continuous film forming property of the oil is lost and the inherent property of the oil to adhere to the solid surfaces such as glass, paper, wood and polyethylene, polypropylene, sand and rocks. Finally, the system becomes homogeneous and all oil washes away.
EXAMPLE 4: To determine whether the property of the bacterial pool herein referred to an EM-30 to disperse and degrade No. 4 Heating oil was unique, additional tests were conducted using several typical crude oils and two oil blends, heating oils No. 4 and No. 6. The rate of dispersion of total crudes and heating oils was determined by inoculating calculated amounts of EM-30 into a Basal Salts aqueous medium supplemented with 1% V/V of the crude oils and heating oils listed in Table X. The inoculated flasks were incubated on a
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The Effect of Temperature, pH, N and P on Dispersion Rates The effect of temperature on the rate of dispersion of No. 4 heating oil by EM-30 was determined by incubating at temperatures from +5 C to 40C with a 5C increment. Samples from each reaction flask were withdrawn at 10-minute intervals and percent dispersion of the oil was determined spectrophotometrically. The results are presented in Table XIV. At higher temperatures, the dispersion appears to be rapid. However, the optimum temperature range appears to be around 25-35C. It becomes difficult to define dispersion at temperatures lower than 10C because of the concomitant oil viscosity decrease and the slowing down of the metabolism of the microorganisms. However, there appears to be no significant change in the dispersion rates between 5 and 15C. These data suggest that the initial dispersion is dependent upon the particle size and amount of dispersant associated with the microbes. Apparently, the rate of initial dispersion by the microbial dispersant is independent of the temperature, however, further increase in the percent dispersion is closely associated with the optimum temperature for the biosynthesis of the dispersant. Additional experiments suggested that the pH of the reaction mixture is not critical. However, assimilable sources of nitrogen and phosphate and trace metals are necessary for the increase in biomass, and associated activities such as dispersion, degradation and transformation of the oil. Similarly, the rate of oil dispersion by microbes is dependent upon the strains which are capable of utilizing some fractions of crude oil as sources of carbon for the growth and for the biosynthesis of the dispersant, size,
52
Effect of nitrogen source To increase lipase production by the fungus and considering that high nitrogen concentrations are typically used for the production of fungal lipases (21), medium A was supplemented with various organic and inorganic nitrogen sources (Table 1). Supplementation was most effective in the case of addition of inorganic salts, with the highest lipolytic activity (12 U/mL) obtained after 72 h with medium F (Fig. 1). However, with KNO3 as the sole source of nitrogen (medium G) the maximum lipolytic activity was relatively low (3.5 U/mL after 72 h). Since growth and lipase production did occur on this medium, KNO3 was included in the calculation of the C/N ratio for all media. Both of the media supplemented only with organic sources (C and D) performed similarly to medium A. In medium E, which contains yeast extract in addition to ammonium sulfate, the lipase production was high (9 U/mL). The specific activity was highest for the medium containing only inorganic nitrogen sources (297 U/mg, medium F), followed by the medium containing the combination of inorganic salts and yeast extract (130 U/mg, medium E). The specific activities attained with the other media were relatively low (59, 10 and 25 U/mg for media A, C and D, respectively). The lower specific activity values obtained for the fungus cultivated in media containing polypeptides could be due to the release of other proteins into the medium. Given that a higher lipolytic activity was obtained in a relatively short fermentation period (72 h) in a medium containing only ammonium sulfate and
53
Effect of carbon source Vegetable oils such as soybean, corn, sunflower, olive, palm and cottonseed oils, amongst others, are cited as inducers of lipase production, comprising, at times, the sole source of carbon in the medium (306-309). To evaluate the effect of carbon source on the production of lipase by P. aurantiogriseum, cultures were done with the addition of corn, soybean, sunflower and olive oils, each separately. For comparison, a culture was also grown with glucose as the sole carbon source. Considering 72 h of fermentation time, the lipolytic activities in the culture broth were approximately 5 U/mL when sunflower, corn and soybean oils were used, compared to an activity of 12.5 U/mL in the presence of olive oil (Fig. 2). Lipolytic activity was not detected when glucose was the sole carbon source, confirming that the presence of an inducer is necessary for P. aurantiogriseum to produce lipases (306). The maximum specific lipase activities obtained in these experiments were: 192 U/mg after 48 h with olive oil; 114 U/mg after 72 h with soybean oil; 220 U/mg after 112 h with sunflower oil and 138 U/mg after 112 h with corn oil. Therefore, although the volumetric activities were quite different in the presence of the various oils, the maximum specific activity did not vary so greatly. However, the time at which this maximum occurred did vary. Fatty acids present in the greatest proportions in these oils are oleic and linoleic acids. Triglycerides of olive oil and corn oil, which were the two best carbon sources for lipase production, contain 28 and 25 % oleic acid, respectively, while those of sunflower oil and soy oil contain 17 and 13 % oleic acid, respectively. On the other hand, sunflower, soy and corn oils have higher percentages of linoleic acid (51, 27 and 33 %, respectively), while olive oil contains only 3 % linoleic acid. Better lipase production appears therefore to be correlated with a higher content of oleic
