Effect of Growth Rate on the Production of β-Galactosidase from Escherichia Coli in Bacillus Subtilis Using Glucose-Limited Exponentially Fedbatch Cultures

Abstract

The effect of specific growth rate (μ) on the production of β-galactosidase from Escherichia coli in Bacillus subtilis was assessed using glucose-limited exponentially fedbatch cultures (EFBC). A B subtilis strain carrying a chromosomal copy of the lacZ gene under the control of the subtilisin (aprE) regulatory region was used in this study. This strain also carries the hpr2 and degU32(Hy) mutations which are known to overexpress the subtilisin (aprE) gene. Constant μ in the range of 0.058–0.35 h −1 were maintained using mineral media and computer-controlled predetermined glucose feeding profiles. At all growth rates tested, a pseudo-steady state in μ was obtained; however, a sudden decrease in the slope of log of biomass concentration against time and accumulation of glucose and acetic acid indicated that control on μ was lost at all growth rates tested. Cultures maintained at a μ value close to the maximum obtained in batch cultures had a specific enzymatic activity relatively constant during the fedbatch phase. In contrast, cultures maintained at a μ value lower than 0.2 h −1 had specific and volumetric β-galactosidase activities that decreased during the fedbatch phase, reaching a value close to zero before glucose feeding was stopped. The maximum β-galactosidase activity after the EFBC phase was obtained at the maximum μ tested whereas it strongly decreased at lower μ values. This suggests that glucose limitation and μ reduction during exponential growth phase represses β-galactosidase production during the exponential and stationary phases.