Methods in Non-Aqueous EnzymologyMunishwar N. Gupta Springer Science & Business Media, 01.07.2000 - 218 Seiten Extending the range of enzymatic catalysis by using non-aqueous media has now developed into a powerful approach in biochemistry and biotechnology. One peculiar feature which distinguishes it from the conventional enzymology (carried out in aqueous buffers) is that the awareness of different parameters that control and influence the behaviour of enzymes in such environments has emerged rather slowly. Science is about being able to repeat what somebody else has done. Absence of knowledge about such well-defined parameters/fac tors has sometimes made some workers rather cautious and diffident about using this approach in their laboratories. But for this, non-aqueous enzymol ogy would be more widely practised. It is these thoughts that made me feel that the availability of some well-defined protocols for various applications invol ving enzymes in non-aqueous environments would further catalyze the growth of this area. Hence this book, in which each chapter has some protocols in a specific area. The protocols are preceded by brief background material. The early chapters, which are of general importance, concern control of water ac tivity and stabilization via immobilization. Some subsequent chapters provide the protocols for transformations involving lipids and carbohydrates, peptide synthesis, and preparation of chiral compounds. The disproportionate focus on lipases is not a coincidence; this class of enzymes has been used more often than others in non-aqueous enzymology. |
Inhalt
1 | |
Enzyme structure in nonaqueous environments | 3 |
Molecular imprinting in anhydrous organic solvents | 5 |
Using antibodies in organic solvents | 6 |
Catalysis by whole cells in organic media | 7 |
Protein folding in nonaqueous media | 8 |
Conclusions | 9 |
Acknowledgments | 10 |
Materials Chemicals | 97 |
Methods 31 Immobilization oflipase | 98 |
Protocol 2 Desymmetrization of mesodiacetates | 100 |
Protocol 3 Resolution of 3hydroxy esters in organic solvents | 101 |
Protocol 4 Online measurement of the conversion in lipasecatalyzed kinetic resolutions in supercritical carbon dioxide | 102 |
Troubleshooting | 103 |
52 Kinetic resolutions of secondary alcohols | 105 |
Acknowledgments | 106 |
14 | |
12 Thermodynamic water activity aw | 15 |
13 Catalytic selectivity and specificity | 16 |
Asymmetric synthesis | 17 |
Materials Chemicals | 20 |
Enzymes | 21 |
Methods 31 Protocols Protocol 1 Addition of water | 23 |
Protocol 3 Salt hydrates | 24 |
Protocol 4 Resolution by lipase catalysed transesterification | 25 |
Troubleshooting | 26 |
Remarks and Conclusions 51 Controlled addition of water or drying | 27 |
52 Saturated salt solution | 29 |
53 Salt hydrates | 30 |
54 Significance of water activity for E and Keq | 32 |
References | 33 |
36 | |
Introduction | 37 |
rigidification of enzymes via multipoint covalent immobilization on preexisting supports | 38 |
creation of hydrophilic nanoenvironments fully surrounding immobilized enzyme molecules | 40 |
13 Immobilizationstabilization of Pig Liver Esterase PLE | 41 |
Methods 31 Protocols Protocol 1 Activation of Agarose gels | 42 |
Protocol 2 Preparation of aldehyde dextran | 43 |
Protocol 3 Immobilization of PLE on glutaraldehydeagarose | 44 |
Protocol 5 Immobilization of poly ethylenimine on PLEagarose | 45 |
41 Multipoint immobilization of proteins on glyoxyl agarose at pH 100 | 46 |
52 Effect of different cosolvents on stability of PLE derivatives | 47 |
53 Effect of cosolvent concentration | 48 |
54 Stability of fully hydrophilized immobilized derivatives | 49 |
Acknowledgments | 50 |
52 | |
Materials Chemicals | 53 |
Enzymes | 54 |
32 Esterification assay octyl oleate | 55 |
Protocol 1 Adsorption on hydrophobic surfaces | 56 |
Protocol 3 Precipitationdrying onto porous silica Celite | 57 |
Troubleshooting | 58 |
52 Hydrophilic vs hydrophobic supports | 59 |
54 Activity vs lipase loading | 60 |
55 Effect of protein treatment on Accurel EP100 catalysts | 61 |
56 Ionexchange resins | 63 |
57 Precipitation drying onto porous silica Celite | 64 |
58 Solgel immobilization | 65 |
59 Effect of water activity Aw on esterification reaction rate | 66 |
Acknowledgments | 67 |
70 | |
Introduction | 71 |
Lipase based hydrolysis | 72 |
Interesterification | 73 |
Materials Chemicals | 74 |
Enzymes | 75 |
Methods 31 Protocols Protocol 1 Surfactant modification of lipases 37 | 76 |
Protocol 3 Palm oil hydrolysis by surfactant modified lipase 37 | 77 |
Protocol 5 Interesterification of triglycerides and fatty acids by lipase in a hollowfiber reactor 40 | 78 |