54
Effect of the nitrogen source concentration For fungi, relatively high nitrogen concentrations (and therefore lower C/N ratios) are typically required in order to favor the production of lipases over the production of other enzymes (306,311,313). To evaluate the effect of the concentration of the nitrogen source, cultures were done with increasing concentrations of ammonium sulfate while the concentration of olive oil was maintained constant, in such a manner as to give C/N ratios of 1, 2.5, 5 and 10. The maximum lipolytic activity obtained with a C/N ratio of 5 was 17 U/mL and with a C/N ratio of 2.5 it was 13 U/mL (Fig. 3). With an even higher C/N ratio of 10, lipase production was relatively poor. With a C/N ratio of 1 the amount of lipase produced was not significant and the mycelial growth in the flask was very poor. The peak volumetric activity was obtained between 72 and 96 h for all C/N ratios. The peak of the specific activity occurred after 48 h with initial C/N ratios of 2.5 and 5, and between 48 and 72 h with an initial C/N ratio of 10. The highest specific activity of 113 U/mg was obtained with a C/N ratio of 5. Reasonably high values of 103 and 83 U/mg were obtained for C/N ratios of 2.5 and 10, respectively.
Effect of the carbon source concentration The effect of the concentration of the carbon source on lipase production was studied with the addition of different concentrations (0.5, 1, 1.5 and 2.0 %) of olive oil. The concentrations of ammonium sulfate and potassium nitrate were maintained constant. The carbon source concentration has a strong influence on the production of lipase by P. aurantiogriseum, as
55
Various cloning and recombinant studies for lipase studies We were able to use PCR to amplify small regions of lipase genes directly from chromosomal DNA. This was achieved by using highly degenerate consensus primers to the oxyanion hole (Jaeger et al., 1994) and active-site regions of lipase genes to amplify fragments of putative lipases. Using genomic-walking PCR (Morris et al., 1995, 1998), we were able to clone a complete lipase gene from amixed environmentalDNA sample and express it in a heterologous host. At the amino acid level, the lipase gene shares less than 20% similarity with any known lipase gene. Expression of the novel lipase gene was highly toxic to the Escherichia coli host, making it unlikely that the gene could have been cloned using an expression library strategy. We conclude that the PCR method described here is useful for lipase gene prospecting, since it allows cloning of genes from organisms that are unculturable (or yet-to-be-cultured) or possess lipase genes whose expression is highly toxic to the heterologous host.
56
Biomass generation and DNA extraction procedure: Environmental biomass was obtained from an olive-oil enriched percolation conducted at 65 C from a natural New Zealand hot spring. High molecular mass DNA ("15 kb) was extracted from B. pumilis biomass and olive oil percolation biomass as described previously (Morris et al., 1998).
Database searching and computational analysis: Lipase gene sequences were obtained using the Entrez search and retrieval system at the National Center for Biotechnology Information (NCBI). Regions with homology to the lipase gene sequences were obtained using blastp at NCBI. Alignment of the olilipase gene with the Bacillus lipase
57
(http :}}www.bionavigator.com}).
Cloning of lipase gene fragments: DNA was extracted from either a pure bacterial strain or from environmental biomass.
Identification of conserved regions within lipase genes Lipases are serine hydrolases and their active site is composed of three residues : a serine, a histidine and a carboxylate residue (Jaeger et al., 1994). Lipase gene alignments show that conservation around the three active-site residues is low (Jaeger et al., 1994), making them dicult targets for genomic prospecting using PCR. However, such alignments reveal a fourth conserved site at a region corresponding to the lipase oxyanion hole (Jaeger et al., 1994). We examined the amino acid sequences of over 70 lipases from bacteria, archaea and eukaryotes containing the [GA] X-S-X-G sequence characteristic of the active site. Lipases possessing the active-site consensus sequence generally also possessed a region homologous to the oxyanion hole region of the Pseudomonas glumae lipase (Jaeger et al., 1994) located 60108 aa upstream of the active site. These results, combined with analysis of available lipase three-dimensional structures, indicated that the activesite consensus sequence and the oxyanion hole were the most promising sites for the design of PCR primers.
