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

Abstract

This study aimed to determine the effects of different feeding strategies in fed-batch cultivations on the production of β-galactosidase fused to a cellulose-binding domain (CBD) by Escherichia coli C41(DE3) using a bench bioreactor. The highest enzyme activities (∼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 β-galactosidase yields per substrate, β-galactosidase yields per biomass, and QP were approximately 950 U/gglucose, 1100 U/gcells, and 540 U/L.h, respectively, using Terrific Broth as culture medium and concentrated feed solution (2 ×). The dairy coproducts were efficient in inducing the expression of the recombinant enzyme, having reached the highest values of β-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 β-galactosidase-CBD employing ricotta whey as an inducer of enzyme expression.