Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 4(11), 103-116, November (2014) Res. J. Chem. Sci.International Science Congress Association 103 Review PaperEnzymatic Bioconversion in Non-conventional Media Gogoi Sumbita Department of Chemistry, Dergaon Kamal Dowerah College, Dergaon, Golaghat-785614, Assam, INDIAAvailable online at: www.isca.in, www.isca.me Received 21st August 2014, revised 13th October 2014, accepted 15th November 2014 AbstractOne strategy for optimizing biocatalysis for the production of flavors compounds, oleochemicals and drug intermediates in pharmaceutical industries is to use non-conventional media, such as non-aqueous heterogeneous systems. In this article, we highlight some of the current trends in biocatalysis in systems, focusing on organic solvent systems, reverse micelles and supercritical fluids. This review also summarizes recent applications of ionic liquids (ILs) as ‘green’ solvents in biocatalytic transformations of commercially important compounds, extractions of a variety of substances, including metal ions, organic and bio-molecules, organosulfur from fuels and gases. For more effective separation of products from ILs, they could also be used along with another ‘green’ technology, supercritical fluid extraction. In addition to their environmentally benign characteristics, ILs have other favourable properties such as hydrophobicity, polarity and selectivity over organic solvents. Keywords: Ionic liquid, supercritical fluid, reverse micelle, green solvent, lipase. Introduction Enzymes need a small amount of water to retain their active three-dimensional conformational state, even when the enzyme is covalently bound to a support. Water contributes to the structural integrity, active site polarity and protein stability. It provides hydrophobic interactions with polar residues on the enzyme molecule, which would otherwise be interacting with each other, creating an incorrect conformational structure. Water can also limit the solubility of hydrophobic substrates around the enzyme. In reactions where the substrates are poorly soluble in water or water is formed as a product, the reaction yields in aqueous system are generally low. The search for new reaction systems with favorable conditions led to enzyme catalysis in non-conventional media. In biocatalysis, the term non-conventional media refers to systems that are solvents other than water. Non-conventional media can be used for biocatalysis with enzymes. In non-aqueous media, the structures of enzymes are more rigid which increased the stability of the enzyme1-3. The use of enzymes in organic solvents can offer advantages such as improved substrate specificity, stereoselectivity, recoverability and low solubility of enzymes4-6. Non-conventional media are of special interest for hydrolases since low water contents can be used in order to favour synthesis reactions, as in case of lipases a large-scale use of solvents able to dissolve hydrophobic solvents to obtain high productivity. In organic media, hydrophobic enzymes such as lipases can be usefully employed to carry out synthetic reactions such as esterification and transesterification reactions since the equilibrium position of the reaction is shifted sufficiently to give a high yield of the synthesis product. Organic solvents may also help to keep a low thermodynamic water activity, which then decrease the thermodynamic barrier for reactions such as the esterification or hydrolysis reaction. Higher conversion yields in non-aqueous systems simplify product recovery. The low solubility of enzymes in organic solvents decreases the amount of enzyme loss via desorption from support material. This enables the use of simple enzyme immobilization techniques such as adsorption which lowers the cost of enzyme preparation10. Strategy for Biocatalysis in Non-Conventional Media Dispersed System: Enzymes used in organic media require some water to achieve good catalytic activity11. In esterification reactions, the initial activity of enzyme exhibits an optimum value at certain water content in the reaction media12. The enzymes do not dissolve in most of the commonly used organic solvents and hence catalysis in general takes place in dispersed media. Successful biocatalysis in non-conventional media has been reported in the main application for ester and peptide synthesis, the resolution of chiral building blocks. Despite the advantages of biocatalysis in organic solvent based system, enzyme stability is lower in hydrophilic solvents (-2.5 logP0), such as acetone and ethers than in hydrophobic solvent (2logP4) such as hexane, heptane or haloalkanes. Hydrophobic solvents do not strip off the essential water layer that stabilizes the biocatalyst while hydrophilic solvents distort this water from the enzyme surface, leading to unfolding of the molecule 13-16. On the contrary, solvent toxicity is a problem for many applications. However, for ester synthesis, the major interest is still in organic liquid solvent systems17-37 represented in table-2 Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(11), 103-116, November (2014) Res. J. Chem. Sci.International Science Congress Association 104 Table-1 NomenclatureS.No. Formula Nomenclature 1. [BMIM][BF 4 ] 1-butyl-3-methylimidazolium tetrafluoroborate 2. [BMIM][BF 6 ] 1-butyl-3-methylimidazolium hexafluoroborate 3. [BMIM] [PF 6 ] 1-butyl-3-methylimidazolium hexafluorophosphate 4. [OMIM][PF 6 ] 1-octyl-3-methylimidazolium tetrafluorophosphate 5. [BMIM][CF 3 SO 3 ] 1-butyl-3-methylimidazolium triflate 6. MTBE methyl tert-butylether 7. [BMIM][NTf] 1-butyl-3-methylimidazolium bis (trifluoromethyl) - sulfonyl) amide 8. [BMIM][dca] 1-butyl-3-methylimidazolium dicyanamide 9. [BDMIM][BF 4 ] 1-butyl-2,3-dimethylimidazolium tetrafluoroborate 10. [MOEMIM][BF 6 ] 1-methoxyethyl-3-methylimidazolium hexafluoroborate 11. [HMIM][BF 4 ] 1-hexyl-3-methylimidazolium tetrafluoroborate 12. [BMIM][OCSO 4 ] 1-butyl-3-methylimidazolium 3-octylsulphate 13. [EMIM][TfO] 1-ethyl-3-methylimidazolium 14. CALB Candida antarctica lipase B 15. CCL Candida cylindracea lipase 16. PPL Porcine pancreas lipase 17. PCL Pseudomonas cepacia lipase 18. CRL Candida rugosa lipase 19. MML Mucor miehei lipase 20. RML Rhizomucor miehei lipase 21. CLEA Cross-linked enzyme aggregate 22. PFL Pseudomonas fluorescens lipase 23. CSL Candida species lipase 24. RDL Rhizopus delemer lipase 25. MJL Mucor javanicus lipase 26. TLL Thermomyces lanuginose lipase 27. AOT Sodium bis(2-ethylhexyl)sulfosuccinate 28. BHDC Benzyl-n-hexadecyldimethyl ammonium chloride Supercritical Fluids (SCF): The use of enzymes in non-conventional media such as supercritical fluids (SCF) has been proposed as a means of improving the activity and utility of such enzymes in anhydrous environments38. Supercritical fluids are mainly carbon dioxide, freons, hydrocarbons or inorganic compound such as SF, NO etc. The most commonly used system is supercritical carbon dioxide (SCCO) which is because of its critical point of 73.8 and 31.1C makes equipment design and reaction set-up relatively simple39. The high diffusivities and low viscosities in SCFs greatly enhance mass transfer of substrates to the enzymes. Solubilization of substrates is generally better compared to that in organic liquid solvents. With the absence of organic solvents, higher purity products can be achieved. Since many ester products are used in food, the favourite choice of supercritical fluid for ester production is carbon dioxide. In these systems, some enzyme activity may be lost during the depressurization of the reaction mixture. The use of high pressure vessels complicates continuous production and scale up. Studies on ester synthesis reactions in supercritical carbon dioxide have resulted in mixed opinions on the economic viability of such systems compared to organic liquid systems40. Separation of products from reactants can be greatly facilitated by the ease with which the solvent power of the SCFs can be adjusted. In supercritical fluids, a small change in pressure or temperature near the critical