Methods in Non-Aqueous Enzymology

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Munishwar 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

Issues and Perspectives
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

for Enzyme Catalysis in NonAqueous Organic Systems
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
Preparation of Enzyme Derivatives with Improved Stability in Organic Media
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
Immobilization of Lipases for Use in NonAqueous Reaction Systems
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
Applications of Enzymes and Membrane Technology in Fat and Oil Processing
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
Strategies for Improving the Lipase Catalyzed Preparation of Chiral Compounds
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
Peptide Synthesis in NonAqueous Media
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
0 Enzyme Selectivity in Organic Media
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
Sugar Transformations Using Enzymes in NonAqueous Media
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
Nterminal Acylation of RNase A and its Characterization
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
Analysis in NonAqueous Milieu Using Thermistors
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
Biomedical Implications
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
Limitations and pitfalls of the modelsapproach
208
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