Troubleshooting | 80 |
Applications 51 Application of modified lipase reactions Hydrolysis activity of surfactant modified lipase | 81 |
Interesterification activity of surfactant modified lipase | 82 |
52 Application of membrane technology | 83 |
Remarks and Conclusions 61 Modification of I i puses | 85 |
64 Modification of lipases by stearic acid | 86 |
Acknowledgments | 87 |
References | 88 |
90 | |
Introduction 11 Lipases | 91 |
12 Choice of solvent system | 92 |
13 Choice of acyl donor | 93 |
14 Influence of immobilization on enantioselectivity and activity | 95 |
15 Kinetic resolution vs asymmetric syntheses | 96 |
110 | |
Introduction | 111 |
Materials Chemicals | 114 |
Solutions | 115 |
32 Fixing initial water activity | 116 |
Reaction media equilibration | 117 |
Separation | 118 |
36 HPLC analysis | 119 |
Protocol 1 Enzymatic synthesis of ZTyrGlyGlyPheLeuNH2 | 120 |
Protocol 2 Enzymatic synthesis of ZTyrGlyGlyPheLeuOEt | 122 |
NH2 OEt _ | 124 |
Troubleshooting | 125 |
51 Synthetic strategy and enzyme selection | 126 |
53 Biocatalyst configuration | 127 |
Substrate carboxyl component | 128 |
Nucleophile structure | 129 |
Acknowledgments | 130 |
133 | |
Materials Chemicals | 134 |
Methods 31 Protocols Protocol 1 General procedure for the preequilibration of the reagents | 135 |
Protocol 3 Oxidation of 4substitutedphenols to 4substitutedotzoqui nones | 136 |
Protocol 6 Transesterification of secphenethyl alcohol Method 61 | 137 |
Remarks and Conclusions | 138 |
Acknowledgments | 144 |
146 | |
Materials Chemicals | 149 |
Methods 31 Adsorption of lipase PS on celite | 150 |
Protocol 2 Enzymatic synthesis of benzyl 604pentenyloxycarbonyl20 levulinylftlactose 16 | 151 |
nic acid Regioselective enzymatic acylation of the benzylamide of lactobio | 152 |
Protocol 4 Regioselective acetylation of rutin 18 | 153 |
Protocol 6 Enzymatic synthesis of allyl pDglucopyranoside 13 | 154 |
Remarks and Conclusions 41 Hydrolasescatalyzed acylation and deacylation of sugars | 155 |
42 Glycosidasescatalyzed synthesis of alkyl glycopyranosides | 157 |
Acknowledgments | 158 |
160 | |
Materials Chemicals | 162 |
Methods 31 Protocols Protocol 1 RNase A modification | 163 |
Protocol 3 Characterization of acylated derivatives | 164 |
Remarks and Conclusions | 165 |
42 Acylated derivatives purification and characterization | 166 |
43 Conclusions | 170 |
Acknowledgments | 171 |
174 | |
11 Fundamentals of calorimetric devices | 175 |
12 Principle of calorimetric measurements | 176 |
13 Selection of specific solvents for thermal biosensing | 178 |
Thermometry coupled to optoacoustic analysis | 179 |
Materials Chemicals | 180 |
Methods Protocol 1 Thermometric sensing | 182 |
Protocol 2 Optoacoustic sensing | 183 |
Remarks and Conclusions 51 General remarks | 184 |
52 Glucose biosensor | 185 |
53 Peroxide biosensor | 186 |
54 Penicillin biosensor | 190 |
55 Biosensors in peptide synthesis | 191 |
56 Fatty acid biosensors | 192 |
References | 193 |
195 | |
Misfolding of proteins | 198 |
21 Effect of the medium and the cosolvent on protein folding | 200 |
Corrective action in cells | 202 |
33 Influence of the pro region | 203 |
35 Mechanism of phairpin formation and folding dynamics | 204 |
Conclusion | 206 |
208 | |
Andere Ausgaben - Alle anzeigen
Häufige Begriffe und Wortgruppen
Accurel acetonitrile acyl acyl donor Adlercreutz adsorption alcohol amino acid antibodies applications aqueous assay biocatalyst Biochem biosensors Biotechnol Bioeng buffer butanoate Candida antarctica catalyst catalyzed Celite Chem Soc chemical chromatography concentration cosolvent covalent derivatives dissolved effect enantiomer enantiomeric enantiomeric excess enantioselectivity enzyme Enzyme Microb Technol esterification esters ethyl fatty acid Figure fractal ganic glycerol groups hexane HPLC hydrolysis hydrophilic hydrophobic immobilized lipase increase interesterification kinetic resolution Klibanov Lett lipase lipase loading lipase-catalyzed lyophilized membrane method miehei misfolded mmol modification modified lipase molecules non-aqueous nucleophile obtained oils organic media organic phase organic solvents oxidase pase peptide synthesis phosphate preparation proteases protein folding Protocol reaction mixture reactor regioselectivity residues reversed micelles RNase salt hydrates samples saturated sodium soluble solution stability stearic acid structure substrate subtilisin surface surfactant temperature Tetrahedron tion transesterification water activity water content yield zyme
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