Reactions In Non-aqueous Media Research Articles

A thermostable organic solvent-tolerant lipase from Brevibacillus sp.: production and integrated downstream processing using an alcohol-salt-based aqueous two-phase system.

Lipases are used for the synthesis of different compounds in the chemical, pharmaceutical, and food industries. Most of the reactions are carried out in non-aqueous media and often at elevated temperature, requiring the use of organic solvent-tolerant thermostable lipases. However, most known lipases are not stable in the presence of organic solvents and at elevated temperature. In this study, an organic solvent-tolerant thermostable lipase was obtained from Brevibacillus sp. SHI-160, a moderate thermophile isolated from a hot spring in the East African Rift Valley. The enzyme was optimally active at 65°C and retained over 90% of its activity after 1 h of incubation at 70°C. High lipase activity was measured in the pH range of 6.5 to 9.0 with an optimum pH of 8.5. The enzyme was stable in the presence of both polar and non-polar organic solvents. The stability of the enzyme in the presence of polar organic solvents allowed the development of an efficient downstream processing using an alcohol-salt-based aqueous two-phase system (ATPS). Thus, in the presence of 2% salt, over 98% of the enzyme partitioned to the alcohol phase. The ATPS-recovered enzyme was directly immobilized on a solid support through adsorption and successfully used to catalyze a transesterification reaction between paranitrophenyl palmitate and short-chain alcohols in non-aqueous media. This shows the potential of lipase SHI-160 to catalyze reactions in non-aqueous media for the synthesis of valuable compounds. The integrated approach developed for enzyme production and cheap and efficient downstream processing using ATPS could allow a significant reduction in enzyme production costs. The results also show the potential of extreme environments in the East African Rift Valley as sources of valuable microbial genetic resources for the isolation of novel lipases and other industrially important enzymes.

Using an eco-friendly deep eutectic solvent for waterless anti-felting of wool fibers

Anti-felting or felting resistance process is a major finishing process for woolen goods which guarantees the dimensional stability of the product in washing and cleaning procedures mainly by covering or removing the scales from the surface of the wool fibers. Today, about 90 percent of anti-felting operations are carried out in a water-based chlorination process which releases tons of chlorine-containing discharge into the environment. Here we introduced deep eutectic solvents (DESs) as green substitutes for non-environmentally friendly sodium hypochlorite for getting anti-felting properties on woolen goods. Response surface methodology (RSM) based on four factors and in three levels, using Box-Behnken design (BBD) was used for designing the experiments and optimizing effective parameters in the anti-felting process of wool fibers using DESs. It was found that a special DES called Reline which is a mixture of choline chloride and urea in a 1:2 M ratio can successfully work as a green anti-felting agent and non-aqueous reaction medium. The shrinkage percentage of the Reline (DES) treated wool was 10% and very close to the 8.56% shrinkage seen on the hypochlorite-treated wool sample compared to more than 32% shrinkage observed on untreated fibers. Reline exhibited better preservation of the physicochemical and mechanical properties of the fibers causing only a 6.04% reduction in the breaking load of wool fibers compared to the hypochlorite-treated sample with a 24.86% reduction in breaking load of fibers. Anti-felted wool using the new Reline agent exhibit much better whiteness and lower yellowness compared to the chlorine-treated fibers. Microscopical observations, reflectance spectrophotometry, and tensile measurements all confirmed the superiority and numerous advantages of using Reline as a green anti-felting agent instead of polluting chlorinated chemicals. Reline usage as a green chemical encompasses other benefits such as reduced water consumption, reduced effluent, and more importantly DES recyclability and reuse.

Recent Progress in Developing a LiOH-Based Reversible Nonaqueous Lithium-Air Battery.

The realization of practical nonaqueous lithium-air batteries (LABs) calls for novel strategies to address their numerous theoretical and technical challenges. LiOH formation/decomposition has recently been proposed as a promising alternative route to cycling LABs via Li2 O2 . Herein, the progress in developing LiOH-based nonaqueous LABs is reviewed. Various catalytic systems, either soluble or solid-state, that can activate a LiOH-based electrochemistry are compared in detail, with emphasis in providing an updated understanding of the oxygen reduction and evolution reactions in nonaqueous media.Weidentify the key factors that can switch the cell chemistry between Li2 O2 and LiOH and highlight the debate around these routes, as well as rationalize potential causes for these opposing opinions. The identities of the reaction intermediates, activity of redox mediators and additives, location of reaction interfaces, causes of parasitic reactions, as well as the effect of CO2 on the LiOH electrochemistry, all play a critical role in altering the relative rates of a series of interconnected reactions and all warrant further investigation.

