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

Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of sp. NR5 UPM β-cyclodextrin glycosyltransferase (CGT-BS) in recombinant . Several rare codons were eliminated and replaced with the ones favored by cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant 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 .