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TITLE
DEDICATION
CERTIFICATE
DECLARATION
ACKNOWLEDGEMENT
ABBREVIATIONS
CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF PLATES
1. INTRODUCTION
1.1. Preface
1.2.Review of literature
1.2.1. Sources of microbial α-amylase
1. Sources of microbial amylase
1.2.2. Production of amylases
2. α-amylase production by microorganisms under submerged fermentation
3. α-amylase production by microorganisms under solid state fermentation
1.2.3. Mutation and recombinant DNA technology in strain improvement
4. Expression of cloned genes for microbial amylases
1.2.4. Purification of α-amylase
5. Purification strategies employed for α-amylase
6. Properties of microbial α-amylase
1.2.5. Application of α-amylase
1.2.5.1. Application of enzyme based detergents
1.2.5.2.Textile industry
1.2.5.3. Clarification of fruit juice for jelly manufacture
1.2.5.4. Use of fungal amylase in baby foods
1.2.5.5. Production of chocolate syrup
1.2.5.6. Production of high conversion syrups
1.2.5.7.Alcohol industry
1.2.5.8. Paper industry
1.2.5.9. Analysis in medicinal and clinical chemistry
1.3. Objectives of the present study
2. MATERIALS AND METHODS
2.1.Source of the Organism
2.1.1. Preparation of the medium
2.1.2. Plating procedure
2.1.3. Isolation, purification and maintenance of amylolytic fungi
2.1.4. Identification of fungi
2.2.Selection of amylase producing fungi for further studies
2.2.1. Media
2.2.2. Preparation of inoculum
2.2.3. Inoculation and incubation
2.2.4. Enzyme recover
2.2.5. Assays
2.2.5.1 α-amylase
2.2.5.2 Protein
2.2.6. α-amylase production under solid state fermentation using natural substrates
2.2.6.1. Microorganism
2.2.6.2. Preparation of solid substrate medium
2.2.6.3. Preparation of inoculum
2.2.6.4. Inoculation and incubation
2.2.6.5. Enzyme recovery
2.2.6.6. Enzyme assay
2.2.7. Impact of process parameters on α-amylase production during solid state fermentation
2.2.7.1 Initial pH of the medium
2.2.7.2. Incubation temperature
2.2.7.3. Moisture content
2.2.7.4. Particle size of the substrate
2.2.7.5. Incubation time
2.2.7.6. Additional carbon source
2.2.7.7. Inorganic nitrogen source
2.2.7.8.Organic nitrogen source
2.2.7.9. Mineral salts
2.2.7.10. Amino acids
2.2.7.11. Surfactants
2.2.7.12. Vitamins
2.2.7.13. Sugar alcohols
2.2.7.14.Sodium chloride concentration
2.2.7.15. Sodium salts of organic acids
2.2.7.16. Organic acids
2.2.7.17. Inhibitors
2.2.8. Effect of storage temperature on α-amylase activity
2.3 Enzyme purification
2.3.1.Ammonium sulphate fractionation
2.3.2. Dialysis
2.3.3. Anion exchange chromatography
2.3.4. Electrophoresis
2.3.4.1. Stock solutions
2.3.4.2. Sample preparation
2.3.4.3. Protein staining
2.3.5. Characterisation of the purified enzyme
2.3.5.1. Effect of pH on enzyme activity and stability
2.3.5.2. Effect of temperature on enzyme activity and stability
2.3.5.3. Substrate specificity
2.3.5.4. Effect of enzymatic inhibitors on α-amylase activity
2.3.5.5. Effect of different metal ions on enzyme activity at a concentration of 1mM
2.3.6. Compatibility with various commercial detergents
3. RESULTS
3.1. Enumeration and isolation of amylase producing fungi
3.1.1. Selection of potent amylase producing fungi
7. Different strains of amylolytic fungi
1. Amylase activity of Penicillium SBSS 1 at different hours of incubation under SSF using wheat bran and SMF on mineral salt starch medium at room temperature
2. Amylase activity of Saccharomyces SBSS 2 at different hours of incubation under SSF using wheat bran and SMF on mineral salt starch medium at room temperature
3. Amylase activity of Rhizopus SBSS 3 at different hours of incubation under SSF using wheat bran and SMF on mineral salt starch medium at room temperature
4. Amylase activity of Mucor SBSS 4 at different hours of incubation under SSF using wheat bran and SMF on mineral salt starch medium at room temperature