Translation of a Phosphine- and Azide-Based Reaction to Chemical Modification of Biomolecules in Ionic Liquid

AbstractThe difference of reaction design principles between traditional, small-molecule synthetic chemistry and biomolecular chemical reactions prevented the simple translation of small-molecule chemistry into biomolecular reactions. One of the key challenges of bioconjugation, or reactions on biomolecules, are the necessity of aqueous solutions as the solvent. In this Synpacts article, we describe our pursuit of using an ionic liquid as a nonaqueous reaction medium to conduct phosphine- and azide-based bioconjugation reactions.

Conversion of Meso-Aryl Substituted Open-Chain Pentapyrroles to Sapphyrins: Synthesis and Electrochemistry

Sapphyrin is a 22-π aromatic pentapyrrolic macrocycle containing one direct link and four bridging methine groups between five pyrrole subunits.1 This macrocycle is an early member of the group of “expanded porphyrins” and was obtained from a pentapyrrole, a byproduct in the synthesis of corroles. A series of open-chain pentapyrroles and sapphyrins with meso-tetraaryl groups were synthesized (Figure 1) and are characterized as to their electrochemistry and protonation reactions in nonaqueous media.2 The protonation-initiated conversion of pentapyrroles to sapphyrins upon oxidation was investigated and cyclic voltammetry was used to measure the redox potentials in CH2Cl2, PhCN, and/or pyridine (Py).REFERENCES1. V. J. Bauer, D. L. J. Clive, D. Dolphin, J. B. Paine, F. L. Harris, M. M. King, J. Loder, S. W. C. Wang, R. B. Woodward, J. Am. Chem. Soc. 1983, 105,6429-6436.2. W. Shan, N. Desbois, V. Blondeau-Patissier, M. L. Naitana, V. Quesneau, C. P. Gros, Y. Rousselin, W. Huang, Z. Ou, K. M. Kadish, Chem. Eur. J., 2017 , 23, 12833-12844.Figure 1 Figure 1

Lipases: Sources, Production, Purification, and Applications.

Background and Sources: Lipase enzyme is a naturally occurring enzyme found in the stomach and pancreatic juice. Its function is to digest fats and lipids, helping to maintain correct gallbladder function. Lipase is the one such widely used and versatile enzyme. These enzymes are obtained from animals, plants and as well as from several microorganisms and are sufficiently stable. These are considered as nature's catalysts, but commercially, only microbial lipases are being used significantly. Applications: They found enormous application in the industries of fat and oil processing, oleochemical industry, food industry, detergents, pulp and paper industry, detergents, environment management, tea processing, biosensors and cosmetics and perfumery. Various recent patents related to lipases have been revised in this review. Conclusion: Lipases are very peculiar as they have the ability to hydrolyse fats into fatty acids and glycerols at the water-lipid interface and can reverse the reaction in non-aqueous media. This natural ability makes it the most widely used enzyme in various industrial applications. This article deals with the immense versatility of lipase enzymes along with the recent advancements done in the various fields related to their purification and mass production in industries.

Synthesis, electrochemistry, protonation and X-ray analysis of meso-aryl substituted open-chain pentapyrroles

Five meso-tetraaryl open-chain pentapyrroles were synthesized and characterized as to their electrochemistry and protonation reactions in nonaqueous media. The investigated compounds are represented as (Ar)4PPyH3 where Ar [Formula: see text],[Formula: see text]-F2Ph, [Formula: see text]-BrPh, Ph, [Formula: see text],[Formula: see text],[Formula: see text]-(OMe)3Ph or [Formula: see text]-MePh and were characterized by UV-vis and 1H NMR spectroscopy, mass spectrometry and electrochemistry. Cyclic voltammetry was used to measure redox potentials, while protonation involving the conversion of (Ar)4PPyH3 to [(Ar)4PPyH5][Formula: see text] was monitored by UV-vis absorption spectroscopy. Equilibrium constants for proton addition were calculated using the Hill equation. One of the pentapyrroles was also structurally characterized. The electrochemical data, protonation constants and crystal structure were then compared with data for previously examined pentapyrroles and analyzed as a function of the solvent properties and nature of substituents on the meso-phenyl rings of the macrocycle.

