PRESENTED BY: BALVEER KAUR M.Sc. II BT {SEM III} PRESENTED TO : PARVEEN PAHUJA. 2-May-15 1

Transcription

1 PRESENTED BY: BALVEER KAUR M.Sc. II BT {SEM III} PRESENTED TO : PARVEEN PAHUJA 2-May-15 1

2 Introduction why are enzymes less active in organic solvents than in water? Modes of using enzymes in organic solvents Fundamentals of non aqueous enzymology Properties of enzymes in organic solvents Advantages Disadvantages Applications 2-May-15 2

3 Enzymes used in their natural aqueous media for production of chemicals & polymers Most of such compounds are insoluble in water, water frequently give unwanted side reactions & degrades organic reagents Such reactions possible only in organic solvents Thermodynamic equilibria of mostly these processes are unfavorable in water Technological utility of enzymes enhanced greatly in organic solvents 2-May-15 3

4 For eg: the proteases α-chymotrypsin & subtilisin have activities times lower in anhydrous octane than in water; the two enzymes are less active still in most other organic solvents. Reasons: Diffusion & accessibility factors Structural changes Substrate desolvation & transition state energy Conformational mobility ph situation 2-May-15 4

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7 Natural enzymes with organic solvent-tolerance are useful for employing in organic solvents. To find organic solvent tolerant enzymes, screening for microorganisms is done. First reported organic solvent-tolerant lipolytic enzyme from an organic solvent-tolerant bacterium, Pseudomonas aeruginosa. Then reported an organic solvent-tolerant proteolytic enzyme from an organic solvent tolerant bacterium, P. aeruginosa 2-May-15 7

8 SOLUBILIZED ENZYME PREPARATIONS SOLID STATE PREPARATIONS 2-May-15 8

9 PEGPPyethylene glycol A mono methoxy- PEG was allowed to react with cyan uric chloride so that 2 PEG molecules were bound to each cyan uric chloride residue. Amino groups on enzymes made a nucleophilic attack in the third activated position. In this way 2 PEG chains linked/ amino group modified Polyacrylates A polymer formed using acrylic acid, methyl methacrylate & 2- ethoxy ethyl methacrylate 2-May-15 9

10 NON COVALENTLY MODIFIED COMPLEXES Enzyme surfactants complexes eg: didodecyl glucosyly glutamate and Aerosol OT Enzyme polymer complexes eg: ethlycellulose, poly vinyl butyral & polyethylene glycol Surfactant coated Nano granules eg; Aerosol OT as surfactant & range of org. solvents soluble in nanogranules are toluene, acetone & ethanol 2-May-15 10

11 ENZYMES IN MICROEMULSIONS Surfactants & solvents eg: aerosol OT, CTAB chloroform Spectroscopic studies eg: Fluorescence & CD Detergent less micro emulsions Eg: Water, hexane & isopropanol 2-May-15 11

12 ENZYMES IMMOBILIZED ON SUPPORTS Immobilization method Mass transfer limitations Influence of pore size Direct effects of the support on the enzyme Effect of additives 2-May-15 12

13 Inorganic supports eg: controlled pore glass & diatomaceous earth (celite) Synthetic polymers eg: polyethene, polypropene, ion exchange resins, cross-linked polystyrene etc. Polysaccharide supports eg: Agarose gels, alginate gels, chitin etc. 2-May-15 13

14 Enzyme powders- lyophilization eg: Resolution of racemic mixtures using hydrolytic enzymes ( Lipase) ph control Inactivation during lyophilization eg: sorbitol ( lyoprotectants) Enzyme crystals : crosslinking with glutaraldehyde & stability increased towards the dimethoxyethane. Active site quantification eg: lyophilized chymotrypsin and subtilisin show that about 65% active site were accessible 2-May-15 14