Characterization of conserved regions within lipase genes Although some homology was detected in most lipases at the oxyanion hole and the active-site consensus region, several factors make the
58
59
60
homologous to those found in Group 1 to determine whether the consensus regions identied were unique for lipases. As shown in Table 3, both regions co-exist in several other a}b hydrolase proteins. Some of these proteins match exactly the consensus sequences found in Group 1 lipases. Therefore, it was concluded that the lipase-prospecting primers would also amplify gene fragments from a selected range of other a}b hydrolases, including dihydrolipoamide acetyltransferases, tropinesterases, lysophospholipases and halogen peroxidases. Testing of lipase-prospecting primers on B. pumilis chromosomal DNA PCR products were obtained for most combinations of the lipase-prospecting primers (Fig. 1a). Sequencing of several of these bands indicated that at least six unique sequences were amplied by the lipaseprospecting primers (Fig. 1a). Three of these amplication products showed homology to known or suspected a}b hydrolases (Table 4) and one of these was nearly identical to the equivalent region of the published B. pumilis lipase (accession no. A34992). To conrm that this gene fragment was derived from a functional lipase gene, primers BPUM1F and BPUM1R (Table 1) were designed to amplify the full-length B. pumilis lipase gene. Cloning of the amplied gene product into the pCR vector (Invitrogen) followed by plating onto lipase tester medium (Kouker&Jaeger, 1987) indicated the amplied lipase gene was functional and expressed under control of the lacZ promoter. Sequencing of the full-length gene indicated that it diered from the published B. pumilis gene at 9 aa positions and was identical to the sequence amplied by the lipase-prospecting primers (data not shown). As predicted, the lipase-prospecting primers also amplied fragments of other a}b hydrolase genes, including a gene with homology to a putative lysophospholipase from Bacillus halodurans (accession no.
61
62
63
64
Expression and characterization of a novel lipase gene product from biomass DNA Although the entire putative lipase gene could be readily amplied using suitable PCR primers, it was found that the gene could not be readily cloned into the pCR cloning vector (Invitrogen) or other plasmids such as pUC18 (Yanisch-Perron et al., 1985) with lac-based promoter systems. In hindsight, this observation was presumably due to the high toxicity of the gene to the host since the lipase gene could be cloned into pET26b() (Novagen) which is characterized by low basal expression levels of genes cloned into the polylinker. The basal level of expression of genes cloned into pET26b() is very low due to a combination of repression by the lacI repressor and tight control of induction by the T7 promoter (Novagen). To minimize fully induced expression levels of the oli-lipase gene, only salt was added to induction cultures, rather than salt and IPTG required for full induction. Despite the tight control of lipase gene expression using this vector, yields of the puried protein using the T7 promoter system were low and microscopic examination of the cells revealed morphological abnormalities in the E. coli host expressing lipase. The puried recombinant oli-lipase was active between 30 and 70C, with an optimal temperature for activity at 4550
65
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Activation and inactivation of the enzyme Cofactors are not required for the expression of lipase activity. Divalent cations, such as calcium, generally stimulate the activity. It has been postulated that this is based on the formulation of calcium salts of long-chain fatty acids. The lipase activity is inhibited drastically by Co++, Ni++, Hg++, and
67
68
69
Take 8 test tubes and add 9ml of water in each test tube and
label it as 10-1, 10-2 up to 10-8. Then take 1ml of water sample from the soil dissolved sample
Take 1ml of sample from the 10-1 test tube and mix it in 10-2 test
tube.
Take 1ml of sample from the 10-2 test tube and mix it in 10-3 test
tube.
Take 1ml of sample from the 10-3 test tube and mix it in 10-4 test
tube.
Take 1ml of sample from the 10-4 test tube and mix it in 10-5 test
tube.
Take 1ml of sample from the 10-5 test tube and mix it in 10-6 test
tube.
Take 1ml of sample from the 10-6 test tube and mix it in 10-7 test
tube.
Take 1ml of sample from the 10-7 test tube and mix it in 10-8 test
tube.
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Preparation of nutrient agar medium: Composition: Luria Bertani agar (LB Agar) Agar agar Prepare the nutrient agar medium for 60ml. The diluted samples were plated on nutrient agar for total viable count. Then sterilize the medium, petriplates, L shaped loop in autoclave for 30 minutes. Pour the nutrient medium into 4 petriplates. After solidification 1ml of sample poured in petriplates spread it with L shaped loop.