point may result in great change in its viscosity and of the diffusivitiy and solubility of compounds dissolved in it. This may leds to control of the rate and enantioselectivity of enzyme catalysed reactions. In enzymatic reactions, in a very limited pressure range near the critical point, interaction between carbon dioxide and enzyme molecules greatly increased with consequent conformational changes in the enzymes, causing active sites to emerge to catalyse stereoselective synthesis. The main advantage of use of supercritical fluids such as carbon dioxide, it is non-toxic, chemically inert and can be removed easily after the reaction. Further, in SCFs due to high diffusion rates, it facilitates the transport phenomena and can increase the bioconversion rate. The high diffusion rate can also facilitate product separation. In addition, the use of supercritical carbon dioxide can have adverse effects on enzymes by decreasing the pH of the Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(11), 103-116, November (2014) Res. J. Chem. Sci.International Science Congress Association 105 Table-2 A literature survey of enzyme catalysed reactions in organic solvent mediaEnzyme Solvent Reaction Ref. PCL Various organic solvents Transesterification reaction between 2--benzyl alcohol and vinyl acetate 17 CRL cyclohexane Resolution of (±) 2,6-dimethyl-1,7-heptandioic acid with n-butanol 18 CALB -hexane Esterification of lauric acid and isopropyl alcohol 19 CALB none Transesterification reaction between ethyl lactate and butanol 20 CALB -hexane Reaction between acetic anhydride and isoamyl alcohol 21 CRL and MML number of organic solvents Transesterification reaction between sec-phenetyhl alcohol and vinyl butyrate 22 Immobilzed CRL -hexane Esterification reaction between isobutyric acid and n-butanol 23 CAL, P. species, CRL -heptane Esterification reaction between tetrahydrofurfurl alcohol and butyric acid 24 Immobilized RML -hexane Esterification reaction between 9,10-dihydroxy steric acid and 1 -octanol 25 Novozym-435 -heptane Transesterification reaction between vinyl acetate and -octanol 26 Novozym 435 Various organic solvent Esterification of lactic acid and glycoside 27 CLEA-Subtilisin Carlsberg Several organic solvents Reaction between -acetyl-L-tyrosine ethyl ester and 1-propanol 28 PPL Various organic solvents Esterification of lauryl alcohol with lauric acid 29 MML -do- Esterification of iso-amyl alcohol with acetic acid 30 Novozym-435 Several organic solvents Synthesis of lauryl palmitate 31 Novozym-435 acetronitrile Synthesis of erythrobyl laurate 32 Novozym-435 Solvent free system Transesterification between ethyl acetate and cinnamoyl alcohol 33 Novozym-435 toluene Synthesis of cinnamyl acetate 34 Rhizopus Oryzae Bi-phasic solvent system Esterification of tuna fish oil fatty acids with butanol 35 Immobilized CRL Bi-phasic solvent system Esterification of lauric acid with butanol 36 CAL Solvent free system Transesterification of isoamyl alcohol with acetate anhydride 37 microenvironment of the enzyme and by the formation of carbamates due to covalent modification of free amino groups at the surface of the protein40,41. Hence, supercritical carbon dioxide medium in the near critical region should trigger the activation of the enzyme by causing movement of its surface groups and creating active sites42. Furthermore, supercritical carbon dioxide is an excellent solvent for non-polar organics. The main limitation of use of carbon dioxide as an analytical extraction solvent is that its polarity is too low to obtain efficient extraction of products, because the analytes lack sufficient solubility or the extractant has a poor ability to displace the analytes from active matrix sites. The use of this type media have been mainly observed for reactions catalyzed by hydrolases, especially lipases43-52 given in table-3. Reverse Micelles: The low water content necessary to favour the synthesis reactions in organic solvents by enzymes can be achieved by micro-encapsulation of the biocatalyst within reverse micelles. In the presence of a certain surfactant in a suitable concentration, reverse micelles form ordered structure readily upon the addition of a small amount of water to a water immiscible hydrophobic solvent53. Reverse miceller systems offer a very large aqueous/ organic solvent interface of approximately 100 mmL-1 of micro emulsion. Due to dynamic structures, the micelles can exchange components between each other and also with bulk organic solvent. This system is highly applicable for biocatalysis because it mimics the natural environment that many enzymes experience within cells. The AOT (Sodium bis 2-ethyl hexyl) sulfosuccinate/Isooctane system is one of the most suitable systems for enzymatic catalysis54-56. Since the reverse micelles formed by this surfactant are very stable over a wide range of concentrations in the absence of co-surfactants. The reversed micellar AOT system is particularly interesting for hydrolases such as lipases, which provide a high interfacial area of contact with the enzyme anchoring at the aqueous side of the AOT interface. Further, lipase catalysed reaction enables the use of hydrophobic substrates, which are readily soluble in the bulk organic phase57 Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(11), 103-116, November (2014) Res. J. Chem. Sci.International Science Congress Association 106 Table-3 Reactions catalyzed by enzymes in supercritical fluidsEnzyme Reaction Remarks Ref Free and immobilized RML, PFL Synthesis of ethyloleate Economy of the process is given in this paper 43 Immobilized MML Hydrolysis of blackcurrant oil Simultaneous extraction and hydrolysis of the oil 44 Immobilized CALB Synthesis of butyl butyrate Combines SCF with membrane technology 42 PCL Transesterification reaction between 1-phenylethanol and vinylacetate Selective towards one isomer of 1-phenyl ethanol, higher reaction rate 45 Immobilized CALB,RML Synthesis of isoamyl acetate Higher initial rate in SCCO 46 Immobilized CALB Synthesis of butyl laurate Productivity dependent on the substrate amount, catalyst remain active after five cycles 47 CLEA-CALB Esterification of isoamyl alcohol with acetic acid Initial activity of the enzyme decreased with increase in pressure, highly stable in SCCO48 Immobilized RML Esterification of oleic acid with 1-octanol Obtained about 93% yield in a continuously operating bioreactor, higher than those obtained In batch mode 49 CRL Regioselective acylation of methyl-6--Trityl glucopyranoside with vinylacetate Obtained 91.4% final conversion 50 Novozym-435 Synthesis of butyl butyrate Reaction followed Ping-Pong Bi-Bi mechanism 51 Novozym-435 Kinetic modeling of decyl acetate Enhanced initial rate at 35C and 100 bar 52 In reverse miceller system, the enzyme may interact with the micellar membrane for which changes in the micellar concentration affect the catalytic activity as the extent of inhibitory interactions between the enzyme and the micelles changes. On the contrary, the enzyme may be dissolved in the aqueous interior pool of the micelles. In that case, the activity of enzyme is independent of micelle concentration. The concentration of micelles in a system is the concentration of surfactant at a constant water-to-surfactant ratio (Wo). With the increase of surfactant concentration at a constant Wo, increases the surface area58. As a result, the number of micelles interact, with a lipase molecule increases undergoing the changes in the secondary structure of lipase upon incorporation in to reverse micellar system from an aqueous system causing decrease in lipase activity. In ionic AOT reverse micellar system, the activity and stability of enzymes are adversely affected due to strong electrostatic interactions between AOT and enzymes59. However, enzymes in reverse micelles formed by some nonionic surfactants have a high activity and stability due to weak interactions between enzyme and the nonionic surfactants60. Nevertheless, the addition of some co-surfactants is still necessary for formation of the non-ionic reverse micelles. A new type of surfactant sodium bis (2-ethylhexyl polyoxyethylene) sulfosuccinate (MAOT), which is structurally designed as a chemically modified AOT, was prepared in the laboratory61. The chemically modified AOT (MAOT)- Isooctane reversed micellar system increased substrate conversion due to decreased electrostatic and hydrophobic interactions between enzyme and MAOT molecules62. At large scale, due to the presence of surfactant and other components such as enzyme and water, it is difficult to recover the product from the reverse micellar system. This problem was overcome by use of membrane reactors in which by continuous operation through ultrafiltration membrane retains the micelle and hence the enzymes while the substrate and product molecules pass freely. Such enzyme bioreactors have been used for ester synthesis63. Table 4 reports some reactions, with potential applications in the food, pharmaceutical and chemical industries. Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(11), 103-116, November (2014) Res. J. Chem. Sci.International Science Congress Association 107 Table-4 Recent examples of enzyme catalysed reactions in reverse micellar systemEnzyme Surfactant Organic solvent Reaction Ref CCL, CSL AOT decane Esterification of () 2-octanol and hexanoic acid 64 RDL AOT Isooctane Esterification of oleic acid and octyl alcohol 65 CRL AOT Isooctane Hydrolysis of Olive oil 62 MJL AOT Isooctane Acylation of doxorubicin 66 TLL AOT isooctane Synthesis of ethyl laurate 67 -chymotripsin AOT -heptane Hydrolysis of 2-naphthyl acetate 68 -chymotripsin BHDC benzene Hydrolysis of 2-naphthyl acetate 69 -chymotripsin AOT n-heptane Hydrolysis of 2-naphthyl acetate 70 Ionic Liquids (ILs): An active area of current research in biotechnology, biocatalysis in non-conventional media, is the use of ionic liquids to improve activity, stability and selectivity of enzymes. Ionic liquids are organic salts, which are liquid over a broad range of temperature and good solvents for wide range of organic, inorganic and polymeric compounds. The first ionic liquid (EtNH x NO) was reported in 1914. However, nowadays the most common ionic liquids for biocatalysis are imidazolium based ionic liquids such as [BMIM][BF] (1-butyl-3-methylimidazolium tetrafluoroborate), [BMIM][BF] (1-butyl-3-methylimidazolium hexafluoroborate) etc. Ionic liquids have low melting point and composed entirely of ions and considered to be highly polar solvents. They possess negligible vapour pressure that can be taken advantage of for the separation of volatile products. Ionic liquids, because of their negligible vapour pressures, have been generally recognized as ‘green’ solvents. Enzymatic reactions based on ionic liquids appear highly promising alternatives for developing ‘green’ chemical processes because of their physical and chemical characteristics. Ionic liquids can selectively dissolve a gas, which makes them potential solvents for gas separations71. Carbon dioxide has relatively high solubility in imidazolium based ILs72,73The solubility of gases, e.g. H, CO, O, is generally good which makes the ionic liquids as attractive solvents for catalytic hydrogenations, carbonylations, hydroformylations and aerobic oxidations. They are immiscible with some organic solvents, such as alkanes, and hence can be used in two-phase systems. The unique properties of ionic liquids are such as they are non-volatile, non-flammable and have excellent chemical and thermal stability. It is possible also to conventionally synthesize them in preparatory scale. The non-volatile nature of ILs provides environmental advantages and is beneficial for plant design due to reduced pressure build-up. One obvious advantage of using ionic liquids over the use of normal organic solvents is that the physical and chemical properties of ionic liquids, including their polarity, hydrophobicity, viscosity and solvent miscibility, can be finely tuned by alternating the organic cation, inorganic anion and attached substituents. Hence, ionic liquids have been referred to as ‘designer’ solvent. This is important because by manipulating the solvent properties, one is allowed to design an ionic liquid for specific reaction conditions, such as to increase the substrate solubility, to modify the enzyme selectivity or to tailor the reaction rate. ILs may provide an ideal solvent for engineering media for biocatalytic reactions because of their advantages. The recent research results indicated that the enantioselectivity and activity of enzymes in ionic liquids were better or comparable to them in organic solvents in some reactions74,75. ILs can also be used as ‘green’ solvents in extraction of variety of substances, including metal ions, organic and biomolecules, organosulfur from fuels and gases76-80. ILs was also used to form two-phase systems with many solvents. The investigations of new biphasic reactions using ILs are of special interest because of the possibility to adjust solubility properties using different cation/anion combinations77. This allows for systematic optimization of a biphasic reaction with regard to product recovery. Ionic liquid biphasic systems were used to separate many biologically important molecules such as carbohydrates and organic acids. Moreover, recently two group of researchers have been employed ionic liquids to whole-cell in situ fermentation and showed its great potential in whole-cell biocatalytic processing due to their low toxicity to microorganisms81,82. In both cases [BMIM][PF] was used in a two phase system as substrate reservoir and / or for in situ removal of the product formed, thereby increasing the productivity of the catalyst. This is useful for systems where organic solvents in combination with an aqueous phase either do not dissolve enough substrate or lead to increased enzyme deactivation83. Table 5 represents some recently reported examples of processes that are carried out in ionic liquids using enzymes. Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(11), 103-116, November (2014) Res. J. Chem. Sci.International Science Congress Association 108 Table-5 Recent examples of reactions catalysed by enzymes in Ionic liquids (ILs) Enzyme ILs Reaction Comments Ref Immobilized esterases, Bacillus Stearothermophilies, Bacilus subtilis Various ILs Transesterification of 1-phenylethanol Higher stability of enzyme compared to organic solvents 84 CRL [BMIM][BF], [MOEMIM][BF 6 ] Acylation of glycosides Higher reaction rates and selectivity than in conventional organic solvents 85 Immobilized CALB [BDMIM][BF] Transesterification using vinylacetate Lipase was recycled for 10 times without losing enantioselectivity and reactivity 86 Immobilized CALB, -chymotripsin Several ILs Transesterification reaction Improved the thermal stability of both the enzymes 87 Free Epoxide Hydrolases Several ILs Stereoselective hydrolysis of epoxides comparable reaction rate and ster selectivity than those in buffer 88 Immobilized PCL [BMIM][BF] Resolution of racemic alcohols Addition of triethyl amine to ILs enhanced the rate of reaction 89 CRL [ BMIM][BF] [HMIM][BF] [BMIM][BF 6 ] Enantioselective hydrolysis ILs as co-solvent markedly enhanced enantioselectivity 90 Immobilized CALB Several ILs Enantioselective acylation Increased reaction rate, decreased enantioselectivity 91 CRL [BMIM][BF], [ONIM][PF 6 ] Esterification of 2-substituted propanoic acids and 1-butanol Higher enantioselectivity than in -hexane 92 Immobilized PCL [BMIM][BF], [BMIM][BF 6 ] Hydrolysis and alcoholysis of 3,4,6- tri--acetyl--glucal High regioselectivity 93 CALB Several ILs Transesterification of ethylbutanoate with 1-butanol Higher activity in [BMIM][BF], [Et 3 MeN][MeSO 4 ] 94 Mandelate racemase, Pseudomonas putida [MMIM][MeSO] [BMIM][OCSO] [BMIM][OCSO [BMIM][O] Kinetic resolution of mendalic acid Reaction rate strongly influenced by aw 95 CAL [BMIM][NTf] Kinetic resolution of rac-2-pentanol vinyl propionate Higher activity than in hexane, greater enantioselectivity greater en enantioselectivi 96 Protease: Papain Alcalase Lipase: Novozym-435 [BMIM][BF] Hydrolysis of amino acid esters Higher enantioselectivity in papain, varied enzyme activity and enantioselectivity with substrate and enzyme used97 CALB Several ILs Transesterification of ethyl butanoate with 1-butanol Activity of CLEA- CALB was twice both in [BMIM][dca] and tert-butyl alcohol than free lipase 98 Lipase: Burkholderia Cepacia Several ILs Acetylation of secondary alcohols by vinyl acetate Increased enantioselectivity and reaction rate in imidazolium-PEG-Alkyl sulfate 99 PPL, CRL [BMIM][BF] [BMIM][PF] Resolution of racemic secondary alcohols by vinyl acetate Higher enantioselectivity and reaction rate 100 CLEA-CRL, Burkholderia cepacia [BMIM][PF] Kinetic resolution of (±)1- phenyl ethanol Higher enantioselectivity 101 CALB Several ILs Transesterification of vinyl butyrate and 1-butanol Higher activity of CALB in ILs containing anions of lower nucleophilicity in water immiscible ILs 102 Bacillus thermocate- Nulatus lipase [BMIM][PF] [BMIM][BF 4 ] Esterification and amidation Best selectivity towards the formation of monoester over the di-ester 103 Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(11), 103-116, November (2014) Res. J. Chem. Sci.International Science Congress Association 109 Enzyme ILs Reaction Comments Ref Immobilized CALB Several ILs Kinetic resolution rac-2-pentanol A membrane bioreactor containing supported liquid Membrane based on ionic liquid was employed 104 Novozym-435 [BMIM][PF] Regioselective acylation of 1--D-Arabinofuranosyl cytosine with vinyl acetate Remarkable enhancement of substrate conversion with a co-solvent mixture of [BMIM][PF6] and pyridine compared to other organic solvents 105 Novozym-435 [BMIM][PF] [EMIM][PF], [BMIM][BF 4 ] Methanolysis of Sunflower oil Both IL and lipase were recycled for four successive reaction cycles without any significant loss of activity 106 Immobilized CALB [BMIM][PF] Production of isoamyl acetate The mixture of IL-enzyme could be recycled for 7 repeated cycles in IL-isoamyl alcohol biphasic System 107 CALB [BMIM][TfO] Synthesis of Palmitoylglucose ester Highest yield of ester at 50 0 C and enzyme Concentration of 50mg/ml 108 Novozym 435 [EMIM][TfN]  Synthesis of caffeic acid phenyl ester  High conversion at 70 0 C  109  Novozym 435 [BMIM][TfO]  Synthesis of mannosyl myristateFull factorial design was used and was found Optimal temperature 80C and substrate molar Ratio of 1/10  110  CALB  [BMPyrr][Dca]  Synthesis of isoamyl acetate  More than 80% of productivity was preserved  111  CALB  [BMIM][FeCl 4 ] Oleic acid to biodiesel  Maximum yield 83.4%, molar ratio 22:1  112  Erbeldinger et al. reported first the use of enzymes in ILs. They used protease thermolysin for the synthesis of the dipeptide Z-aspartame113. The enzyme stability was increased in IL, but the rate of reaction was comparable with those found in conventional organic solvents. The protease -chymotrypsin was also used to carry out some transesterification reactions of -acetyl-L-phenylalanine ethyl ester or -acetyl-L-tyrosine ethyl ester to transform into their corresponding propyl esters114-116. To carry out these reactions, Laszlo and Compton114 used [OMIM][PF] (1-octyl-3-methylimidazolium tetrafluoro phosphate) and [BMIM][PF] and compared the results with those obtained in other organic solvents. They found that as in polar organic solvents, a certain amount of water is necessary to maintain enzymatic activity. The reaction rates were also comparable in both ILs and organic solvents. Iborra and coworkers115 compared stability of -chymotrypsin in different ILs through the transesterification of -acetyl-L-tyrosine ethyl ester with 1-propanol. In the ionic liquids tested, the stability of enzyme increased which led to higher final product concentration. The majority of enzymes reported to be active in ILs belong to the class of lipases. Sheldon and co-workers first demonstrate the potential use of ILs for lipase catalysis116. They compared the reactivity of CALB in ILs, such as [BMIM][PF] and [BMIM][BF] with conventional organic solvents. Lipases and esterases are usually used for the kinetic resolution of racemates by esterification, transesterification or hydrolysis reactions. Schofer et al. studied the kinetic resolution of 1-phenylethanol for a set of eight different lipases and two esterases in ten different ILs with methyl-tert-butylether (MTBE) as reference using vinylacetate as acyl donor117. For the lipases from Pseudomonas and Alcaligenes species, an improved enantioselectivity was observed in [BMIM][NTf] as compared with MTBE as solvent. The best results were obtained in CALB in [BMIM][CFSO], [BMIM] [NTf] and [OMIM][PF]. Other groups investigating the same system reported good activities in these ILs118-120. Park and Kazlauskas demonstrated the influence of additional washing steps upon the enzyme activity118. Persson and Bornscheuer investigated the same system catalysed by esterases from Bacillus subtilis and Bacillus stearothermophilus and two lipases (CALB and Pseudomonas sp.), reported no activity of the lyophilized powder of the esterases in ILs84. When immobilized onto Celite, higher specific activity and enantioselectivity was obtained which is comparable to those in conventional organic solvents such as n-hexane, MTBE and vinyl acetate and for the two lipases. The stability of esterase from . stearothermophilus at 40C was considerably increased in the ionic liquids [BMIM][BF] and [BMIM][PF] as compared to -hexane and MTBE. The lipase catalysed enantioselective transesterification of racemic alcohols was also studied for three lipases using vinyl acetate as acyl donor in two ILs and hexane as reference100. In the presence of catalytic amounts of organic bases such as triethylamine or pyridine, both the rate and enantioselectivity of the reaction was increased by CALB compared to hexane. No reaction was observed in PPL or CRL. Earlier studies of the lipase from Pseudomonas sp. revealed that the water content of the reaction medium had a strong influence Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(11), 103-116, November (2014) Res. J. Chem. Sci.International Science Congress Association 110 on enzyme activity121. To compare the enzyme activity and selectivity in solvents of different polarities independently of the water content, it is necessary to evaluate the water activity (a) in these solvents. Eckstein et al. used the method of water activity equilibration over saturated salt solutions and found that the enantioselectivity of the lipase is less influenced by the water content or temperature when the reaction is performed in [BMIM] [NTf 122. Barahona et al. studied the effect of water activity on the immobilized candida antarctica lipase B catalyzed esterification of geraniol in [BMIM][PF] 95. The reaction rate was lower than in hexane. The concentration based equilibrium constant for the reaction in [BMIM][PF] was dependent on the water activity in the system, which is about 20 times lower than in hexane. Iborra et al. reported that for the resolution of rac-2-pentanol using CALB at 2% (v/v) water content, the use of ionic liquid, [BMIM][NTf] is more effective than hexane74. Both the synthetic activity and selectivity of the process is dependent on the water activity, although it has no affect on the enantioselectivity of the enzyme. Sheldon and coworkers investigated the structure and activity of CALB on seven different ILs and tert-butyl alcohol as reference through a simple transesterification reaction of ethyl butanoate with1-butanol94. They reported that in [BMIM][BF] and [BMIM][PF], the reaction rate was comparable with that in tert-butyl alcohol. However, in ionic liquids containing alkylsulfate, nitrate and lactate anions, which dissolved CALB, the reaction rate was about ten times slower than in [BMIM][BF]. An exception was the [EtMeN][MeSO], as dissolved CALB maintains its activity in this solvent. The denaturation of CALB was observed upon dissolution in ILs in which the activity was low, whereas the conformation of enzyme dissolved in [EtMeN][MeSO] closely