15 WATER : Amount of water associated with the enzyme key determinant of the properties of enzymes. Effect of water on enzyme activity Water content in typical non aqueous enzyme system is usually as low as o.o1%. Small variation in water content changes the enzyme activity. Amount of water required for catalysis dependent on enzyme eg: lipases are highly active when few molecules are associated subtilisin & chymotrypsin - < 50 molecules of water/ enzyme molecule making an enzyme more hydrophobic by chemical modification can reduce the requirement of water for enzyme 2-May-15 15

16 Effect of water on protein mobility Water acts as plasticizer to increase the flexibility polarizability increases mobility also increases. Active site mobility increases upon addition of water eg: For subtilisin the increase in active site flexibility increases active site polarity 2-May-15 16

17 SOLVENT : solvent not only directly or indirectly affects the enzyme activity & stability but also changes the specificity. Effect of solvent on enzyme active centers: Solvent can affect the activity by disrupting the total number of active sites. Active site conc. of chymotrypsin in water not affected by addition of 3 dipolar solvents: 32% dioxan,14% acetone & 13% acetonitrile but only 2/3 of this is catalytically active in dry octane. Eg: active site of chymotrypsin in organic media is disrupted around 42% 2-May-15 17

18 Effect of solvent on substrates & products Solvents can also effect the conc. of substrates & products in aqueous layer around the enzyme & then affect the enzyme activity. 2-May-15 18

19 Substrate specificity. Enantio selectivity Chemo selectivity Regioselectivity Rigidity Enhanced substrate stability Ligand induced enzyme memory 2-May-15 19

20 Binding energy of an enzyme with substrate determined by the difference btw energy of ES complex & energy of enzyme & substrate in solution, binding is always influenced by solvent eg: substrate specificity of α-chymotrypsin, esterase, & subtilisin changed upon replacement of reaction medium with an organic solvent. The reversal of specificity in solvents was due to lack of hydrophobic interaction in non aqueous media. In fact, the substrate specificity of α-chymotrypsin in octane was reversed compared to that in water. Similar results with PEG modified chymotrypsin,trypsin & subtilisin in benzene. 2-May-15 20

21 Enzymatic enantio- and prochiral selectivities can be greatly influenced, and sometimes reversed in organic solvents Example : The enantioselectivity of α- chymotrypsin in the transesterification of methyl 3-hydroxy-2- phenylpropionate with propanol has been studied. The enzyme strongly prefers the S-enantiomer of the substrate in some solvents, the R-enantiomer is more reactive in others. Few methods exist that affect the enantio selectivity of enzymatic reactions : site directed mutagenesis, use of enantio selective inhibitors, coenzyme analogs, temperature & water miscible co solvents. 2-May-15 21

22 Enantio selectivity of the enzyme was lower in the solvents with higher hydrophobicity. Eg: Enantio selectivity of subtilisin, elastase, trypsin & α-chymotrypsin were lower in organic solvents different from that in water. 2-May-15 22

23 Ability to discriminate btw chemically distinct functional groups. Eg. Aspergillus niger lipase catalyzed acylation of 6- amino 1- hexanol proceeded with preference for hydroxyl group. This unexpected selectivity allowed the authors to produce monoesters of amino alcohols in good yield. Chemo selectivity of the enzyme affected by the reaction medium eg: the chemo selectivity of pseudomonas sp. Lipase in the acylation of N-αbenzoyl-L-lysinol with trifluoroethyl butyrate varied from 1.1 in tertbutyl alcohol to 21 in 1,2-dichloroethane 2-May-15 23

24 Few studies of effect of media on regioselectivity of enzymes. Rudio et.al reported that the reaction rates of P. cepacia lipase catalyzed transesterification of 9 with butanol in organic solvents differed significantly. 2-May-15 24

25 Organic solvents lack water s ability to engage in multiple hydrogen bonds,& have lower dielectric constants, leading to stronger intraprotein electrostatic interactions leading to rigidity. Addition of small quantities of water or glycerol or ethylene glycol helps increase flexibility. 2-May-15 25