A Green Process for Synthesis of Geraniol Esters by Immobilized Lipase from Candida Antarctica B Fraction in Non-Aqueous Reaction Media: Optimization and Kinetic Modeling

Abstract Enzymatic synthesis of molecules such as flavors, perfumes and fragrances has a great commercial advantage of being marketed as “natural” and also it offers exquisite selectivity of enzymes that can be superior over chemical catalysis. The current work focuses on the enzymatic synthesis of geranyl acetate as model compound, including optimization of reaction conditions such as nature of catalyst, reaction media, speed of agitation, mole ratio and temperature. A variety of esters were also synthesized. Geraniol was esterified with various acids, aromatic esters and vinyl esters in 1:4 molar ratio. Among all vinyl ester was the best giving in good yield (77–100 %) as compared to aromatic esters (5–82 %) and acids (7–31 %). Novozym 435 was found to be most active catalyst with ~96 % conversion and 100 % selectivity in 60 min at 55 °C in n-heptane as solvent for geranyl acetate. The maximum reaction rate was estimated (Vmax = 0.2712 mol L−1 min-1) by using the double reciprocal plot. It is a ternary complex (ordered bi-bi) mechanism with inhibition by geraniol.

Di(hydroperoxy)cycloalkanes Stabilized via Hydrogen Bonding by Phosphine Oxides: Safe and Efficient Baeyer–Villiger Oxidants

The syntheses of two new representatives of di(hydroperoxy)cycloalkane adducts of phosphine oxides, Cy3PO·(HOO)2C(CH2)4 and Cy3PO·(HOO)2C(CH2)6, and the new hydroperoxy(hydroxy)alkane adduct Cy3PO·(HOO)(HO)C(CH2)5 are described. Different synthetic routes were explored. The formation of the adducts proceeds from hydrogen peroxide adducts of phosphine oxides in the presence of cyclic ketones. Additionally, the direct addition of Cy3PO to free di(hydroperoxy)cycloalkanes results in the desired adducts. A mechanism for the formation is proposed, which is supported by generating and identifying the monoperoxy adduct Cy3PO·(HOO)(HO)C(CH2)5. All adducts are fully characterized with single-crystal X-ray crystallography, NMR, and IR spectroscopy. All adducts are safe and solid and have shelf lifetimes of months. They are highly soluble in organic solvents, which allows for homogeneous reactions in nonaqueous media. This was demonstrated by selectively oxidizing Ph2P–PPh2 to the moisture-sensitive Ph2P(O)–P(O)Ph2....

Water-Retaining Polymers in Organic Solvent Increase Lipase Activity for Biodiesel Synthesis

Background: Biocatalyzed esterification reactions in non-aqueous media require water removal materials and optimized enzyme formulations to improve reaction rate and conversion. We have tested a lipase biocatalysis process using anionic polymeric chains of polyacrylate as super-absorbent of water in the conversion of methyl oleate from oleic acid and methanol and in toluene as reaction medium. The water removal material that we used also for the enzyme immobilization is commercially named Aquasorb 3005K2 (herein called Aquasorb). Methods and findings: The synthesis of methyl oleate was catalyzed by Candida rugosa lipase or lipase G from Penicillium camemberti in the presence or absence of Aquasorb. The simple addition of Aquasorb to the reaction media of the lipase (commercial sample) catalyzed reaction allowed an enzyme activity 20% and 40% higher than that in the absence of Aquasorb for lipase from C. rugosa and lipase G, respectively. Furthermore, for the commercial form of lipase G, when used in the presence of Aquasorb, it was obtained a higher conversion of esterification (93% instead of 60%). Aquasorb was used also as support for the adsorption of the enzyme (via hydrogel formation followed by lyophilization) producing a lipase that was 100% and 60% more active than the enzyme dissolved in buffer in the absence of Aquasorb and lyophilized. However, these lipase formulations showed only about 20 % activity compared to when dry Aquasorb was simply added to the non-aqueous reaction medium. Conclusions: The use of Aquasorb appears beneficial as additive in esterification reactions (e.g., for biodiesel production) catalyzed by lipases in non-aqueous reaction media.

