The aim of this work was to develop an effective fed-batch feeding strategy to enhance recombinant glucose isomerase (r-GI) production by recombinant Escherichia coli BL21 (DE3) pLysS on an industrially relevant feedstock without the application of an exogenous inducer. Following the batch operation (0 < t < 7H), the effects of pulse and/or continuous feeding of hydrolyzed beet molasses were investigated under five different feeding strategies. The two most promising strategies with respect to r-GI activity were (i) PM-0.05, designed with one pulse feed (t = 7H) followed by a continuous feed and (ii) 2PM(F)-0.05, designed with two consecutive pulse feeds (t = 7 and 10H) followed by a continuous feed. The continuous feeding of molasses for both fermentation strategies employed the same precalculated feeding rate, (o) = 0.05H(-1). The maximum r-GI activities exhibited by PM-0.05 and 2PM(F)-0.05 were 29,050 and 30,642U dm(-3), respectively. On the one hand, compared to PM-0.05 r-GI activity reached its maximum within a shorter cultivation time (t(max) = 2H) at 2PM(F)-0.05, which could be preferable in terms of manufacturing costs and possible risks; on the other hand, PM-0.05 is a simpler fermentation regime compared to 2PM(F)-0.05 with respect to manipulations that should be considered in large-scale production. (C) 2017 International Union of Biochemistry and Molecular Biology, Inc.