Improved extracellular secretion of β-cyclodextrin glycosyltransferase from Escherichia coli by glycine supplementation without apparent cell lysis

Abstract

The use of an effective inducer feeding strategy without causing cell lysis presents significant advantage to enhance the secretion of an enzyme to the culture medium of Escherichia coli. The cgt gene encoding β-cyclodextrin glycosyltransferase (β-CGTase) was cloned into pQE30xa as an N-terminal His-tagged protein and transformed into E. coli. The induction strategy was applied towards enhancing the extracellular secretion of the recombinant β-CGTase by increasing permeability of the outer membrane of E. coli. The supplementation of 1.2 mM glycine following 2 h of fermentation at 37°C enhanced the activity of β-CGTase to 38.295 U/mL, which was approximately 1.3-fold higher than the control (without induction). Further flow cytometry analysis was adopted as a rapid and highly reproducible approach to determine the effect of glycine supplementation on the viability of E. coli cells. The supplementation of glycine did not contribute to apparent cell lysis, with no adverse effects on cell viability, hence indicating the effectiveness of glycine in enhancing the extracellular secretion of β-CGTase. The recombinant β-CGTase was then purified through a combination of diafiltration and Ni-NTA affinity chromatography with 18.4-fold increase in purity. An effective glycine feeding strategy could enhance the extracellular secretion of β-CGTase without adverse effects on cell viability. This strategy could be applied potentially to enhance the secretion of a recombinant protein to the culture medium from E. coli cells without having cell lysis.

Combined Optimization of Codon Usage and Glycine Supplementation Enhances the Extracellular Production of a β-Cyclodextrin Glycosyltransferase from Bacillus sp. NR5 UPM in Escherichia coli

“…However, by taking into account the theoretical extinction coefficient for the oxidized and reduced CGT-BS protein of 142,810 and 142,560 M −1 •cm −1 , respectively (Table 2) with the absorbance of the purified (ultrafiltration) protein fraction was 0.109, the purified protein concentration was calculated to be 0.06 mg/mL. Nonetheless, the purity of codon optimized protein (cocgt-BS) obtained in this study was not much different with the resultant recombinant cgt-BS protein (non-optimized codon) in the previous study [19]. Similarly, the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) also showed the expected With the addition of glycine using the optimized feeding strategy as discussed earlier, the β-CGTase activity increased gradually and reached its highest secretion of 65.524 U/mL at 12 h of cultivation, which was increased by 1.7-fold in comparison to a single approach of codon usage optimization and 5.6-fold as compared to the wild type strain when using soluble starch as a substrate [31].…”

“…However, by taking into account the theoretical extinction coefficient for the oxidized and reduced CGT-BS protein of 142,810 and 142,560 M −1 •cm −1 , respectively (Table 2) with the absorbance of the purified (ultrafiltration) protein fraction was 0.109, the purified protein concentration was calculated to be 0.06 mg/mL. Nonetheless, the purity of codon optimized protein (cocgt-BS) obtained in this study was not much different with the resultant recombinant cgt-BS protein (non-optimized codon) in the previous study [19]. Similarly, the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) also showed the expected band size of the purified protein at approximately 80 kDa (Supplementary Figure S1), in correlation with the theoretical molecular weight predicted using ExPASY's ProtParam online tool (Table 2).…”

“…In a previous study, the individual supplementation of glycine into the culture media was shown to enhance the α-CGTase secretion from 2.4 to 32 U/mL at 48 h of cultivation [20]. It is even more impressive that the addition of glycine as an inducer did not cause apparent effects on bacterial cell viability [19], which indicates that the secretion of recombinant β-CGTase into the culture medium was not due to cell lysis. Since the optimization of either codon usage or glycine supplementation could enhance the secretion of enzyme into the culture medium, we are hypothesizing that the combined optimization could further improve the extracellular expression of β-CGTase even better than a single approach.…”

“…Glycine was found to cause morphological changes, such as an enlargement of spheroidal morphology in E. coli, as it is integrated into the nucleotide-activated peptidoglycan precursors, which leads to the increased permeability of host cell [23,24]. The glycine added into the culture medium could replace the alanine residues in the peptide component of the peptidoglycan of E. coli cell wall, resulting in a more loosely cross-linked peptidoglycan and therefore, enhancing the permeability of outer membrane of E. coli [25], without causing bacterial cell lysis [19,24].…”

“…However, the rate and amount of materials transported across the cell membrane can be changed by altering the cell membrane permeability [18]. Glycine has been particularly shown to act as the best inducer in enhancing the secretion of recombinant β-CGTase as compared to Triton X-100 and xylose [19], even though all of these inducers were reported to play a role in increasing the permeability of cell membrane, which allowed protein translocation across the membrane.…”