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TITLE
DEDICATION
CERTIFICATE 1
CERTIFICATE-2
DECLARATION
ACKNOWLEDGEMENT
CONTENTS
ABBREVIATIONS
1. INTRODUCTION
2. REVIEW OF LITERATURE
Classification of Microbial Proteases
Serine Proteases
Thiol Proteases
Acid (Carboxyl) Proteases
Metallo Proteases
Commercial Applications
Extracellular Alkaline Protease Production by Bacteria
Screening and Selection of Strains
Methods of Production
Bacterial Alkaline Protease Production by Submerged Fermentation
Table 1.Bacteria in which the extracellular alkaline protease production by SmF has been studied
Factors influencing alkaline proterase production by bacteria by submerged fermentation
Growth phase
Carbon source
Nitrogen source
Temperature
pH
Mineral salts
Age of inoculum
Size of inoculum
Agitation
Aeration
Incubation period
Two phase fermentation systems
Fed batch fermentation
Bacterial Alkaline Protease Production by Solid State Fermentation
Bacillus amyloliquefaciens
Bacillus licheniformis
Pseudomonas sp.
Purification and Characterization of Bacterial Alkaline Proteases
Table 2. Bacterial sources from which the extracellular alkaline serine proteases have been isolated, purified and studied
Molecular mass
Isoelectric point
Optimum pH and temperature for activity
Effect of metal ions on activity
pH and themostability
Stability towards detergents
Km value
3. SCREENING AND SELECTION OF STRAINS FOR ALKALINE PROTEASE PRODUCTION
Section A - Screening and Selection of Strain for Alkaline Protease Production by Submerged Fermentation
MATERIALS AND METHODS
Bacterial strains
Enzyme assay
Selection of high yielding strains
Identification of the highest yielding strains
Monitoring the stability of the highest yielding strains
RESULTS
Selection of high yielding strains
Table 3. Extracellular alkaline protease production by the top five high yielding strains and NCIM strains
Identification of the highest yielding strains
Table 4. Characters shown by Bacillus sp. K 25
Stability of the highest yielding strains
DISCUSSION
Section B - Screening and Selection of Strain for Alkaline Protease Production by Solid State Fermentation
MATERIALS AND METHODS
Bacterial strains
Selection of high yielding strains
Step 1 -Preliminary SSF studies
Step 2 -Detailed SSF studies
identification of the highest yielding strain
Monitoring the stability of the highest yielding strain
RESULTS
Selection of high yielding strains
Preliminary SSF studies
Table 5. Alkaline protease production by top five high yielding strains and NCIM strains in preliminary SSF studies
Detailed SSF studies
Table 6. Alkaline protease production by the high yielding strains in solid state fermentation systems with different moisture levels, incubated for different periods
Identification of the highed yielding strain
Stability of the highest yielding strain
DISCUSSION
4. OPTIMIZATION OF CONDITIONS FOR THE PRODUCTION OF ALKALINE PROTEASES
Section A - Optimization of Conditions for the Production of Alkaline Protease by Bacillus sp. K 25 by the Submerged Fermentation Method
MATERIALS AND METHODS
Growth phase
Temperature of Incubation
Carbon and nitrogen sources
Salts
Age of lnoculum
Size of inoculum
Agitation
Incubation period
RESULTS
Effect of growth phase
Fig. 1. Time course of growth and alkaline protease production by Bacillus sp. K 25
Effect of temperature of incubation
Fig. 2. Effect of temperature of incubation on alkaline protease production by Bacillus sp. K 25
Effect of pH
Fig. 3. Effect of initial pH of medium on alkaline protease production by Bacillus sp. K 25
Effect of carbon and nitrogen sources
Table 7. Effect of different carbon sources on alkaline protease production by Bacillus sp. K 25
Table 8. Effect of various nitrogen sources on alkaline protease production by Bacillus sp. K 25
Table 9. Effect of using starch and soyabean meal at different concentrations on alkaline protease production by Bacillus sp. K 25
Effect of salts
Table 10. Effect of using different concentrations of sodium chloride on alkaline protease production by Bacillus sp. K 25
Table 11. Effect of different salts on alkaline protease production by Bacillus sp. K 25
Table 12. Effect of different concentrations of calcium chloride on alkaline protease production by Bacillus sp. K 25
Table 13. Effect of age of inoculum on alkaline protease production by Bacillus sp. K 25
Effect of size of inoculum
Table 14. Effect of inoculum size on alkaline protease production by Bacillus sp. K 25
Effect of agitation
Table 15. Effect of agitating the culture on alkaline protease production
Effect of period of incubation
Table 16. Effect of incubation period on alkaline protease accumulation in the culture of Bacillus sp. K 25
DISCUSSION
