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.

Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM β-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli. Several rare codons were eliminated and replaced with the ones favored by E. coli cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant E. coli following optimization of glycine supplementation enhanced the secretion of β-CGTase activity up to 2.2-fold at 12 h of cultivation as compared to the control. β-CGTase secreted into the culture medium by the transformant reached 65.524 U/mL at post-induction temperature of 37 °C with addition of 1.2 mM glycine and induced at 2 h of cultivation. A 20.1-fold purity of the recombinant β-CGTase was obtained when purified through a combination of diafiltration and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. This combined strategy doubled the extracellular β-CGTase production when compared to the single approach, hence offering the potential of enhancing the expression of extracellular enzymes, particularly β-CGTase by the recombinant E. coli.