Kinetic analysis of carboxymethyl cellulase production in bacillus megaterium (pCK108)

Abstract

Kinetics of carboxymethyl cellulase (EC 3.2.1.4) production by recombinant cell, $\mbox{\underline{Bacillus}}\quad\mbox{\underline{megaterium}}$ (pCK108), was investigated in a chemically defined medium. The gene expression of plasmid pCK108 was surveyed at different culture conditions. The ratio of specific growth rate of plasmid-absent to plasmid-carrying cells was determined to 1.31. Phenotypic instability of plasmid in Bacillus megaterium, originated from segregation during cell growth. The optimal temperature supporting cell growth was determined to be 30℃ at which the plasmid pCK108 could be stably maintained. Cellular plasmid contents, cellulase production rate and the efficiency of gene expression increased significantly with the increase of temperature from 30℃ to 44℃. At temperatures higher than 38℃, however, the plasmid stability decreased sharply to 60% level at the culture time of 30 hours. The optimal pH supporting cell growth and plasmid stability was determined to be 7.0. The production of the cellulase was not stimulated by sophorose that has been recognized as strong inducer for the production of fungal cellulase. Carboxymethyl cellulose did not also induce the biosynthesis of the cellulase. The cellulase production with glucose as carbon source, occurred after the cessation of exponential growth, in the mode of non-growth associated. By slow utilizable carbon source such as lactose, however, the enzyme production was also occurred during cell growth. The production of carboxymethyl cellulase from $\mbox{\underline{Bacillus}}\quad\mbox{\underline{megaterium}}$ (pCK108) was regulated by carbon catabolite repression. In batch culture with lactose as sole carbon source (50 g/l), the cellulase was produced to 7.5 U/ml. The enzyme production in continuous culture was favored when the growth rate was a relatively small fraction of the maximum growth rate under the prevailing culture condition. The gene expression level was higher at lower dilution r...