2-May-151

PRESENTED BY: BALVEER KAUR

M.Sc. II BT {SEM III}

130181118

PRESENTED TO : PARVEEN PAHUJA

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

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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

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For eg: the proteases α-chymotrypsin & subtilisin have activities 104-105-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

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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

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SOLUBILIZED ENZYME PREPARATIONS

SOLID STATE PREPARATIONS

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PEGPPyethylene glycolA 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

PolyacrylatesA polymer formed using acrylic acid, methyl methacrylate &

2- ethoxy ethyl methacrylate

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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

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ENZYMES IN MICROEMULSIONS

Surfactants & solvents eg: aerosol

OT , CTABchloroform

Spectroscopic studies eg:

Fluorescence & CD

Detergent less micro emulsions

Eg:Water, hexane &

isopropanol

ENZYMES IMMOBILIZED ON SUPPORTS

Immobilization method

Mass transfer limitations

Influence of pore size

Direct effects of the support on the enzyme

Effect of additives

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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.

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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

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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 contentchanges the enzyme activity.

Amount of water required for catalysis – dependent onenzyme eg: lipases are highly active when few molecules areassociated

subtilisin & chymotrypsin - < 50 molecules of water/enzyme molecule

making an enzyme more hydrophobic by chemical modification can reduce the requirement of water for enzyme

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Effect of water on protein mobility

Water acts as plasticizer to increase theflexibility – polarizability increases – mobilityalso increases.

Active site mobility increases upon addition ofwater eg: For subtilisin the increase in activesite flexibility – increases active site polarity

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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%

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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.

Substrate specificity .

Enantio selectivity

Chemo selectivity

Regioselectivity

Rigidity

Enhanced substrate stability

Ligand induced enzyme memory

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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.

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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.

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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.

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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-α-benzoyl-L-lysinol with trifluoroethyl butyrate varied from 1.1 in tertbutyl alcohol to 21 in 1,2-dichloroethane

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Few studies of effect of media on regioselectivity of enzymes.

Rudio et.al reported that the reaction rates of P. cepacialipase catalyzed transesterification of 9 with butanol in organic solvents differed significantly.

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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.

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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.

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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

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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

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

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Inactivation of enzymes.

Labour & cost-intensive preparation of biocatalysts in covalently modified systems.

Mass-transfer limitations in case of heterogeneous systems or viscous solvents.

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PRODUCTION OF INTERMEDIATES OF HERBICIDES & PHARMACEUTICALS.

PRODUCTION OF ESTER FUELS.

PRODUCTION OF POLYPHENOLS.

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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.

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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

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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

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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 vapourpressure and, therefore, may be easier to efficiently reuse than organic solvents

Ionic liquids are widely investigated for applications in organo-metallic catalysis

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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

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Gupta, M. N. (1992) Enzyme function in organic solvents.

A.M Klibanov, A. M. (1988) Enzymatic Catalysis in Non-aqueous Solvent.

NET sources: http://biowiki.ucdavis.edu/Biochemistry/Catalysis/E

NZYME_CATALYSIS_IN_ORGANIC_SOLVENTS users.unimi.it/ScDotChi/documents/lezioni/riva_ser

gio/Riva%20_Organic%20solvents%20_%207_%20fundamentals.pdf

syncozymes.com/chinese/bioresource/Enzyme Immobilization-Papers/trends biotechnol,1997,15,97-101.pdf

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