Then incubate it for 24hrs at 340c. The dominant organisms were isolated and individually streaked on tributyrin agar plates.
The formation of halo zone around the colony on tributyrin agar was considered as the positive colony.
Sub culturing and storage of cultures Preparation of nutrient broth: LB Broth is dissolved in 25ml of distilled water. Take 5 test tubes and 5ml of nutrient broth in each test tube.
In one test tube inoculate the colony from the 10-6 culture plate. Then in another test tube inoculate the colony from 10-8 culture plate.
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culture plate.
Take another test tube and inoculate the colony which is similar in
Prepare the smear in these two slides and air dry it. Then add crystal violet and allow it to dry for 2 minutes and wash with distilled water. Then add grams iodine and allow it to dry for 30sec and wash it with distilled water. Then decolorized with acetone. Then counter stain with saffranin.
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Observation: Gram +ve bacilli was observed 10-6 and 10-8 colony. Gram ve cocci was observed in 10-8 with anti-biotic inoculated culture.
Preparation of nutrient medium: Composition: 1. Nutrient broth 2. Cacl2 3. Agar agar. 4. SDS (sodium dodecyl sulphate) In 5ml of hot water SDS is dissolved. Then take nutrient broth, Cacl2, agar are dissolved in 45ml of water. Autoclave the medium for 30 minutes. After autoclave add SDS along the wall of flask and mix carefully with out bubbles. Pour the medium to the petriplates and grow the organism by streak plate method.
Incubate the petriplates for 24hrs at 360c. In one plate inoculate the colony with 10-6 and 10-8 from broth
culture.
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broth culture.
Preparation of broth: LB agar is dissolved in 50ml of water. Take 5 test tubes; in each test tube 10ml of LB agar is poured. Then inoculate the colony from the culture plates.
Incubate it for 24hrs at 360c.
DNA isolation Steps: 1. Lysis of cells. 2. Precipitation of proteins. 3. Precipitation of nucleic acids. 4. Purification of nucleic acids.
Types of lysis: 1. Physical lysis like sonication, grinding, etc. 2. Chemical lysis i. SDS for denaturing the proteins ii. Placing cells in hypotonic solution. 3. Enzymatic lysis.
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DNA isolation by phenol choloroform method. DNA isolation by lysis buffer protocol. DNA isolation by rapid method. DNA isolation by helini ready template A & B method. DNA isolation by solution A,B,C method. Protocol 1: a. Take 1.5ml bacterial culture into sterilize appendab tube. b. Centrifuge at 10000 rpm.
c. Discard the supernatant and add 600 l of lysis buffer.
temperature.
f. Add 200 l of sodium acetate solution and mix the contents of the tube
by vortexing for 1 minute which precipitates the proteins. g. Centrifuge the tubes at 10000 rpm for 5 minutes. h. After centrifugation transfer the supernatent into the fresh tube.
i.
Add 600 l of isopropanol to the supernatant mix the solution well and incubate in -200c for 30 minutes and recover the precipitated DNA by centrifugating at 10000 rpm for 2 minutes.
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and chelates the DNA from enzymatical degradation. Composition of lysis buffer: 1. Tris chloride 2. EDTA 3. SDS Composition of sodium acetate: 1. 5 molar of sodium acetate 2. Glacial acetic acid 3. Distilled water
Protocol 2:
a. Take 1.5ml of bacterial culture in sterilized appendab tube. b. Centrifuge at 10000 rpm for 10 minutes c. Discard the supernatant.
d. To the pellet add 320 l of solution-A (TRIS & EDTA) and 80ml of
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times and incubate at room temperature for 5 minutes. h. Centrifuge at 10000 rpm for 10 minutes. i. Transfer the supernatant into a fresh tube. Now supernatant contains DNA & RNA. Add equal volumes of ice cold iso propanol. Gently invert and incubate it at room temperature for 5 minutes. j. Then centrifuge at 10000 rpm for 15 minutes. k. Discard the supernatant.
l.
To the pellet add 70% ethanol of 200 l without disturbing the pellet keep for 1 minute.
m. Centrifuge at 10000 rpm for 1 minute. n. Discard the supernatant and leave the pellet to an air dried the ethanol by inverting it on a paper for 10 minutes.
o. Suspend the pellet in 50 l of TE buffer and dissolve it.