Comparative Study of Special Features of the Oxygen Reaction (Molecular Oxygen Ionization and Evolution) in Aqueous and Nonaqueous Electrolyte Solutions (a Review)

Studies of the oxygen reaction, including the oxygen ionization and evolution processes occurring at typical electrode materials in aqueous and nonaqueous electrolytes, are analyzed. A connection between the problematics of the oxygen electrode reaction in nonaqueous media and the developing of novel batteries, in the first place, Li–O2 batteries, is emphasized. Unlike aqueous solutions, the oxygen reduction in aprotic electrolytes was shown to occur without breaking of the O–O bond; it is accompanied by formation of poorly soluble product of two-electron reaction (Li2O2) in the pores of positive electrode. The effect of the solvent donor number and the anion composition on the oxygen reduction mechanism and the lithium peroxide deposit structure is described. A marked reduction of the Li2O2 oxidation overvoltage when passing from carbonaceous materials to platinum-containing catalysts in the positive electrode is elucidated; in the latter case, the effect of electrocatalyst type upon the Li2O2 formation reaction is somewhat reduced. The elucidation of the contribution of processes occurring at the free and lithium-peroxide-covered electrode surface during the oxygen reaction for wide variety of active materials is formulated as the main basic problem of the future research.

Influence of β-octabromination on free-base triarylcorroles: Electrochemistry and protonation-deprotonation reactions in nonaqueous media

Electrochemical and acid-base properties of four free-base triarylcorroles were examined in nonaqueous media. These compounds are represented here as (tdcc)H[Formula: see text], (tpfc)H[Formula: see text], (Br[Formula: see text]tdcc)H[Formula: see text] and (Br[Formula: see text]tpfc)H[Formula: see text], where tdcc and tpfc are the trianions of tris(2,6-dichlorophenyl)corrole and tris(pentafluorophenyl)corrole, respectively. Different spectroscopic and electrochemical properties were observed for the [Formula: see text]-brominated corroles as compared to the non-brominated derivatives, due in part to the corrole ring distortion and in part to the strong electron-withdrawing properties of the Br groups. The brominated free-base corroles are easier to deprotonate than the non-brominated corroles in solution, which was confirmed by electrochemistry and spectroelectrochemistry as well as protonation/deprotonation reactions of the compounds with acid or base in PhCN. The electrochemistry of the protonated and deprotonated corroles is presented and comparisons made with previously published data for other protonated and deprotonated free-base corroles under the same solution conditions.

Hydration-aggregation pretreatment for drastically improving esterification activity of commercial lipases in non-aqueous media.

We investigated a novel, simple method for activating lipases in non-aqueous reaction media. Lipase powders were suspended in n-fatty alcohols and were then hydrated by adding a small amount of water. A paste-like aggregate was recovered from the mixture followed by lyophilization for obtaining activated lipases as dry powders. Lipase activity was evaluated for esterification between myristic acid and methanol in n-hexane. The activated lipases exhibited high esterification activity depending on the experiment conditions during hydration-aggregation pretreatment such as the amount of added water, the temperature, the pH of added buffer solutions, and the carbon chain length of the n-fatty alcohols used as pretreatment solvents. Various commercial lipases from different origins could be activated by this method. Changes in lipase conformation induced by the hydration-aggregation pretreatment were studied based on fluorescence and Fourier-transform infrared spectroscopy.

Electrochemistry and Protonation of Open-Chain Pentapyrroles and Sapphyrins with Highly Electron-Withdrawing Meso-Substituents

A series of open-chain pentapyrroles and sapphyrins with highly electron-withdrawing meso-tetraaryl groups were synthesized and characterized as to their electrochemistry and protonation reactions in nonaqueous media. The chemical conversion of pentapyrroles to sapphyrins upon oxidation in acid media is known but it was not known if sapphyrins could be electrosynthesized from pentapyrroles under the appropriate solution conditions. This was investigated in the present study which compares the redox behavior and acid-base properties of pentapyrroles to that of sapphyrins having the same meso-substituents. The investigated compounds were each characterized by UV−vis and 1H NMR spectroscopy, mass spectrometry and electrochemistry, in addition to spectroelectrochemistry. Structures of the investigated compounds are shown in Chart 1 and are represented as (Ar)4PpyH3 and (Ar)4SapH3. Cyclic voltammetry was used to measure redox potentials and spectroelectrochemistry was used to monitor the products of each redox reaction. The protonation processes were monitored by UV-vis absorption spectroscopy as protons were added to the investigated compounds in the form of trifluoroacetic acid (TFA). Equilibrium constants were calculated using both the Hill equation and the Mole Ratio method. The electrochemical data, the protonation constants and previous reported mechanisms for reduction and oxidation of pentapyrroles and sapphyrins with other meso-tetraaryl groups were then analyzed as a function of the solvent properties and para-substituents on the four phenyl rings of compounds in the two series. Figure 1

