Recombinant production in Escherichia coli of a -galactosidase fused to a cellulose-binding domain using low-cost inducers in fed-batch cultivation

This study aimed to determine the effects of different feeding strategies in fed-batch cultivations on the production of beta-galactosidase fused to a cellulose-binding domain (CBD) by Escherichia coli C41(DE3) using a bench bioreactor. The highest enzyme activities (similar to 17,000 U/L) were obtained in fed-batch cultivations controlled by DO-stat and induced with lactose (5 g/L) at 18 h of cultivation. Under these cultivation conditions, the maximum values of beta-galactosidase yields per substrate, beta-galactosidase yields per biomass, and Q(P) were approximately 950 U/g(glucose), 1100 U/g(cells), and 540 U/L.h, respectively, using Terrific Broth as culture medium and concentrated feed solution (2 x). The dairy copmducts were efficient in inducing the expression of the recombinant enzyme, having reached the highest values of beta-galactosidase-CBD activity after 40 h of cultivation. The maximum enzyme activities obtained when using whey permeate or ricotta whey as inducers were 22,950 or 24,640 U/L, respectively. Using the optimal temperature of E. coli growth before induction and ricotta whey as an inducer increased enzyme productivity in 50%. Plasmid construction showed high stability (>94%) throughout E. coli cultivation. This study demonstrated the feasibility of using the DO-stat feeding strategy to produce beta-galactosidase-CBD employing ricotta whey as an inducer of enzyme expression.