5. Amylase activity of Neurospora SBSS 5 at different hours of incubation under SSF using wheat bran and SMF on mineral salt starch medium at room temperature
6. Amylase activity of Mucor SBSS 6 at different hours of incubation under SSF using wheat bran and SMF on mineral salt starch medium at room temperature
7. Amylase activity of Aspergillus SBSS 7 at different hours of incubation under SSF using wheat bran and SMF on mineral salt s arch medium at room temperature
8. Amylase activity of Aspergillus SBSS 8 at different hours of incubation under SSF using wheat bran and SMF on mineral salt starch medium at room temperature
9. Amylase activity of Penicillium SBSS 9 at different hours of incubation under SSF using wheat bran and SMF on mineral salt starch medium at room temperature
10. Amylase activity of Neurospora SBSS 10 at different hours of incubation under SSF using wheat bran and SMF on mineral salt starch medium at room temperature
8. Amylase production by the top ten ranked strains (strains which showed high levels of amylolytic activity in terms of maximal clearing zones on starch agar medium) under submerged fermentation using mineral salt starch medium for 96 hours at room temperature
9. Amylase production by the top ten ranked strains (strains which showed high levels of amylolytic activity in terms of maximal clearing zones on starch agar medium) under solid state fermentation using wheat bran for 96 hours at room temperature
3.2. Impact of process parameters on α-amylase production during solid state fermentation
3.2.1. Effect of initial pH of the medium
10. Effect of pH on α-amylase production by Penicillium janthinellum under SSF using wheat bran
3.2.2. Effect of incubation temperature
11. Effect of pH on a-amylase production by Penicillium janthinellum under SSF using wheat bran
11. Effect of temperature on α-amylase production by Penicillium janthinellum urder SSF using wheat bran
12. Effect of temperature on a-amylase production by Penicillium janthinellum under SSF using wheat bran.
3.2.3. Effect of moisture content
12. Effect of moisture content on α-amylase production by Penicillium janthinellurr under SSF using wheat bran
3.2.4. Effect of particle size
13. Effect of moisture content on α-amylase production by Penicillium janthinellum under SSF using wheat bran
13. Effect of particle size on α-amylase production by Penicillium janthinellum under SSF using wheat bran
14. Effect of particle size on α-amylase production by Penicillium janthinellum under SSF using wheat bran
3.2.5. Effect of incubation time
14. Effect of incubation time on α-amylase production by Penicillium janthinellum under SSF using wheat bran
3.2.6. Effect of additional carbon source
15. Effect of incubation time on α-amylase production by Penicillium janthinellum under SSF using wheat bran
15. Effect of additional carbon source on α-amylase production by Penicillium janthinellum under SSF using wheat bran
16. Effect of additional carbon source on α-amylase production by Penicillium janthinellum under SSF using wheat bran
3.2.7. Effect of inorganic nitrogen source
16. Effect of inorganic nitrogen source on α-amylase production by Penicillium janthinellum under SSF using wheat bran
3.2.8. Effect of organic nitrogen source
17. Effect of inorganic nitrogen source on α-amylase production by Penicillium janthinellum under SSF using wheat bran
17. Effect of organic nitrogen source on α-amylase production by Penicillium janthinellum under SSF using wheat bran
18. Effect of organic nitrogen source on α-amylase production by Penicillium janthinellum under SSF using wheat bran
3.2.9. Effect of mineral salts
18. Effect of mineral salts on α-amylase production by Penicillium janthinellum under SSF using wheat bran