26 Reason for enhanced thermo stability- Rigidity of molecules. Covalent processes such as deamination, peptide hydrolysis & cysteine decomposition require water. Ex:- porcine pancreatic lipase, lysozyme, chymotrypsin, mitochondrial cytochrome oxidase & ATPase. 2-May-15 26

27 Subtilisin lyophilized from aqueous solution containing various competitive inhibitors was 100 times more active in anhydrous solvents than the enzyme lyophilized in the absence of ligands Ligand-induced enzyme memory disappears when the enzyme is re-dissolved in water 2-May-15 27

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29 One of the imp property 'molecular memory' effect that leads to high conformational rigidity in organic solvents For example, lyophilized -chymotrypsin first dissolved in water and then diluted 100-fold with t-amyl alcohol has a specific activity of greater magnitude that of the same lyophilized enzyme directly suspended in that solvent containing the same 1% of water. As extra water is added to this suspension, presumably erasing the memory 2-May-15 29

30 When substrates have greater solubility in organic solvents Reduced risk of microbial growth Enhanced thermo-stability Relative ease of product recovery from organic solvents More energy efficient downstream processing when volatile solvents are used Ability to carry out new reactions impossible in water because of kinetic or thermodynamic restrictions Insolubility of enzymes in organic media 2-May-15 30

31 Inactivation of enzymes. Labour & cost-intensive preparation of biocatalysts in covalently modified systems. Mass-transfer limitations in case of heterogeneous systems or viscous solvents. 2-May-15 31

32 PRODUCTION OF INTERMEDIATES OF HERBICIDES & PHARMACEUTICALS. PRODUCTION OF ESTER FUELS. PRODUCTION OF POLYPHENOLS. 2-May-15 32

33 Enantiopure 2-chloro- and 2-bromo-propionic acids, used as intermediates for the synthesis of phenoxypropionic herbicides and of some pharmaceuticals have been obtained from yeast lipase catalysed enantioselective butanolysis in anhydrous solvents. 2-May-15 33

34 Production from coal-derived alcohols and fatty acids Phenolic tars from coal gasification wastes were converted to alcohol by treating with ethylene oxide and the intermediate alcohols were esterified with the fatty acids in a nonaqueous lipase system. Phenoxyethyl esters thus formed could be substituted for diesel fuels 2-May-15 34

35 Deals with peroxidase- catalyzed polymerization of phenols. Polyphenols thus formed are used as conventional phenol- formaldehyde resins as adhesives Also as laminates and photographic developers 2-May-15 35

36 Ionic liquids can be defined as salts that do not crystallize at room temperature Ionic liquids are possible green replacements for organic solvents because have no vapour pressure and, therefore, may be easier to efficiently reuse than organic solvents Ionic liquids are widely investigated for applications in organo-metallic catalysis 2-May-15 36

37 Enzyme activities in ionic liquids are generally comparable or sometimes higher than those observed in organic solvents In ionic liquids enhanced thermal and operational stabilities and regio- or enantioselectivities have been observed Ionic liquids permit to carry out enzyme-catalyzed reactions in non-aqueous media on polar substrates such as peptides, sugars, nucleotides, and biochemical intermediates A serious drawback of ionic liquids is represented by the fact that product isolation is more complex, especially for non-volatile materials 2-May-15 37

38 Gupta, M. N. (1992) Enzyme function in organic solvents. A.M Klibanov, A. M. (1988) Enzymatic Catalysis in Non-aqueous Solvent. NET sources: NZYME_CATALYSIS_IN_ORGANIC_SOLVENTS users.unimi.it/scdotchi/documents/lezioni/riva_ser gio/riva%20_organic%20solvents%20_%207_%20fund amentals.pdf syncozymes.com/chinese/bioresource/enzyme Immobilization-Papers/trends biotechnol,1997,15, pdf 2-May-15 38

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