resembled the native one. The CLEA of CALB was twice as active in [BMIM][dca] that deactivated the free enzyme98. The stability and activity of CALB in ILs was also studied through transesterification of 1-butanol with vinyl butyrate102. They studied the reaction in nineteen different 1,3-dialkylimidazolium based ionic liquids and in hexane as reference solvent. They reported that in water-immiscible ILs, the enzymatic activity and selectivity were higher than that obtained in hexane. However, in water-miscible ILs, the activity was lower than in the reference solvent. CALB exhibited greater stability in water-immiscible ILs than in water-miscible ILs. One of the main advantages of ILs that makes them ‘green’ is the low volatility, which creates challenges for product separation and recovery. If the product is volatile, back distillation may be used to remove the product from IL123. If the product is of hydrophilic nature in a hydrophobic IL, water may be used to remove the product from IL124. However, one of the main drawbacks of IL technique is the difficulty of recovery of non-volatile or low volatility products. In such cases, to overcome this limitation, another type of ‘green’ solvent, supercritical fluids (SCFs), has been adapted for product recovery from ILs125. Among SCFs, SCCO is volatile and non-polar, hence easily forms two-phase systems with non-volatile and polar ILs. The principle of product recovery by these biphasic systems is based on the solubility of SCCO in the ILs to transfer organic products to the SCCO-rich phase and the insolubility of the IL in SCCO. More recently, the biphasic IL / SCCO system have been used in metal catalysed and enzyme catalysed reactions. This biphasic system offers advantages that enzyme catalyst are soluble and stable in ILs, but have low solubility in SCCO. Moreover, many organic compounds are soluble in SCCO, offering easy separation of products from ILs and the process can be designed as batchwise or continuous operations126-131. A number of biocatalytic reactions were achieved successfully in IL/SCCO biphasic system114,132-133. The kinetic resolution of rac-1-phenylethanol with vinyl propionate catalysed by free and immobilized CALB in IL/SCCO biphasic system was studied at 10 Mpa and at 120 and 150134. Both free and immobilized CALB were able to catalyze specifically the synthesis of (R)-1-phenylethylpropionate and excellent activity, stability and enantioselectivity were reported in continuous operation. Iborra et al.135 demonstrated the dynamic kinetic resolution of the same system in different IL/SCCO biphasic systems by simultaneously using both immobilized CALB and silica modified with benzenosulphonic acid (SCX) catalysts at 40C and 10Mpa. SCX was reported to be an efficient heterogeneous chemical catalyst for the racemization of ( S)-1-phenylethanol in [EMIM][NTf], [BTMA][NTf] and [BMIM][PF]. Coating both CALB and SCX with ILs greatly improved the efficiency of the process in SCCO, providing good yield of (R)-1-phenylethylpropionate (78%) with excellent enantioselectivity (ee = 91-98%) in continuous operation. The biphasic IL/alkane system have also been used in some enzyme catalysed reactions136-138. A continuously operated shaped microreactor was used for CALB catalysed synthesis of isoamyl acetate in 1-butyl-3-methyl pyridinium dicyanamide/n-heptane two phase system 136 and 48.4gm-3s-1 of isoamyl acetate was produced, which was almost three fold better as compared to the intensely mixed batch process. The synthesis of ferulic acid oleylalcohol ester was studied in IL/isooctane system 138. Both considerable bioconversion and volumetric productivity were reported in [HMIM][PF] and [OMIM][ PF] mediated system. Under optimized reaction conditions of 60C, 150 mg of Novozym 435 and 100 mg molecular sieve, upto 48.50 mg/ml productivity of ester was reported for [HMIM][PF6]/isooctane (0.5ml/1.5ml) system with a substrate concentration of ferulic acid of 0.08mmol/ml and oleyl alcohol of 0.32 mmol, while an optimum volumetric productivity of 26.92 mg/ml was obtained. Conclusion This review summarizes some of the current advances in the field of non-aqueous biocatalysis in heterogeneous solvent systems, including ionic liquids (ILs) which offer new possibilities for the application of solvent engineering to Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(11), 103-116, November (2014) Res. J. Chem. Sci.International Science Congress Association 111 biocatalytic reactions. Biocatalysis in ionic liquids is an exciting area of research which holds considerable potential for industrial applications. In some cases, biocatalyst can have profound effect on activities and selectivities in ionic liquid media. Furthermore, ILs is designer solvents. Hence, for a specific system, ILs can be achieved by a suitable combination of cation and anion. References 1.Linko Y. Y., Lamsa M., Huhtala A. and Rantanen O., Lipase biocatalysis in the production of esters, J Am Oil Chem Soc, 72, 1293-1299 (1995) 2.Turner N. A. and Vulfson E. N., At what temperature can enzymes maintain their catalytic activity?, Enzym Microb Technol, 27, 108-113 (2000) 3.Knezevic Z., Bobic S., Milutinovic A., Obradovic B., Mojovic L. and Bugarski B., Alginate-immobilized lipase by electrostatic extrusion for the purpose of palm oil hydrolysis in lecithin / isooctane system, Process Biochem, 38, 313-318 (2002) 4.Klibanov A. M. and Zaks A., Enzyme-catalysed processes in organic solvents, Proc Natl Acad Sci USA, 82, 3192-3196 (1985) 5.Rubio E., Fernandez M.A. and Klibanov A. M., Effect of solvent on enzyme regioselectivity, J Am Chem Soc, 113, 695-696 (1991) 6.van Tol J.B.A., Stevens R.M.M., Veldhuizen W. J., Jongejan J.A. and Duine J.A., Do organic solvents affect the catalytic property of lipase? Intrinsic kinetic parameters of lipases in ester hydrolysis and formation in various organic solvents, Biotechnol Bioeng, 47(1), 71-81 (1995) 7.Parida S. and Dordick J.S., Tailoring lipase specificity by solvent and substrate chemistries, J Org Chem, 58, 3238-3244 (1993) 8.Khmelnitsky Y.L., Levashov A.V., Klyachko N.L. and Martinek K., Engineering biocatalytic systems in organic media with low water content, Enzym Microb Technol, 10, 710-724 (1988) 9.Halling P.J., Organic liquids and biocatalysis: Theory and Practice, TIBtech, 7(1), 50-51 (1989) 10.Wehtje E., Adlercreutz P. and Mattiasson B., Improved activity retention of enzymes deposited on solid supports, Biotechnol Bioeng, 41, 171-178 (1993) 11.Zaks A. and Klibanov A.M., Enzymatic catalysis in non-aqueous solvents, J Biol Chem,263, 3194-3201 (1988) 12.Wehtje E., Adlercreutz P. and Mattiasson B., Formation of C–C bonds by mandelonitrile lyase in organic solvents, Biotechnol Bioeng,36(1), 39-46 (1990) 13.Hazarika S., Goswami P., Dutta N.N. and Hazarika A.K., Ethyl oleate synthesis by Porcine pancreatic lipase in organic solvents, Chem Eng J, 85(1), 61-68 (2002) 14.Gogoi S., Hazarika S., Dutta N.N. and Rao P.G., Esterification of lauric acid with lauryl alcohol using cross-linked enzyme crystals: Solvent effect and kinetic study, Biocatal Biotransform, 24(5), 343-351 (2006)15.Sztajer H., Lunsdorf H., Erdmann H., Menge U. and Schmid R., Biochim Biophys Acta,1124, 253-261 (1992)16.Azevedo A.M., Prazers D.M.F., Cabral J.M.S. and Fonseca L.P., Stability of free and immobilized peroxidase in aqueous-organic solvent mixtures, J Mol Catal B: Enzym,15(2), 147-153 (2001) 17.Hazarika S., Goswami P. and Dutta N.N., Lipase catalyzed transesterification of 2--benzylglycerol with vinyl acetate: solvent effect, Chem Eng J, 94(1), 1-10 2003) 18.Hedenstrom E., Edlund H. and Lund S., Stereoselective esterification of 2,6-dimethyl-1,7-heptanedioic acid catalysed by Candida rugosa lipase, J Mol Catal B: Enzym, 23(1), 53-59 (2003) 19.Sekeroglu G., Fadiloglu S. and Ibanoglu E., Production and characterization of Isopropyl Laurate using immobilized lipase, Turkish J Eng Env Sci, 28(3), 241-247 (2004) 20.Pirozzi D. and Greco G., Activity and stability of lipase in the synthesis of butyl lactate, Enzym Microb Technol, 34(1), 94-100 (2004) 