Biochemical and biocalorimetric investigations to enhance the production of penicillin G acylase from Bacillus badius and recombinant Bacillus badius PAC expressed IN E coli DH5and#945;

Abstract

Penicillin G acylase (PGA) is a commercially important enzyme that cleaves penicillin G to 6-Amino penicillanic acid (6-APA) and phenyl acetic acid (PAA). 6-APA is the key intermediate in the production of various semi-synthetic penicillins. PGA is also used in the hydrolysis of ß lactam antibiotics that account for 60 percent of the world s antibiotic sales: (out of a total 5×107 kg/year). The focus of this thesis is on investigating enhancement of the secretion of PGA in parental strain B. badius as well as the recombinant construct B. badius pac in E. coli DH5and#945; by optimizing the growth parameters, medium compositions and cultivation conditions. Extensive experiments were conducted in batch mode in shake flask, fermentor and bioreaction calorimeter. A detailed calorimetric investigation on PGA production was carried out to enable generation of thermo kinetic data possible for commercial application. The reaction calorimetric studies coupled with respirometric studies suggested that the enzyme activity of the species B. badius was calorimetrically traceable. The three phases of growth were distinctly noticeable in the metabolic heat time curve. The increase in enzymatic activity with restricted growth confirmed the intracellular nature of the secretion process. The biocalorimetric study on the growth of E. coli DH5and#945; containing B. badius pac gene did indicate that the production of PGA could be monitored and reliable information on the growth dynamics of the organism can be achieved. Metabolic heat, enzyme activity, substrate utilization rate and oxygen uptake rate correlated well with each other. The heat yields due to biomass growth, 15.26 kJ/g, substrate consumption, 16.52 kJ/g and oxygen uptake, 413.2 kJ/mol helped us to understand the energetics of the organism under study. The oxycalorific coefficient agreed well with what had been reported hitherto and suggested that the process was aerobic. The biocalorimetric data generated would be useful for design and scale-up of fermentors. newline newline newline