Properties of antimony triiodide photodecomposition and Lewis acidity

Antimony triiodide is a versatile compound that is not studied in detail in the current Inorganic Chemistry literature at the undergraduate level. In this work we propose a simple microscale synthesis describing two other interesting properties which are not reported in the common chemical literature: photo-oxidation and acid–base reaction in non-aqueous media. The experiment can be successfully and safely achieved in a two-hour lab class at the sophomore level.

Isolation, purification and characterization of a salt-active and organic-solvent-thermostable phenylalanine dehydrogenase from Bacillus nanhaiensis DSF-15A2

BackgroundAs phenylalanine dehydrogenase (EC 1.4.1.20, PheDH) activity and stability was still diminished in organic solvent and biocatalytic reaction in non-aqueous media has presented as a great alternative to chemical synthesis and biosynthesis, suggesting that it is crucial to obtain a PheDH with high organic solvent-tolerant. ResultsIt is the first case when thermo-solvent tolerant PheDH producing bacterium was isolated from a marine sample. The BnPheDH’ stability could be significantly improved by 1M NaCl, which was analyzed by molecular dynamics simulation, and it showed better stability in methanol, ethanol, ethylene glycol, ethylene glycol monoethyl ether and acetone. The kcat/Km in 30% (v/v) methanol and ethylene glycol monomethylether was 1.15- and 1.35-fold than that of in the aqueous phase, indicating that the catalytic efficiency was significantly improved by the methanol and ethylene glycol monomethylether. ConclusionsThe results indicated that the affinity of enzyme, substrate and co-factor and the catalytic efficiency can be significantly improved by organic solvents, suggesting that the BnPheDH had great application value in organic phase or aqueous-organic phase catalytic reaction, and it would be used as an robust biocatalyst to synthesize synthons in the food and pharmaceutical industries.

A Journey toward Sulfolane Microemulsions Suggested as Inert, Nonaqueous Reaction Media.

Recently, it turned out that nanostructured reaction media containing highly inert solvents as tetrahydrothiophen-1,1-dioxide (sulfolane) are beneficial for strongly oxidizing or reductive reactions. Because of their ability of solubilizing polar and nonpolar solvents with a large nanostructured interface in particular microemulsions provide such interesting reaction media. Starting from the pseudoternary microemulsion H2O-n-octane-C12E4/C12E5 (polyoxyethylene n-alkyl ether), water was successively replaced by the highly inert tetrahydrothiophen-1,1-dioxide (sulfolane). We found that an increasing sulfolane content drives the system beyond the tricritical point. Replacing the already long chain surfactants C12E4 and C12E5 by a mixture of the even longer chain surfactants C18E6 and C18E8, we were able to prepare nonaqueous sulfolane microemulsions for the first time. We also teach how in a second step the phase behavior of the hydrophilic sulfolane-n-octane-C18E8 system can be tuned at constant temperature (as required by the reaction conditions) by addition of the hydrophobic cosurfactant 1-octanol (C8E0). The change in curvature that occurs by adding 1-octanol is demonstrated measuring the size of reverse micelles by DLS. We found that the radius varies from at least 8 to 16 nm, a suitable sizes for inverse nanoreaction vessels.

Dihydrogen bond intermediated alcoholysis of dimethylamine-borane in nonaqueous media.