21.Romero M.D., Calvo L., Alba C., Daneshfar A. and Ghaziaskar S., Enzymatic synthesis of isoamyl acetate with immobilized Candida antarctica lipase in -hexane, Enzym Microb Technol, 37(1), 42-48 (2005) 22.Castillo B., Pacheco Y., Al-Azzam W., Griebenow K., Devi M., Ferrer A. and Barletta G., On the activity loss of hydrolases in organic solvents I. Rapid loss of activity of a variety of enzymes and formulations in a range of organic solvents, J Mol Catal B: Enzym, 35, 147-153 2005) 23.Yadav G.D. and Lathi P.S., Kinetics and mechanism of synthesis of butyl isobutyrate over immobilized lipases, Biochem Eng J, 16, 245-252 (2003) 24.Yadav G.D. and Devi K.M., Immobilized lipase-catalysed esterification and transesterification reactions in non-aqueous media for the synthesis of tetrahydrofurfuryl butyrate: comparison and kinetic modeling, Chem Eng Sci, 59, 373-383 (2004) 25.Awang R., Basri M., Ahmad S. and Salleh A.B., Lipase-catalysed esterification of palm-based 9,10-dihydroxystearic acid and 1-octanol in hexane – a kinetic study, Biotechnol Lett26(1), 11-14 (2004) 26.Yadav G.D. and Trivedi A.H., Kinetic modeling of Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(11), 103-116, November (2014) Res. J. Chem. Sci.International Science Congress Association 112 immobilized lipase catalysed transesterification of n-octanol with vinyl acetate in non-aqueous media, Enzym Microb Technol, 32, 783-789 (2003) 27.Wei D., Gu C., Song Q. and Su W., Enzymatic esterification for glycoside lactate synthesis in organic solvent, Enzym Microb Technol, 33, 508-512 (2003) 28.Shah S., Sharma A. and Gupta M.N., Cross-linked protein-coated microcrystals as biocatalysts in nonaqueous solvents, Biocatal Biotransform, 26(1), 1-6 (2008) 29.Gogoi S, Pathak MG, Dutta A and Dutta NN, Porcine pancreas lipase catalysed synthesis of lauryl laurate in organic solvent media: A Kinetic Study, Indian J Biochem and Biophysics, 45(3), 192-197 (2008) 30.Gogoi S. and Dutta N.N., Kinetics and mechanism of esterification of isoamyl alcohol with acetic acid by immobilized lipase Indian J Chem Technol, 16(3), 209-215 (2009) 31.Syamsul K.M.W., Salina M.R., Siti S.O., Hanina M.N., Basyaruddin M.A.R. and Jusoff K., Green synthesis of Lauryl Palmitate via lipase catalysed reaction, World Applied Sci J, 11(4), 401-407 (2010) 32.Park K.M., Lee D.E., Sung H., Lee J. and Chang P.S., Lipase catalysed synthesis of erythorbyl laurate in acetonitrile, Food Chemistry, 129(2), 59-63 (2011) 33.Geng B., Wang M., Qi W., Su R. and He Z., Cinnamyl acetate synthesis by lipase catalysed transesterification in a solvent free system, Biotechnol. Applied Biochem., 59(3) , 270-275 (2012) 34.Yadav G.D. and Devendran S., Lipase catalysed synthesis of cinnamyl acetate via transesterification in non-aqueous medium, Process Biochem, 47(4), 496-502 2012) 35.Bhandari K.., Chaurasia S.P., Dalai A.K., Gupta A. and Singh K., Kinetic study on enzymatic esterification of tuna fish oil fatty acids with butanol, J Mol Catal B: Enzym, 94(2), 104-110 (2013) 36.Shankar S., Agarwal M. and Chaurasia S.P., Study of reaction parameters and kinetics of esterification of lauric acid with butanol by immobilized Candida Antarctica lipase, Indian J Biochem Biophysics, 50(6), 570-576 2013) 37.Siong L.S., Azudin N.Y. and Shukor S.R.A., Process Modelling of isoamyl acetate synthesis catalysed by lipase from Candida Antarctica in a solvent free system, J Engg Sci, 10(1), 59-71 (2014) 38.Ceria E and Palocci C, Lipases in supercritical fluids, Methods Enzymol, 286(5-6), 495-508 (1997)39.Adlercruetz P., Biocatalysis in non-conventional media, In Applied Biocatalysis, ed by Straathof AJJ and Adlercruetz P. Harwood Academic Publishers, Amsterdam, 295-316 (2000) 40.Chulalaksananukul W., Condoret J.S. and Combes D’, Geranyl acetate synthesis by lipase-catalysed transesterification in supercritical carbon dioxide, Enzym Microb Technol, 15(7), 691-698 (1993) 41.Lozano P., Villora G., Gomez D., Gayo A.B., SanchezConesa J.A., Rubio M. and Iborra J.L., Membrane reactor with immobilized Candida antarctica lipase B for ester synthesis in supercritical carbon dioxide, J Supercrit Fluids, 29(2), 121-128 (2004) 42.Ikushima Y., Advances in colloid and interface Science, 71, 259-280 (1997)43.Knez Z. and Habulin M., Compressed gases as alternative enzymatic reaction solvents: A short review, J Supercrit Fluids, 23(1), 29-42 (2002) 44.Sovova H. and Zarevucka M., Lipase catalysed hydrolysis of blackcurrant oil in supercritical carbon dioxide, Chem Eng Sci 58, 2339-2350 (2003) 45.Celia E., Cernia E., Palocci C., Soro S. and Turchet T., Tuning Pseudomonas cepacia lipase (PCL) activity in supercritical fluids, J Supercrit Fluids, 33(2), 193-199 2005) 46.Romero M.D., Calvo L., Alba C., Habulin M., Primozic M. and Knez Z., Enzymatic synthesis of isoamyl acetate with immobilized Candida antarctica lipase in supercritical carbon dioxide, J Supercrit Fluids, 33(1), 77-84 (2005) 47.Guni T., Paolucci-Jeanjean D., Belleville M.P. and Rios G.M., Enzymatic membrane reactor involving a hybrid membrane in supercritical carbon dioxide, J Mem Sci, 297(1), 98-103 (2007) 48.Dijkstra Z.J., Merchant R. and Keurentjes J.T.F., Stability and activity of enzyme aggregates of Calb in supercritical CO, J Supercrit Fluids, 41(1), 102-108 2007) 49.Laudani C.G., Habulin M., Knez Z., Della Porta G. and Reverchon E., Immobilized lipase-mediated long-chain fatty acid esterification in dense carbon dioxide: bench scale packed-bed reactor study, J Supercrit Fluids, 41(1), 74-81 (2007) 50.Palocci C., Falconi M., Chronopoulou L. and Cernia E., Lipase-catalysed regioselective acylation of tritylglycosides in supercritical carbon dioxide, J Supercrit Fluids, 45(1), 88-93 (2008) 51.Verma M.N. and Madras G., Kinetics of synthesis of butyl butyrate by esterification and transesterification in supercritical carbon dioxide, J Chem Technol Biotechnol., 83, 1135-1144 (2008) 52.Olivera M.V., Rebocho S.F., Ribriro A.S., Macedo E.A. Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(11), 103-116, November (2014) Res. J. Chem. Sci.International Science Congress Association 113 and Loureiro J.M., Kinetic modeling of decyl acetate synthesis by immobilized lipase catalysed transesterification of vinyl acetate with decanol in supercritical carbon dioxide, J Supercrit Fluids, 50(2), 138-145 (2009) 53.Tsai S.W. and Chiang C.L., Kinetics, mechanism and time course analysis of lipase-catalysed hydrolysis of high concentration olive oil in AOT-isooctane reverse micelles, Biotechnol Bioeng, 38, 206-211 (1991) 54.Alves J.R.S., Fonseca L.P., Ramalho M.T. and Cabral J.M.S., Optimization of Penicillin acylase extraction by AOT/isooctane reversed micellar systems, Biochem Eng , 15(1), 81-86 (2003) 55.Carvelho C.M.L. and Cabral J.M.S., Reverse micelles as reaction media for lipases, Biochemie, 82, 1063-1085 2000) 56.Krieger N., Tapia M.A., Melo E.H.M., Lima-Filho J.L., Aires-Barros M.R. and Cabral J.M.S., Purification of the Penicillium citrinum lipase using AOT reversed micelles, J Chem Technol Biotechnol, 69(1), 77-85 (1997) 57.Marhuenda-Egea F.C., Piera-Velazquez S., Cadenas C. and Cadenas E., Reverse micelles in organic solvents: a medium for the biotechnological use of extreme halophilic enzymes at low salt concentration, Archaea, 1(2), 105-111 (2002) 58.Brown E.D., Yada R.Y. and Marangoni A.G., The dependence of the lipolytic activity of Rhizopus arrheizuslipase surfactant concentration in aerosol-OT/isooctane reverse micelles and its relationship to enzyme structure, Biochim Biophys Acta, 1161(1), 66-72 (1993) 59.Stamatis H., Xenakis A., Dimitriadis E. and Kolisis F.N., Catalytic behaviour of Pseudomonas cepacia lipase in w/o microemulsions, Biotechnol Bioeng, 45(1), 33-41 1995) 60.Yamada Y., Kuboi R. and Komasawa I., Increased activity of Chromobacterium viscosum lipase in aerosol-OT reverse micelles in the presence of non-ionic surfactants. Biotechnol Prog, : 468-472 (1993). 