Title of Thesis

Exploitation of Agro-Industrial Wastes for the Microbial Production on Intracellular and Extracellular Enzyme Penicillin-G-Acylase by Fermentation

Author(s)

Nasira Bashir

Abstract
Penicillinase deficient strains of E. coli and B. megaterium were isolated from the local habitat for the production of intra-cellular and extra-cellular enzyme penicillin-GAcylase (PGA), respectively. After screening, the best enzyme producing strain of E. coli (PCSIR-102) and B. megaterium (5-B) were mutagenized by UV, MNNG, SDS and EMS reagents. The strain of E. coli (MNNG-37) gave higher enzyme PGA activity (231 IU mg-1) in fermentation medium of composition: Peptone 2%, KH2PO4 0.3%, K2HPO4 0.7%, MgSO4.7H2O 0.02%, sodium-L-glutamate 0.5% and yeast extract 0.5%. at pH 6.0, 30 0C temperature for 50 hours. The strain of B. megaterium (MNNG-9) gave more higher enzyme PGA activity (329 IU mg-1) in fermentation medium of composition (g/l): yeast extract 2.0; peptone 5.0; sodium chloride 5.0 (Oxoid) at pH 7.0, temperature 37 0C for 45 hours.
Strain of B. megaterium (MNNG-9) after treatement with EMS was further mutagenized with in MNNG (Two-stage mutagenization) which gave higher enzyme activity of strain M-9 (525 IU mg-1) in molasses medium of composition (g l-1): CaCl2.2H2O 0.05; K2HPO4 1; MgSO4.7H2O 0.5; phenyl acetic acid 10; molasses 12 and Soytone 30 at pH 7.0, temperature 37 0C for 30 hours.
The specific activity of PGA by E. coli (PCSIR-102) and B. megaterium (5-B) was also studied by using different substrates like wheat bran, rice hulls and defatted oil seed cakes of soybean, sunflower or cotton by solid substrate fermentation. Low activity of PGA was found as compared to the submerged fermentation.
The cells of B. megaterium (M-9) were immobilized in calcium alginate beads. The specific activity of PGA was (505 IU mg-1) after 15 hours of fermentation at 30 0C. However, the enzyme PGA produced from B. megaterium (M-9) was immobilized giving better enzyme activity as compared to the immobilized whole cells and free cells.
88% of the enzyme PGA from B. megaterium (M-9) was recovered from the fermentation broth. The level of purification was also confirmed with the help of Fast Performance Liquid Chromatography.

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1.1 Historical Background

1.2 Name and Discovery
1.3 Structure
1.4 Mechanism of Action
1.5 Origin and Properties
1.6 Selection of Strain and Raw Material
1.6.1 Production and trade statistics
1.6.2 Composition of sugar cane molasses
1.7 Strain Improvement
1.8 Surface Response Methodology
1.9 Enzyme Immobilization
1.10 Hydrolysis of Penicillin-G-Acylase
1.11 Isolation of 6-APA
1.12 Applications and Uses
1.13 Objectives and Targets

2.1 Structure

2.2 Isolation, Selection and Screening
2.3 Enzyme Production Improvement
2.4 Mutagenesis
2.5 Immobilization
2.6 Kinetics
2.7 Recovery and Purification
2.8 Applications and Uses

3.1 Methodology

3.2 Fermentation Techniques
3.3 Analysis
3.4 Microscopic Examination

4.1 Isolation of Microorganisms

4.2 Selection of PGA producing strains of E. coli
4.3 Optimization of cultural conditions for the production of PGA by E. coli (PCSIR-102) in shake flasks
4.4 Selection of PGA producing strains of B. megaterium
4.5 Optimization of cultural conditions for the production of PGA by B. megaterium

4.6 Physical Mutagenesis of E. coli (PCSIR-102) by UV-light
4.7 Physical Mutagenesis with UV-light by B. megaterium (5-B)
4.8 Mutagenesis of strain E. coli (PCSIR-102) by MNNG treatment
4.9 Mutagenesis of strain B. megaterium (5-B) by MNNG treatment
4.10 Chemical mutagenization with Sodium dodecyl sulfate (SDS) by using the strains of E. coli (PCSIR-102) and B. megaterium (M-9) for the production of enzyme penicillin-G-acylase (PGA)
4.11 Effect of Molasses medium on the activity of PGA by E. coli (MNNG-37)
4.12 Effect of Molasses medium on the activity of PGA by B. megaterium (MNNG-9)
4.13 Optimization of cultural conditions for the PGA production from the mutant strain of B. megaterium (M-9) by using Surface Response Methodology
4.14 Optimization of cultural conditions for the PGA production from the mutant strain of E. coli (MNNG-37) by using Surface Response Methodology
4.15 Fermentation Profile of B. megaterium (M-9) In 14-Liter Glass Stainless Steel Fermenter
4.16 Production of PGA from E. coli (PCSIR-102) and B. megaterium (M-9) by Solid Substrate Fermentation
4.17 Immobilized Fermentation of B. megaterium (M-9) for the production of PGA 230
4.18 Immobilization of Extra-cellular enzyme Penicillin-G-Acylase (PGA) from B. megaterium (M-9)
4.19 Purification of Penicillin-G-acylase from B. megaterium (M-9)
4.20 Laboratory Scale Production of PGA by Penicillinase Resistant strain of B. megaterium (M-9)
4.21 Acheivements
4.22 Conclusion
4.23 Summary

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