Heterologous Expression of Cyclodextrin Glycosyltransferase my20 in Escherichia coli and Its Application in 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid Production

Abstract

In this study, the cgt gene my20, which encodes cyclodextrin glycosyltransferase (CGTase) and was obtained by the metagenome sequencing of marine microorganisms from the Mariana Trench, was codon optimized and connected to pET-24a for heterologous expression in Escherichia coli BL21(DE3). Through shaking flask fermentation, the optimized condition for recombinant CGTase expression was identified as 20°C for 18 h with 0.4 mM of isopropyl β-D-L-thiogalactopyranoside. The recombinant CGTase was purified by Ni2+-NTA resin, and the optimum pH and temperature were identified as pH 7 and 80°C, respectively. Activity was stable over wide temperature and pH ranges. After purification by Ni2+-NTA resin, the specific activity of the CGTase was 63.3 U/mg after 67.3-fold purification, with a final yield of 43.7%. In addition, the enzyme was used to transform L-ascorbic acid into 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G). The maximal AA-2G production reached 28 g/L, at 40°C, pH 4, 24 h reaction time, 50 g/L donor concentration, and 50 U/g enzyme dosage. The superior properties of recombinant CGTase strongly facilitate the industrial production of AA-2G.

Microbial Cyclodextrin Glycosyltransferases: Sources, Production, and Application in Cyclodextrin Synthesis

“…The purified CGTase exhibited optimum activity at pH 5 and 95 °C, thermostability even at 100 °C, and converted starch to β-CD majorly [20]. Song et al [54] reported the cloning of codon-optimized CGTase encoding gene (my20) procured from the metagenome sequencing of marine microorganisms (obtained from Mariana Trench) in a pET24a vector and its subsequent expression in E. coli BL21 (DE3). Recombinant CGTase was maximally expressed at 20 °C after 18 h postinduction by using 0.4 mM IPTG, with the enzyme exhibiting optimal activity at pH 7 and 80 °C [54].…”

“…For example, CGTase from Thermoanaerobacter thermosulfurigenes EM1 has a temperature optimum of 80-85 °C [92], CGTase from Thermoanaerobacter sp. has a temperature optimum of 80 °C [54], and CGTase from Bacillus stearothermophilus NO2 has an optimum enzyme activity at 70 °C [84]. Moreover, recombinant CGTase from Pyrococcus furiosus DSM3638 expressed in E. coli exhibited a temperature optimum of 95 °C [20].…”