Dimethylamine-borane (DMAB) acid/base properties, its dihydrogen-bonded (DHB) complexes and proton transfer reaction in nonaqueous media were investigated both experimentally (IR, UV/vis, NMR, and X-ray) and theoretically (DFT, NBO, QTAIM, and NCI). The effects of DMAB concentration, solvents polarity and temperature on the degree of DMAB self-association are shown and the enthalpy of association is determined experimentally for the first time (-ΔH°assoc = 1.5-2.3 kcal/mol). The first case of "improper" (blue-shifting) NH···F hydrogen bonds was observed in fluorobenzene and perfluorobenzene solutions. It was shown that hydrogen-bonded complexes are the intermediates of proton transfer from alcohols and phenols to DMAB. The reaction mechanism was examined computationally taking into account the coordinating properties of the reaction media. The values of the rate constants of proton transfer from HFIP to DMAB in acetone were determined experimentally [(7.9 ± 0.1) × 10(-4) to (1.6 ± 0.1) × 10(-3) mol(-1)·s(-1)] at 270-310 K. Computed activation barrier of this reaction ΔG(‡theor)298K(acetone) = 23.8 kcal/mol is in good agreement with the experimental value of the activation free energy ΔG(‡exp)270K = 21.1 kcal/mol.

Impact of substituents and nonplanarity on nickel and copper porphyrin electrochemistry: first observation of a Cu(II)/Cu(III) reaction in nonaqueous media.

Electrochemical studies of the oxidation of dodecasubstituted and highly nonplanar nickel porphyrins in a noncoordinating solvent have previously revealed the first nickel(III) porphyrin dication. Herein, we investigate if these nonplanar porphyrins can also be used to detect the so far unobserved copper(III) porphyrin dication. Electrochemical studies of the oxidation of (DPP)Cu and (OETPP)Cu show three processes, the first two of which are macrocycle-centered to give the porphyrin dication followed by a CuII/CuIII process at more positive potential. Support for the assignment of the CuII/CuIII process comes from the linear relationships observed between E1/2 and the third ionization potential of the central metal ions for iron, cobalt, nickel, and copper complexes of (DPP)M and (OETPP)M. In addition, the oxidation behavior of additional nonplanar nickel porphyrins is investigated in a noncoordinating solvent, with nickel meso-tetraalkylporphyrins also being found to form nickel(III) porphyrin dications. Finally, examination of the nickel meso-tetraalkylporphyrins in a coordinating solvent (pyridine) reveals that the first oxidation becomes metal-centered under these conditions, as was previously noted for a range of nominally planar porphyrins.

Isolation, Purification and Characterization of Secondary Structure and Kinetic Study of Lipase from Indian Major Carp, Catla catla (Catla)

Lipases are ubiquitous enzymes which catalyze the hydrolysis of fats into fatty acids and glycerol at the waterlipid interface and reversing the reaction in non-aqueous media. Lipases occupy a place of prominence among biocatalysts owing to their novel and multifold applications in oleochemistry, organic synthesis, detergent formulation and nutrition.Lipase was extracted and isolated from the alimentary canal and digestive gut of Catla catla (catla). The tissue was homogenized in a ratio of 1:3 with starting buffer (0.01 M TrisHCl, pH 7.2). The crude extract thus obtained was precipitated using ammonium sulphate (20-80%). Excess salt was removed by Dialysis and the resultant dialysate (Desalted Enzyme) was subjected to DEAE-Cellulose column for Ion Exchange Chromatography at a flow rate of 0.5 ml/min. Elution was carried out by a step gradient of NaCl (100-800 mM) in the starting buffer. Active fractions were pooled as Purified Fraction (PF) and were used for physical characterization of pH, temperature and effect of calcium on the enzyme activity, structural characterization, molecular characterization and for kinetic studies. The Purified Fraction (PF) showed final specific activity of 1438.72 U/mg. The optimum pH was 7.8 and the optimum temperature was found to be 20EšC. Melting Temperature (Tm) value was 42EšC and the activation energy of purified lipase was 34.82 KJ/mol/K. Thermal stability of lipase was found to be at 20EšC. Lipase activity retained up to 3 h of incubation with 10 mM and 20 mM of CaCl2 in starting buffer. This shows that Calcium has enhancing property from the denaturation of enzyme. Michaelis-Menten constant (Km) of lipase from Indian major carp, catla for the hydrolysis of pNPP was 6.695 mM. Turnover number (kcat) of lipase (catla) was 0.0022 s-1. Catalytic efficiency (kcat/Km) of lipase was 0.0003412 s-1 mM-1. SDS-PAGE of purified lipase (PF) revealed a homogenous single band with molecular mass of 70 kDa. Secondary structural arrangement of α helices and β strands of purified lipase results 48.51% and 9.74%, respectively using Circular Dichroism.