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Title
DEDICATION
CERTIFICATE 1
CERTIFICATE-2
DECLARATION
ACKNOWLEDGEMENT
Abbreviations
CONTENTS
List of Figures
List of Plates
Preface
1 General Introduction
1.1 Pectic enzymes
1.1.1 Acidic pectinases
1.1.2 Alkaline pectinases
1.1.3 Industrial production of pectic enzymes
1.1.3.1 Submerged fermentation
1.1.3.2 Semisolid fermentation
1.1.3.3 Extraction and recovery
1.2 Pectic enzymes-classification
1.2.1 Esterases
1.2.1.1 Pectin methyl esterase
1.2.2 Hydrolases
1.2.2.1 Endo-polygalacturonase (Endo-PG)
1.2.2.2 Exo-polygalacturonase (Exo-PG)
1.2.2.3 Oligo galacturonate hydrolase
1.2.2.4 Endo poly methyl galacturonase
1.2.3 Pectin lyases
1.2.3.1 Endo polygalacturonate lyase (Endo-PGL)
1.2.3.2 Exo polygalacturonate lyase (Exo-PGL)
1.2.3.3 Oligo galacturonate lyase (OGL)
1.2.3.4 Endo poly methyl galacturonate lyase (Endo PMGL)
1.3 Regulation of pectinase biosynthesis
1.4 Phytopathogenicity of pectic enzymes
1.5 Pectic polysaccharides-the substrate
1.5.1 Structure and composition
1.5.2 Occurrence and properties
1.5.3 Commercial pectin and applications
1.6 Objectives of the present study Section A
2 Isolation of pectinolytic fungi
2.1 Introduction
2.2 Review of literature
2.3 Materials and methods
2.3.1 Primary screening
2.3.2 Secondary screening
2.3.2.1 Estimation of polygalacturonase
2.3.2.2 Estimation of pectin lyase
2.4 Results
2.4.1 Primary screening
2.4.2 Secondary screening
I Secondary screening for polygalacturonase producing fungi
II. Secondary screening for pectin Iyase producing fungi
2.5 Discussion
3 Impact of physical and chemical parameters in the production of polygalacturonase and pectin Iyase
3.1 Introduction
3.2 Review of literature
3.3 Materials and methods
3.3.1 Comparison of solid state and submerged fermentation
3.3.2 Effect of initial pH on enzyme production
3.3.3 Effect of temperature on enzyme production
3.3.4 Comparison of wheat bran and inert support-poly styrene beads as solid substrates
3.3.5 Effect of particle size of wheat bran on enzyme production
3.3.6 Effect of incubation time on enzyme production
3.3.7 Effect of carbon sources on enzyme production
3.3.8 Effect of nitrogen sources on enzyme production
3.3.9 Effect of salts on enzyme production
3.4 Results
3.4.1 Comparison of submerged and solid state fermentation
3.4.2 Effect of initial pH on enzyme production
III. Effect of initial pH on polygalacturonase production from Penicillium citrinum under SSF
3.4.3 Effect of temperature on enzyme production
IV. Effect of initial pH on pectin lyase production from Penicillium citrinum under SSF
V Effect of temperature on polygalacturonase production from Penicillium citrinum under SSF
3.4.4 Comparison of wheat bran and poly styrene beads as solid substrates
VI Effect of temperature on pectin Iyase production from Penicillium citrinum under SSF
3.4.5 Effect of particle size of wheat bean on enzyme production
VII. Effect of particle size of wheat bran on polygalacturonase production from Penicillium citrinum under SSF
3.4.6 Effect of incubation time on enzyme production
Vlll Effect of particle size of wheat bran on pectin Iyase production from Penicillium citrinum under SSF
IX. Effect of incubation time on polygalacturonase production from Penicillium citrinum under SSF
X Effect of incubation time on pectin Iyase production from Penicillium citrinum under SSF
3.4.7 Effect of carbon sources on enzyme production
XI. Effect of carbon sources on polygalacturonase production from Penicillium citrinum under SSF
