Enzymes do not notice all water molecules in solution, just the ones that are ... Filter off enzyme, evaporate the solvent and separate product from remaining ...
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
Biocatalysis in organic solvents Improving enzymes by using them in monophasic organic solvents • Alexander Klibanov MIT, Cambridge, USA • NATURE (2001) 409: 241-246
Biocatalysis in organic solvents Principle • Enzymes do not notice all water moleculesin solution, just the ones that are nearby • Replace bulk water with organic solvent • Moist, solid enzyme, suspended in organic solvent
Biocatalysis in organic solvents Advantages • Easy downstream processing Filter off enzyme, evaporate the solvent and separate product from remaining starting material • No need for immobilisation • Shift thermodynamic equilibria • Change enzyme selectivity
Biocatalysis in organic solvents Solid enzymes • Catalytically active when water content < 2% Precautions • Stirring, shaking or sonication needed for S-diffusion • Optimise micro-pH around enzyme • Control water activity • Use correct hydrophilicity organic solvent
Biocatalysis in organic solvents Diffusional limitation • Crystalline, lyophilized, precipitated or adsorbed enzyme • Sufficient mobility required to allow minor conformational changes (formation ES-complex) • Substrate channeling occurs when sufficient agitation is provided
Biocatalysis in organic solvents Lyophilization • Dehydration may change enzyme structure • Use of lyoprotectants, such as sugars, PEG, certain inorganic salts, substrate-resembling ligands and crown ethers • Activation up to four orders of magnitude
Biocatalysis in organic solvents Effect of pH Fig. 5.8 • pH measurements not easy • Ionization sate of enzyme determines its conformation, activity and selectivity • Employ solid enzymes that have been recovered from lyophilization or precipitation from a buffer at their pH optimum: pH memory
Biocatalysis in organic solvents Effect of water • How much water is required to retain catalytic activity? • How can we define the amount of water in the reaction mixture? • How can we control water activity in the reaction mixture?
Biocatalysis in organic solvents How much water is required to retain catalytic activity? • Enzyme dependent (chymotrypsin, tyrosinase) • Tightly bound water remains present, even after lyophilization • Often an optimal water content can be found Fig. 5.9
Biocatalysis in organic solvents Reaction rate vs. stability • Low rate at low water content Fig. 5.9 • Stability high at low water content Fig. 5.10 • Water is involved in inactivation reactions • Water increases enzyme flexibility unfolding
Biocatalysis in organic solvents How can we define the amount of water in the reaction mixture? • Concentration or volume percentage not useful • Degree of hydration (enzyme bound water) • Thermodynamic water activity aw
Biocatalysis in organic solvents Water activity • Pv (water-solvent) / Pv (water-water) • Determines how much water is bound to the enzyme • Determines the catalytic activity to a great extent • Determines the effect of water on the chemical equilibrium position
Biocatalysis in organic solvents Fixed water activity • Allows to investigate the influence of solvent on enzyme catalysis • Corrects for different degree of hydration due to different solubility (cf. hexane - ethyl acetate) • At a known water activity the hydration of enzyme is fixed
Biocatalysis in organic solvents How can we control water activity in the reaction mixture? • Pre-equilibration of both enzyme and substrate solution in atmospheres of controlled water activity (before mixing) • Range of water activities can be obtained by using different saturated salt solutions
Biocatalysis in organic solvents The nature of the organic solvent • Apolar water-immiscible solvents (log P > 4) • Laane et al. 1987 Fig. 5.11 • No clear correlation in the region 1 < log P < 4 • Influence diëlectric constant Fig. 5.12
Biocatalysis in organic solvents Influence diëlectric constant • Low reduced enzyme flexibility • Medium optimal flexibility and activity • High polar solvent strips enzyme water layer
Biocatalysis in organic solvents Hydrophobic active sites like hydrophobic substrates • Rate enhancement of enzymes in waterbecause the substrate wants to partition from water into the active site • In organic solvent the substrate is no longer squeezed out of the medium owing to the hydrophobic effect and the energetic advantages of partioning drop lower rates
Biocatalysis in organic solvents Energy of the transition state • Many enzymes form charged tetrahedral reaction intermediates (polar transition state) • Stabilised by internally bound water • Shielded from solvent • Diminished activity in organic solvent
Biocatalysis in organic solvents Probing of biochemically significant enzyme intermediates • Generation and stabilization of horseradish peroxidase compound II in neat benzene solution at room temperature • Patricia Mabrouk JACS (1995) 117, 2141-2146 • PEGylated enzyme: highly soluble !
Biocatalysis in organic solvents Effects on enzyme selectivity • Change in substrate, enantiomeric, prochiral, regio- and chemoselectivities • Substrate recognition • Proteases: driving force of enzyme-substrate binding is hydrophobic interactions between the side chain of amino-acid substrate and enzyme active site
Biocatalysis in organic solvents Effects on regio-and chemoselectivity • Lipases • Preference for distinct functional group in substrate molecule • Acylation of hydroxyl group in relation to amino group favored in organic solvent
Biocatalysis in organic solvents Effects on enzyme selectivity • Change in product specificity • Hydroxylation efficiency of vanillyl-alcohol oxidase with 4-alkylphenols • Organic solvent influences access of water to the active site
Biocatalysis in organic solvents Medium engineering Tailoring the medium composition to optimise the reaction yield • Effects on enzyme activity and stability • Requirements for water, pH and buffer capacity • Solubility and stability substrate and products • Recovery of products, separation of biocatalyst and removal of substrate
Biocatalysis in organic solvents Protein engineering Site-directed mutagenesis Random mutagenesis • Minimisation of surface charges, enhancement of internal polar interactions, disulfide bonds etc. • Change of substrate specificity, binding affinity or stereospecificity