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Protocol 3: a. Take 1ml of bacterial culture in sterilized appendab tube. b. Centrifuged at 10000 rpm for 10 minutes. c. Discard the supernatant.
d. Dissolve the pellet in 100 l of ready template A.
g. Vortex the sample for 1 minute. h. Keep the solution 5 minutes for room temperature.
i.
j. Centrifuge at 10000 rpm for 5 minutes. k. Add equal volumes of P:C:I to the supernatant. l. Centrifuge at 10000 rpm for 5 minutes. m. Then transfer the aqueous layer into fresh tube. n. Then add equal volumes of C:I to the aqueous layer o. Then centrifuge at 10000 rpm for 5 minutes. p. Transfer the aqueous layer into fresh tube.
q. To that add 3 molar potassium acetate or 160 l ice cold iso propanol.
78
s. Centrifuge at 10000 rpm for 5 minutes. t. Discard the supernatant. u. Wash the pellet with 90% ethanol or 70% ethanol. v. Air dry the pellet w. Dissolve the pellet with TE buffer.
Protocol 4: a. Take 1ml of bacterial culture in sterilized appendab tube. b. Centrifuged at 10000 rpm for 10 minutes. c. Discard the supernatant.
d. Dissolve the pellet in 100 l of ready template A.
g. Vortex the sample for 1 minute. h. Keep the solution 5 minutes for room temperature.
i.
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PCR amplification of 16s rDNA gene: By general microbial base characterization we cant say the species and strain number. It is tie consuming and very laborious. 16s rDNA is unique for microbes. But by using the 16s rDNA gene we can say much information about the species and strain number also. Here we used to have idea about the Tm, Annealing temperature, forward primer sequence, reverse sequence etc.
94
2min 45 sec
45 sec 62c
3min
80
45 sec
Molecular weight based confirmation of amplified product: After successful amplification of 16s rDNA gene we can reveal the molecular weight and it will be confirmed with molecular weight marker.
Purification of amplified product: The amplified product should not be suitable for sequencing, because it have the chemicals like Mg+2, assay buffer, Taq DNA polymerase, dNTPs and other contaminants. So before subjecting this wehave to purify the DNA from all chemicals. In all the products Nal based purification is the best for removing the contaminants for effective sequence analysis.
Sequence of amplified product: The purified DNA was sent for sequencing by autoradiography.
NCBI BLAST analysis: The sequenced DNA was subjected to Bioinformatics Blast and this provides the information about the species and strain level information about our organism.
Phylogenetic tree construction: This provides the very close information abouhowfar organism relates with other organism available in the database.
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RESULT
For the molecular identification of isolated strains 16S-rDNA profiles were obtained with Taq I Polymerase. The product was then
82
Analysis of the Genotypic and Phenotypic Groups The biological significance, lipases hold tremendous potential for exploitation in biotechnology. They posses the unique feature of acting at the aqueous and non aqueous interface which distinguishes them from esterases (Verger, 1997; Schmidt and Verger, 1998). The concept of lipase interfacial activity evolved from restriction of their catalytic activity to interface between lipid and water. The catalytic activity of lipases depends largely on the aggregated state of substrates. Experimental evidences suggest that the activation involves unmasking and structuring of enzyme-active-site, through conformational changes, that require presence of oil-in water droplets. Recent studies on the structure of several lipases have provided some clues for understanding their hydrolytic activity, interfacial activation and stereo selectivity of lipases (Kazlauskas and Bornscheuer, 1998). Enzymes such as proteases and carbohydrases have been used industrially for a number of years and corner the largest share of the world wide enzyme market. Whilst lipases at present account for less than 5% of the market, this share has the potential to increase dramatically via a wide range of different applications. The lipases catalyze wide range of reactions, including hydrolysis, inter-esterification, alcholysis, acidolysis, esterification and aminolysis. They catalyse the hydrolysis of fatty acid ester bond in the
83
Sequencing results were obtained in a SEQ 44 personal sequencing system. The results were submitted to GenBank and the sequence was blasted to the NCBI Genbank. From the BLAST sequence Phylogenetic tree was constructed.
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85
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89
90
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In this study, soil contaminated with oils collected within different olive oil mills in Andhra Pradesh. The strains were screened for the presence of lipase extracellular enzyme activities. For enzyme screening SDS was used as substrate. In total 3 lipase (0.02% used as substrate), activities were detected. All of the isolates were Gram (+), endospore forming rods, thus they were identified as ..
Taq I was used for 16S- rDNA . By choosing the universal primers for 16S rRNA gene, partial sequence analysis was carried out. Sequencing results were submitted to GenBank and the sequence was blasted against the Genbank sequence and pylogenitic tree was constructed. Lipases produced by the isolated strains can be studied in respect of enzyme activity, purification and production. The genes coding for these enzymes can also be cloned to obtain recombinant thermoacidophilic enzymes.
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