61.Maria P.D., Sinisterra J.V., Montero J.M., Lotti M., Valero F. and Alcantara A.R., Acyl transfer strategy for the biocatalytical characterization of Candida rugosalipases in organic solvets, Enzym Microb Technol, 38(2), 199-208 (2006) 62.He Z.M., Wu J.C., Yao C.Y. and Yu K.T., Lipase-catalysed hydrolysis of olive oil in chemically-modified AOT/isooctane reverse micelles in a hollow fiber membrane reactor, Biotechnol Lett, 23, 1257-1262 (2001) 63.Serralheiro M.L.M., Prazeres D.M.F. and Cabral J.M.S., Continuous production and simultaneous precipitation of a di-peptide in a reversed micellar membrane reactor, Enzym Microb Technol 24, 507-513 (1997) 64.Karlsson S., Backlund S., Eriksson S. and Hedstrom G., Enzymatic esterification and transesterifications in AOT-based gels with different composition, Colloid Surf B: Biointerface, 9(1), 67-72 (2001) 65.Naoe K., Ohsa T., Kawagoe M. and Imai M., Esterification by Rhizopus delemar lipase in organic solvent using sugar ester reverse micelles, Biochem Eng , 9(1), 67-72 (2001) 66.Altreuter D.H., Dordick J.S. and Clark D.S., Optimization of ion-paired lipase for non-aqueous media: acylation of doxorubicin based on surface models of fatty acid esterification, Enzym Microb Technol, 31(1), 10-19 2002) 67.Fernandes M.L.M., Kriger N., Baron A.M., Zamora P.P., Ramos L.P. and Mitchell D.A., Hydrolysis and synthesis reactions catalysed by Thermomyces lanuginosa lipase in the AOT/Isooctane reversed micellar system, J Mol Catal B: Enzym,30(1), 43-49 (2004) 68.Falcone R.D., Biasutti M.A., Correa N.M., Silber J.J., Lissi E. and Abuin E., Effect of the Addition of a Nonaqueous Polar Solvent (Glycerol) on Enzymatic Catalysis in Reverse Micelles. Hydrolysis of 2-Naphthyl Acetate by Chymotrypsin, Langmuir, 20, 5732-5737 2004) 69.Moyamo F., Falcone R.D., Mejuto J.C., Silber J.J. and Correa N.M., Cationic Reverse micelles create water with super hydrogen bond donor capacity for enzymatic catalysis: Hydrolysis of 2-naphthyl acetate by Chymotrypsin, Chemistry:A European Journal, 16, 8887-8893 (2010)70.Moyamo F., Setien E., Silber J.J. and Corrrea N.M., Enzymatic hydrolysis of N-Benzoyl-L-Tyrosine P-nitroanilide by Chymotrypsin in DMSO-water/AOT/n-heptane reverse micelles.A interfacial effect on the enzymatic activity, Langmuir 29, 8245-8254 (2013)71.Brennecke J.F. and Maginn E.J., Purification of gas with liquid ionic compounds, US patent 6579 343 (2003) 72.Anthony J.L., Maginn E.J. and Brennecke J.F., Solubilities and thermodynamic properties of gases in the ionic liquid 1--butyl-3-methylimidazolium hexafluorophosphate, J Phy Chem B 106, 7315-7320 2002) 73.Cadena C., Anthony J.L., Shah J.K., Morrow T.I., Brennecke J.F and Maginn E.J., why is CO so soluble in imidazolium-based ionic liquids? J Am Chem Soc126,5300-5308 (2004) 74.Noel M., Lozano P., Vaultier M. and Iborra J.L., Kinetic resolution of rac-2-pentanol catalysed by Candida antarctica lipase B in the ionic liquid, 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide, Biotechnol. Lett.,26(3), 301-306 (2004) Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(11), 103-116, November (2014) Res. J. Chem. Sci.International Science Congress Association 114 75.Hongwei Y., Jinchuan W. and Bun C.C., Kinetic resolution of ibuprofen catalysed by Candida rugosalipase in ionic liquids, Chirality,17(1), 16-21(2005) 76.Zhao H., Shuqian S. and Ma P., Use of ionic liquids as green solvents for extractions, J Chem Technol Biotechnol, 80, 1089-1096 (2005) 77.Chun S., Dzyuba S.V. and Bartsch R.A., Influence of structural variation in room-temperature ionic liquids on the selectivity and efficiency of comparative alkali metal salt extraction by a crown ether, Analytical Chemistry,73, 3737-3741 (2001) 78.Dai S., Ju Y.H. and Barnes C.E., Solvent extraction of strontium nitrate by a crown ether using room-temperature ionic liquids, Journal of the Chemical Society, Dalton Transactions, , 1201-1202 (1999)79.Vidal S., Neiva Correia M.J., Marques M.M., Ismael M.R. and Angelino Reis M.T., Studies on the use of ionic liquids as potential extractants of phenolic compounds and metal ions, Separation Science and Technology,39, 2155-2169 (2004) 80.Matsumoto M., Mochiduki K., Fukunishi K. and Kondo K., Extraction of organic acids using imidazolium-based ionic liquids and their toxicity to Lactobacillus rhamnosus,Sep Purif Technol, 40(1), 97-101 (2004) 81.Howarth J., James P. and Dai J., Immobilized baker’s yeast reduction of ketones in an ionic liquid [bmim] PFand water mix, Tetrahedron Lett, 42, 7517-7519 (2001)82.Matsumoto M., Mochiduki K., Fukumishi K. and Kondo K., Extraction of organic acids using imidazolium-based ionic liquids and their toxicity to Lactbacillus rhamnosus Sep Purif Technol, 40, 97-101 (2004) 83.Wasserscheid P., Welton T. (Eds): Ionic Liquids in synthesis. Wiley-VCH, Weinheim, 2002. 84.Persson M. and Bornscheuer U.T., Increased stability of an esterase from Bacillus stearothermophilus in ionic liquids as compared to organic solvents, J Mol Catal B: Enzym, 22(1), 21-27 (2003)85.Kim M.J., Choi M.Y., Lee J.K. and Ahn Y., Enzymatic selective acylation of glycosides in ionic liquids: significantly enhanced reactivity and regioselectivity, J Mol Catal B: Enzym, 26(2), 115-118 (2003) 86.Itoh T., Nishimura Y., Ouchi N., Hayase S., 1-butyl-2,3-dimethylimidazolium tetrafluoroborate: the most desirable ionic liquid solvent for recycling use of enzyme in lipase –catalysed transesterification using vinyl acetate as acyl donor, J Mol Catal B: Enzym, 26(1), 41-45 2003) 87.Lozano P, Diego TD, Carrie D, Vaultier M and Iborra JL, Enzymatic ester synthesis in ionic liquids, J Mol Catal B: Enzym, 21(1), 9-13 (2003) 88.Kaar J.L., Jesionowski A.M., Berberich J.A., Moulton R. and Russell A.J., Impact of ionic liquid physical properties on lipase activity and stability, J Am chem. Soc125, 4125-4131 (2003) 89.Rasalkar M., Potdar M.K. and Salunke M.M., Pseudomonas cepacia lipase-catalysed resolution of racemic alcohols in ionic liquid using succinic anhydride: role of triethylamine in enhancement of catalytic activity, J Mol Catal B: Enzym, 27, 267-270 (2004) 90.Mohile S.S., Potdar M.K., Harjani J.R., Nara S.J. and Salunke M.M., Ionic liquids: efficient additives for Candida rugosa lipase-catalysed enantioselective hydrolysis of butyl 2-(4-chlorophenoxy)propionate, J Mol Catal B: Enzym, 30(2), 185-188 (2004) 91.Irimescu R. and Kato K., Lipase catalysed enantioselective reaction of amines with carboxylic acids under reduced pressure in non-solvent system and in ionic liquids, Tetrahedron Lett, 45, 523-525 (2004) 92.Ulbert O., Frater T., Belafi-Bako K. and Gobicza L., Enhanced enantioselectivity of Candida rugosa lipase in ionic liquids as compared to organic solvents, J Mol Catal B: Enzym, 31(1), 39-45 (2004) 93.Nara S.J., Mohile S.S., Harjani J.R., Naik P.U. and Salunke M.M., Influence of ionic liquids on the rates and regioselectivity of lipase-mediated biotransformations on 3,4,6-tri--acetyl glucal, J Mol Catal B: Enzym, 28(1), 39-43 (2004) 94.Lau R.M., Sorgedrager M.J., Carrea G., Rantwijk F.V., Secundo F. and Sheldon R.A., Dissolution of Candida antarctica lipase B in ionic liquids: effects on structure and activity, Green Chem, , 483-487 (2004)95.Kaftzik N., Kroutil W., Faber K. and Kragl U., Mandelate racemase activity in ionic liquids: scopes and limitations, J Mol Catal A: Chem, 214(2), 107-112 (2004)96.Noel M., Lozano P., Vaultier M. and Iborra J.L., Kinetic resolution of rac-2-pentanol catalysed by Candida antarctica lipase B in the ionic liquid, 1-butyl-3-methylimidazolium bis [(trifluoromethyl)sulfonyl]amide, Biotechnol Lett, 26, 301-306 (2004)97.Liu Y-Y, Lou W-Y, Zong M-H, Xu R., Hong X and Wu H, Increased enantioselectivity in the enzymatic hydrolysis of amino acid esters in the ionic liquid 1-butyl-3-methyl-imidazolium tetrafluoroborate, Biocatal Biotransform, 23(1), 89-95 (2005)98.van Rantwijk F., Secundo F. and Sheldon R.A., Structure and activity of Candida antarctica lipase B in ionic liquids, Green Chem, 282-286 (2006)99.Itoh T., Matsushita Y., Abe Y., Han S.H., Wada S., Morimoto M. and Hirose Y., Increased enantioselectivity and remarkable acceleration of lipase-catalysed Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(11), 103-116, November (2014) Res. J. Chem. Sci.International Science Congress Association 115 transesterification by using an imidazolium PEG-Alkyl sulfate ionic liquid, Chem Eur J, 12, 9228-9237 (2006)100.Wu X-M, Xin J-Y, Sun W. and Xia C-G, Environmentally friendly, efficient resolution of racemic secondary alcohols by lipase-catalysed enantioselective transesterification in ionic liquids in the presence of organic bases, Chem Biodev, , 183-188 (2007)101.Shah S. and Gupta M.N., Kinetic resolution of (±)-1phenylethanol in [Bmim][PF] using high activity preparations of lipases, Bioorg Med Chem Lett, 17, 921-924 (2007)102.De Los Rios A.P., Hernandez-Fernandez F.J., Martinez F.A., Rubio M. and Villora G., The effect of ionic liquid media on activity, selectivity and stability of Candida antarctica lipase B in transesterification reactions, Biocatal Biotransform, 25, 151-156 (2007)103. Martin J.R., Nus M., Sinisterra Gago J.V. and Sanchez-Montero J.M., Selective esterification of phthalic acids in two ionic liquids at high temperatures using a thermostable lipase of Bacillus thermocatenulatus: A comparative study, J Mol Catal B: Enzym, 52-53, 162-167 (2008)104. Hernandez-Fernandez F.J., De Los Rios A.P., Tomas-Alonso F., Gomez D. and Villora G., On the development of an integrated membrane process with ionic liquids for the kinetic resolution of rac-2-pentanol, J Mem Sci, 314, 238-246 (2008)105. Tan Z-Y, Wu H. and Zong M-H, Novozym 435-catalysed regioselective benzoylation of 1--D-arabinofuranosylcytosine in a co-solvent mixture of MIM. PF and pyridine, Biocatal Biotransform,25, 408-413 (2007)106. Sunitha S., Kanjilal S., Reddy P.S. and Prasad P.B.N., Ionic liquids as a reaction medium for lipase-catalysed methanolysis of sunflower oil, Biotechnol Lett,29, 1881-1885 (2007)107.Feher E., Illeova V., Kelemen-Horvath I., Belafi-Bako K., Polakovic M. and Gubicza L., Enzymatic production of isoamyl acetate in an ionic liquid-alcohol biphasic system, J Mol Catal B: Enzym, 50(1), 28-32 (2008)108.Liang J., Zeng W., Yao P. and Wei Y., Lipase catalysed regioselective synthesis of palmitoylglucose in ionic liquids, Advances Biol Chem, , 226-232 (2012)109.Ha S.H., Anh T.V., Lee S.H. and KOO Y.M., Effect of ionic liquids on enzymatic synthesis of caffeic acid phenethyl ester, Bioprocess Biosyst Engg, 35, 235-240 (2012)110.Galonde N., Brostaux Y., Richard G., Nott K. and Jerome C., Use of response methodology for the optimization of the lipase catalysed synthesis of mannosyl myristate in pure ionic liquid, Process Biochem, 48, 1914-1920 (2013)111.Novak U and Znidarsic-Plazl, Integrated lipase catalysed isoamyl acetate synthesis in a miniaturized system with enzyme and ionic liquid recycle, Green Process Synth,, 561-568 (2013) 112.Mohammad Fauzi AH, Amin NAS and Mat R, Esterification of oleic acid to biodiesel using magnetic ionic liquid: Multi-objective optimization and kinetic study, Applied Energy,114, 809-818 (2014)113. Erbeldinger M., Mesiano A.J., Rssel A.J., Enzymatic catalysis of formation of Z-aspartame in ionic liquid-an alternative to enzymatic catalysis in organic solvents, Biotechnol Prog,16, 1129-1131 (2000)114. Laszlo J.A. and Compton D.L., -Chymotripsin catalysis in imidazolium-based ionic liquids, Biotechnol Bioeng,75(2), 181-186 (2001)115. Lozano P., de Diego T., Guegan J.P., Vaultier M. and Iborra J.L., Stabilization of -Chymotripsin by ionic liquids in transesterification reactions, Biotechnol Bioeng,75, 563-569 (2001)116. Eckstein M., Sesing M., Kragl U. and Adlercreutz P., At low water activity -chymotripsin is more active in an ionic liquid than in non-ionic organic solvents, Biotechnol Lett, 24, 867-872 (2002)117. Lau R.M., van Rantwijk F., Seddon K.R., Sheldon R.A., Lipase-catalysed reactions in ionic liquids, Organ Lett, , 4189-4191 (2000)118. Schofer S.H., Kaftzik N., Wasserscheld P. and Kragl U., Enzyme catalysis in ionic liquids: lipase catalysed kinetic resolution of 1-phenethanol with improved enantioselectivity, Chem Commun, , 425-426 (2001)119. Park S. and Kazlauskas R.J., Improved preparation and use of room temperature ionic liquids in lipase-catalysed enantio- and regioselective acylations, J Org Chem, 66, 8395-8401(2001)120. Kim K.W., Song B., Choi M.Y. and Kim M.J., Biocatalysis in ionic liquids: markedly enhanced enantioselectivity of lipase, Org Lett,, 1507-1509 (2001)121. Lozano P., de Diego T., Carrie D., Vaultier M. and Iborra J.L., Over-stabilization of Candida antarcticalipase by ionic liquids in ester synthesis, Biotechnol Lett, 23, 1529-1533 (2001)122. Goderis H.L., Ampe G., Feyten M.P., Fouwe B.L., Guffens W.M., van Cauwenbergh S.M., Tobback P.P., Lipase-catalysed ester exchange reactions in organic media with controlled humidity, Biotechnol Bioeng, 30, 258-266 (1986)123. Eckstein M., Wasserscheid P. and Kragl U., Enhanced enantioselectivity of lipase from Pseudomonas sp. at high Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(11), 103-116, November (2014) Res. J. Chem. Sci.International Science Congress Association 116 temperatures and fixed water activity in the ionic liquid 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide, Biotechnol Lett, 24, 763-767 (2002) 124. Keim W., Waffenschmidt H. and Wasserscheid P., Stabilization of homogeneous catalysts for recycle during distillative product separation using an ionic liquid, DE 19 901 524 (Germany) (2000) 125. Huddleston J.G, Willlauer H.D., Swatloski R.P., Visser A.E. and Rogers R.D., Room-temperature ionic liquids as novel media for clean liquid-liquid extraction, Chem Comm 16: 1765-1766 (1998) 126. Kragl U., Eckstein N. and Kaftzil N., Enzyme catalysis in ionic liquids, Curr Opin Biotechnol, 13, 565-571 (2002)127. Brown R.A., Pollet P., McKoon E., Eckert C.A., Liotta C.L. and Jessop P.G., Asymmetric hydrogenation and catalyst recycling using ionic liquid and supercritical carbon dioxide, J Am Chem Soc, 123, 1254-1255 (2001)128. Liu F., Abrams M.B., Baker R.T. and Tumas W., Phase-separable catalysis using room temperature ionic liquids and supercritical carbon dioxide, Chem Comm,, 433-434 (2001)129. Sellin M.F., Webb P.B. and Cole-Hamilton D.J., Continious flow homogeneous catalysis: hydroformylation of alkenes in supercritical fluid-ionic liquid biphasic mixtures, Chem Comm,, 781-782 (2001)130. Webb P.B., Sellin M.F., Kunene T.E., Williamson S., Slawin A.M.Z. and Cole-Hamilton D.J., Continuous flow hydroformylation of alkenes in supercritical fluid-ionic liquid biphasic systems, J Am Chem Soc,125, 15577-15588 (2003)131. Boesmann A., Francio G., Janssen E., Solinas M., Leitner W. and Wasserscheid P., Activation, tuning and immobilization of homogeneous catalysts in an ionic/compressed CO continuous-flow system, Angew chem. Int Edn, 40, 2697-2699 (2001)132. Lozano P., De Diego T., Carrie D., Vaultier M. and Iborra J.L., Continuous green biocatalytic processes using ionic liquids and supercritical carbon dioxide, Chem Comm,, 692-693 (2002)133. Reetz M.T., Wiesenhofer W., Francio G. and Leitner W., Biocatalysis in ionic liquids: batchwise and continuous flow processes using supercritical carbon dioxide as mobile phase, Chem Commun, , 992-993 (2002)134. Lozano P., De Diego T., Carrie D., Vaultier M. and Iborra J.L., Lipase catalysis in ionic liquids and supercritical carbon dioxide at 150C, Biotechnol Prog,19, 380-382 (2003)135. Lozano P., De Diego T., Larnicol M., Vaultier M. and Iborra J.L., Chemoenzymatic dynamic kinetic resolution of rac-1-phenylethanol in ionic liquids and ionic liquids/supercritical carbon dioxide systems, Biotechnol Lett,28, 1559-1565 (2006)136.Pohar A., Plazl I., Znidarsic-Plazl P., Lipase-catalysed synthesis of isoamyl acetate in an ionic liquid/n-heptane two-phase system at the microreactor scale, Lab Chip , 3385-3390 (2009) 137.Fan Y., Qian J., Lipase catalysis in ionic liquids/supercritical carbon dioxide and its applications, J Mol Catal B: Enzym,66(1), 1-7 (2010)138.Chen B., Liu H., Guo Z., Huang J., Wang M., Xu X. and Zheng L., Lipase-catalysed esterification of Ferulic acid with oleyl alcohol in ionic liquid/isooctane binary systems, J Agric Food Chem,59, 1256-1263 (2011)