Effective approach to greatly enhancing selective secretion and expression of three cytoplasmic enzymes in Escherichia coli through synergistic effect of EDTA and lysozyme

Abstract

An effective approach to greatly enhancing the selective secretion and expression of recombinant cytoplasmic enzymes in Escherichia coli was successfully developed through the synergistic effect of ethylenediaminetetraacetate (EDTA) and lysozyme. The method was applied to two endoglucanases (EGs) and an amylase. The optimal culture conditions of temperature and concentration of isopropyl-β-D: -1-thiogalactopyranoside (IPTG) were 23-30 °C and 0.2 mM, respectively, under which the three enzymes could be expressed in active form. Among all the chemicals tested, EDTA was found to be most suitable for enhancing the secretion of EG-I-1A into the medium. Addition of lysozyme alone had little influence on the secretion and expression. In contrast, on the basis of the addition of 5 g EDTA/L at the induction time of 12 h, the simultaneous addition of 0.15 g lysozyme/L further significantly increased the secretion and expression of the three enzymes, demonstrating the synergistic effect of EDTA and lysozyme. The production of EG-I-1A in the culture medium by adding 5 g EDTA/L and 0.15 g lysozyme/L under the optimal culture conditions of 23 °C and 0.2 mM IPTG was over 260-fold higher than that without EDTA and lysozyme under the standard conditions of 37 °C and 1 mM IPTG. In summary, the advantage of this novel cultivation approach for secretion was that not only did it selectively enhance the secretion of the proteins of interest, but also greatly increased the expression of the three enzymes by over 80 %.

Secretion of recombinant thermo-alkali-stable endoxylanase of polyextremophilic Bacillus halodurans TSEV1 and its utility in generating xylooligosaccharides from renewable agro-residues

“…The effect of EDTA-lysozyme combination for secreting xylanase selectively into the extracellular medium at 30 • C after overnight incubation could be attributed to their role in permeabilizing cell membrane [30]. The temperature is another important parameter known to affect the production of soluble recombinant proteins in E. coli [31].…”

Challenges and advances in the heterologous expression of cellulolytic enzymes: a review

“…One example of temperature-related expression variability in E. coli is the cellulase from the springtail of Cryptopygus antarcticus , which is best expressed as a soluble protein at 10°C [ 44 ]. Another potential problem is the low secretion of recombinant cellulases in E. coli , which can be improved by ethylenediaminetetraacetic acid (EDTA) treatment [ 45 ] or by the expression of cellulases as OsmY fusion proteins [ 46 ]. OsmY is induced by hyperosmotic stress [ 47 ] and supports the transport of the fusion proteins over the outer membrane [ 46 ], where the fused protein gets cleaved off.…”

Periplasmic and extracellular production of cellulase from recombinant Escherichia coli cells

“…Secretion also gives the expressed enzyme greater access to the substrate that is not adequately taken up by cells, such as cellulosic biomass, and opens the path to consolidated bioprocessing (CBP) of cellulose, whereby cost reduction is achieved by combining cellulose hydrolysis and fermentation in a single microorganism. Secretion of recombinant cellodextrinase to the periplasmic space in an E. coli mutant with leaky outer membrane for cellodextrin improved endoglucanase secretion using ethylenediaminetetraacetic acid (EDTA) treatment . Moreover, expression of cellulases as OsmY fusions is one of the few successful examples towards CBP in recombinant E. coli …”

“…320 www.soci.org Z Yıldırım, E Çelik Secretion of recombinant cellodextrinase to the periplasmic space in an E. coli mutant with leaky outer membrane for cellodextrin 13 improved endoglucanase secretion using ethylenediaminetetraacetic acid (EDTA) treatment. 14 15 -17 In this study, an endoglucanase (Cel5A) from Fusarium graminearum, previously shown to be a highly active enzyme among 348 cell wall degrading enzymes of plant pathogens, 18 was investigated for extracellular production and compared with periplasmic production. For the extracellular production, Cel5A was fused to YebF, a native E. coli protein that is naturally secreted.…”