3.2.10. Effect of amino acids
19. Effect of mineral salts on α.-amylase production by Penicillium janthinellum under SSF using wheat bran
19. Effect of amino acids on α-amylase production by Penicillium janthinellum under SSF using wheat bran
20. Effect of amino acids on α-amylase production by Penicillium janthinellum under SSF using wheat bran
3.2.11. Effect of surfactants
20. Effect of various surfactants on α-amylase production by Penicillium janthinellum under SSF using wheat bran
3.2.12. Effect of vitamins
21. Effect of various surfactants on α-amylase production by Penicillium janthinellum under SSF using wheat bran
21. Effect of vitamins on α-amylase production by Penicillium janthinellum under SSF using wheat bran
22. Effect of vitamins on α-amylase production by Penicillium janthinellum under SSF using wheat bran
3.2.13. Effect of sugar alcohols
22. Effect of sugar alcohols on α-amylase production by Penicillium janthinellum under SSF using wheat bran
3.2.14. Effect of NaCl concentration
23. Effect of sugar alcohols on α-amylase production by Penicillium janthinellum under SSF using wheat bran
23. Effect of NaCI concentration on α-amylase production by Penicillium janthinellum under SSF using wheat bran
24. Effect of NaCl concentration on α-amylase production by Penicillium janthinellum under SSF using wheat bran
3.2.15. Effect of sodium salts of organic acids
24. Effect of sodium salts of organic acids on α-amylase production by Penicillium janthinellum under SSF using wheat bran
3.2.16. Effect of organic acids
25. Effect of sodium salts of organic acids on α-amylase production by Penicillium janthinellum under SSF using wheat bran
25. Effect of organic acids on α-amylase production by Penicillium janthinellum under SSF using wheat bran
3.2.17. Effect of various inhibitors
26. Effect of organic acids on α-amylase production by Penicillium janthinellum under SSF using wheat bran
26. Effect of various inhibitors on α-amylase production by Penicillium janthinellum under SSF using wheat bran
27. Effect of various inhibitors on α-amylase production by Penicillium janthinellum under SSF using wheat bran
3.3. Effect of storage temperature on α-amylase activity
27. Effect of storage temperature on α-amylase activity of Penicillium janthinellum
28. Effect of storage temperature on α-amylase production by Penicillium janthinellum under SSF using wheat bran
3.4. Purification of α-amylase
29. Elution profile- DEAE column chromatography
28. Purification of α-amylase produced by Penicillium janthinellum
1. Polyacrylamide gel electrophoresis (Native PAGE)
2. SIDS Polyacrylamide gel electrophoresis (SDS-PAGE)
3.5. Enzyme characteristics
3.5.1. Effect of temperature on enzyme activity and stability
30. Effect of temperature on α-amylase activity
3.5.2. Effect of pH on enzyme activity and stability
31. Effect of temperature on α-amylase stability
3.5.3. Substrate specificty
32. Effect of pH on α-amylase activity
33. Effect of pH on α-amylase stability
3.5.4. Effect of enzyme inhibitors on enzyme activity
34. Substrate specificity of α-amylase produced by Penicillium janthinellum
3.5.5. Effect of different metal ions on amylase activity at a concentration of 1 mM
35. Effect of enzyme inhibitors on amylase activity
3.6.Compatibility with various commercial detergents
36. Effect of different metal ions on amylase activity
37. Effect of detergents on activity of α-amylase from Penicillium janthinellum in presence and in absence of 20mM CaCI2 during 30 minutes contact time
4. DISCUSSION
4.1. Isolation, purification and maintenance of amylolytic fungi
4.2. α-amylase production under solid state fermentation using wheat bran
4.3. Purification and characterisation of α-amylase
4.4. Compatibility with various commercial detergents
4.5. Conclusion
5. SUMMARY
6. REFERENCES
APPENDIX