XII. Effect of carbon sources on pectin Iyase production from Penicillium citrinum under SSF
3.4.8 Effect of various nitrogen sources on enzyme production
XIII. Effect of nitrogen sources on polygalacturonase production from Penicillium citrinum under SSF
3.4.9 Effect of salts on enzyme production
XIV. Effect of nitrogen sources on pectin lyase production from Penicillium citrinum under SSF
XV Effect of salts on polygalacturonase production from Penicillium citrinum under SSF
3.5 Discussion
XVI. Effect of salts on pectin lyase production from Penicillium citrinum under SSF
4 Purification and characterisation of polygalacturonse
4.1 Introduction
4.1.1 Removal of microbial cells and other solid matter
4.1.2 Aqueous biphasic systems
4.1.3 Ultra filtration
4.1.4 Concentration by precipitation
4.1.5 Heat treatment
4.1.6 Chromatography
4.1.6.1 Ion exchange chromatography
4.1.6.2 Affinity chromatography
4.1.6.3 Gel permeation chromatography
4.2 Review of literature
4.3 Materials and methods
4.3.1 Ammonium sulfate fractionation
4.3.2 Dialysis
4.3.3 Concentration
4.3.4 Gel filtration chromatography
4.3.5 Electrophoresis
4.3.6 Estimation of total proteins
4.3.7 Characterization of purified polygalacturonase
4.3.7.1 Substrate specificity of polygalacturonase
4.3.7.2 Substrate concentration of polygalacturonase
4.3.7.3 Optimum temperature and stability of polygalacturonase
4.3.7.4 Optimum pH and stability of polygalacturonase
4.3.7.5 Effect of various heavy metals on polygalacturonase
4.3.7.6 Effect of salts on polygalacturonase
4.3.7.7 Total carbohydrate content in polygalacturonase
4.3.7.8 Mode of action of polygalacturonase
4.4 Results
4.4.1 Purification of polygalacturonase
XVII. Elution profile of polygalacturonase
4.4.2 Characterisation studies
4.4.2.1 Substrate specificity
1 Native PAGE
2 SDS- PAGE
XVIII. Substrate specificity of polygalacturonase
4.4.2.2 Substrate concentration
XIX. Lineweaver - Burk plot
4.4.2.3 Effect of temperature and stability of polygalacturonase
4.4.2.4 Effect of pH and stability of polygalacturonase
XX. Optimum temperature and thermal stability of polygalacturonase
4.4.2.5 Effect of heavy metals on polygalacturonase
XXI. Optimum pH and pH stability of polygalacturonase
XXII. Effect of HgCl2 on polygalacturonase
XXIII. Effect of CdSO4 on polygalacturonase
4.4.2.6 Effect of salts on polygalacturonase
XXIV. Effect of ZnSO4on polygalacturonase
XXV. Effect of CaCl2on polygalacturonase
XXVI. Effect of NaCl on polygalacturonase
4.4.2.7 Total carbohydrate
4.4.2.8 Mode of action
XXVII. Effect of NH4CI on polygalacturonase
XXVIII. Mode of action of polygalacturonase
4.5 Discussion
5 Applications of polygalacturonase
5.1 Bio conversion of orange peel by polygalacturonase
5.1.1 Introduction
5.1.2 Review of literature
5.1.3 Materials and methods
5.1.4 Results
XXIX. Conversion of pectin by polygalacturonase from Penicillium citrinum
5.1.5 Discussion
5.2 Polygalacturonase in fruit juice industry
5.2.1 Introduction
5.2.2 Review of literature
5.2.3 Materials and methods
5.2.4 Results
XXX Effect of temperature on cloudiness of orange juice by polygalacturonase of Penicillium citrinum and commercial pectinase
XXXI. Cloud stability and pectin degradation of orange juice by polygalacturonase from Penicillium citrinum and commercial pectinase at 44°C
5.2.5 Discussion
XXXII. Comparison of polygalacturonase from Penicillium citrinum and commercial pectinase in pectin degradation and clarification of lemon juice at various incubation time and temperature at 40° C
6 Summary and Conclusion
6.1 Summary
6.2 Conclusion
BIBILIOGRAPHY