Section B - Optimization of Conditions for the Production of Alkaline Protease by Bacillus pumilus K 242 by the Solid State Fermentation Method
MATERIALS AND METHODS
Solid substrate
Particle size
Moisture level, Temperature of incubation and Incubation period
pH moistening solution
Supplementation with carbon sources
Supplementation with nitrogen sources
Supplementation with sodium chloride
Age of inoculum
Size of inoculum
Medium volume: Flask volume
RESULTS
Effect of different solid substrates
Table 17. Alkaline protease production by Bacillus pumilus K 242 by SSF, using different commercially available substrates
Effect of size of particles of wheat bran
Table 18. Effect of using wheat bran of different particle size on alkaline protease production by Bacillus pumilus K 242
Effect of moisture level, temperature of incubation and incubation period
Table 19. Effect of moisture level, temperature of incubation and incubation period on alkaline protease production by Bacillus pumilus K 242
Effect of pH of moistening solution
Fig. 4. Effect of pH of moistening solution on alkaline protease production by Bacillus pumilus K 242
Effect of extra carbon sources
Table 20. Effect of supplementation of wheat bran medium with different carbon sources on alkaline protease production by Bacillus pumilus K 242
Effect of extra nitrogen sources
Table 21. Effect of supplementation of solid substrate medium with different nitrogen sources on alkaline protease production by Bacillus pumilus K 242
Effect of sodium chloride
Table 22. Effect of incorporating sodium chloride at different concentrations into the moistening solution, on alkaline protease production by BaciIIus pumilus K 242
Effect of age of lnoculurn
Table 23. Effect of age of inoculum on alkaline protease production by Bacilus pumilus K 242
Effect of size of inoculurn
Table 24. Effect of inoculum size on alkaline protease production by Bacillus pumilus K 242
Effect of the ratio, medium volume: flask volume
Table 25. Effect of varying the ratio medium volume: flask volume on alkaline protease production by Bacillus pumilus K 242
DISCUSSION
5. PURIFICATION OF ALKALINE PROTEASES
Section A - Purification of Alkaline Protease from the Culture Supernatant of Bacillus sp. K 25
MATERIALS AND METHODS
RESULTS
Fig. 5. Elution profile of alkaline protease of Basillus sp. K 25 from CM cellulose column
Table 26. Purification of extracellular alkaline protease of Bacillus sp. K 25
Fig. 6. Elution profile of alkaline protease of Bacillus sp. K 25 from Sephadex G- 100 column
Plate 1. Native polyacrylamide gel electrophoresis of purified alkaline protease of Bacillus sp. K25
DISCUSSION
Section B - Purification of Alkaline Protease from the Bacterial Bran Extract of Bacillus pumilus K 242
MATERIALS AND METHODS
RESULTS
Fig. 7. Elution profile of proteases of Bacillus pumilus K 242 from DEAE Sephadex A-50 column
Fig. 8. Elution profile of alkaline protease of Bacillus pumilus K 242 from Sephadex G- 100 column
Plate 2. Native polyacrylamide gel electrophoresis of purified alkaline protease of Bacillus pumilus K 242
Table 27. Purification of extracellular alkaline protease from the bacterial bran extract of Bacillus pumilus K 242
DISCUSSION
6. CHARACTERIZATION OF ALKALINE PROTEASES
MATERIALS AND METHODS
Effect of pH on activity
Effect of temperature on activity
Effect of inhibitors on activity
Effect of metal ions on activity
Effect of pH on stability
Effect of temperature on stability
RESULTS
Effect of pH on activity
Fig. 9a. Effect of pH on caseinolysis by protease K 25
Fig. 9b. Effect of pH on caseinolysis by protease K 242
Effect of temperature on activity
Fig. 10. Effect of temperature on caseinolysis by protease K 25 andprotease K 242
Effect of inhibitors
Table 28. Effects of inhibitors on the activity of proteases K 25and protease K 242
Effect of metal ions on activity
Table 29. Effect of metal ions on the activity of proteases K 25 and K 242
pH stability
Fig. 11. Effect of pH on the stability of protease K 25 and protease K 242
Thermostability
Fig. 12. Effect of temperature on the stability of protease K 25 andprotease K 242
DISCUSSION
7. STUDIES ON SUITABILITY OF PROTEASES OF BACILLUS SP. K 25 AND BACILLUS PUMILUS K 242 AS DETERGENT ENZYMES
MATERIALS AND METHODS
Stability in presence of commercial detergents
Ability of proteases to act on insoluble substrate in presence of commercial detergents
RESULTS
Table 30. Stability of proteases of BaciIIus sp. K 25, Bacillus pumiIus K 242 andBadus licheniformis NCIM 2042 in commercial detergents
Table 31. Activity of proteases of Bacillus sp. K 25, Bacilus pumilus K 242and Bacillus licheniformis NCIM 2042 on blue case in-PAG in presence of different commercial detergents
DISCUSSION
8. SUMMARY AND CONCLUSION
BIBLIOGRAPHY