Beet molasses based exponential feeding strategy for thermostable glucose isomerase production by recombinant Escherichia coli BL21 (DE3)

Abstract

BACKGROUND: The effects of pretreated beet molasses based feeding strategies on thermostable glucose isomerase (GI) production by recombinant Escherichia coli BL21 (DE3) pLysS were investigated. RESULTS: The thermostable GI encoding gene of Thermus thermophilus (xylA) was recombined with pRSETA vector, and the pRSETA::xylA obtained was transferred into E.coli BL21 (DE3) pLysS and used for GI production. The highest soluble GI activity was obtained at t = 30 h, as A = 16 400 U L−1 (20.6 U mg−1 protein) under molasses based fed‐batch operation, with a specific growth rate µ = 0.1 h−1 (M‐0.1); on the other hand, the highest cell concentration was obtained at µ = 0.15 h−1 operation as 9.6 g L−1 at t = 32 h. The highest oxygen uptake was 4.57 mol m−3 s−1 at M‐0.1 operation. CONCLUSIONS: Molasses based fed‐batch operations were more successful in terms of cell concentration and thermostable enzyme production due to the existence of a natural sugar inducer, galactose, in the molasses composition. This study demonstrates the significance of proper feeding strategy development for over‐production of enzymes by recombinant E. coli strains. © 2012 Society of Chemical Industry

Recombinant protein production by sucrose‐utilizing Escherichia coli W: untreated beet molasses‐based feeding strategy development

“…For the expression of the xyl A gene, E. coli W (NRRL B‐766) was used. E. coli XL1‐Blue carrying pRSETA vector (Invitrogen Life Technologies, USA) was used for the propagation of pRSETA:: xyl A plasmid . E. coli cells, harboring recombinant plasmids, were maintained on LB agar slants that contained (g L −1 ): yeast extract, 5.0; tryptone, 10.0; NaCl, 10.0; and agar, 15.…”

“…The cell concentrations based on dry weights were measured with a UV‐Vis spectrophotometer (Thermo Spectronic, Heλios‐α, Waltham, MA) using a calibration curve obtained at 600 nm. Intracellular soluble GI activity was determined by measuring the conversion of D‐glucose into D‐fructose, as described previously . One unit of GI activity was defined as the activity in which 1 µmol of D‐fructose per min at 80°C was formed.…”

“…Related with the use of molasses as the carbon source, since E. coli strains, except E. coli W, cannot utilize sucrose, molasses is pretreated such that sucrose is hydrolysed into glucose and fructose and then used as the carbon source. In the literature, there are a few studies reporting the use of pretreated (hydrolysed) molasses for r‐protein production by E. coli . There is no work in the literature reporting the use of molasses for r‐protein production without any treatment in E. coli ‐based fermentations by sucrose‐utilizing E. coli W.…”

“…In this work, for the production of glucose isomerase (D‐xylose ketol isomerase, GI, EC 5.3.1.5) that catalyses the reversible isomerization of D‐glucose to D‐fructose, pRSETA:: xyl A plasmid carrying GI encoding gene of Thermus thermophilus ( xyl A ) was transferred into E. coli W. Thereafter, the influences of beet molasses feeding strategies with and without additional nitrogen and phosphate sources, on the cell growth, substrate utilization, GI and by‐product formation profiles were investigated, systematically. Moreover, the substrate uptake rates and yield coefficients of the bioprocess were calculated and presented.…”

Improvement and characterization of a hyperthermophilic glucose isomerase from Thermoanaerobacter ethanolicus and its application in production of high fructose corn syrup

“…The lifetime productivity of TNGI at 90 °C is 1169 kg fructose/kg enzyme, which is 5.6 times higher than that of SMGI [5]. In recent years, over-production of recombinant hyperthermophilic GIs has been achieved in Escherichia coli strains [1,2,4]. Akdağ and Çalık reported the highest production of GI reached 4364.1 U/L using the exponential feeding strategy [1].…”