Efficient extracellular recombinant production and purification of a Bacillus cyclodextrin glucanotransferase in Escherichia coli

Abstract

BackgroundCyclodextrin glucanotransferases (CGTases) catalyze the synthesis of cyclodextrins, cyclic oligosaccharides composed of glucose monomers that find applications in the pharmaceutical, food, and cosmetic industries. An economic application of these industrially important enzymes requires their efficient production and recovery. In this study, the effect of Sec-type signal peptides on the recombinant expression of a CGTase derived from Bacillus sp. G825-6 was investigated in Escherichia coli BL21(DE3) using a codon-adapted gene. In addition, a novel purification method for the CGTase using starch adsorption was developed.ResultsExpression vectors encoding N-terminal PelB, DacD, and the native Bacillus sp. G825-6 CGTase signal peptides (SP) were constructed for the recombinant CGTase. With the DacD SP derived from E. coli, a 3.9- and 3.1-fold increase in total enzyme activity was obtained compared to using the PelB and the native CGTase SP, respectively. DacD enabled a 7.3-fold increase of activity in the extracellular fraction after induction for 24 h compared to the native CGTase SP. After induction for 48 h, 75% of the total activity was detected in the extracellular fraction. By a batch wise adsorption to starch, the extracellular produced CGTase could be purified to homogeneity with a yield of 46.5% and a specific activity of 1637 U/mg.ConclusionsThe signal peptide DacD promoted the high-level heterologous extracellular expression of a recombinant CGTase from Bacillus sp. G825-6 with a pET20b(+) vector in E. coli BL21(DE3). A protocol based on starch adsorption enabled a fast and efficient purification of the recombinant enzyme.

Domain shuffling of cyclodextrin glucanotransferases for tailored product specificity and thermal stability

“…A previously constructed CGTase expression vector pET20b(+):: dacD‐cgt encoding for the 671 amino acids of the mature G825 CGTase (Fig. S1) and the pelB sequence substituted with dacD was used for the expression . A synthetic DNA fragment ( geoT ) (Fig.…”

“…S2) encoding for the 679 amino acid of the mature CGTase derived from the G. stearothermophilus ET1 CGTase with substitutions S33T, E64D, N180S, K204R, L214I, V241I, F266Y, S400A, A457G, A471S, N609D, L617M, and I654T was cloned into the expression vector pET20b(+):: dacD‐geoT between BamHI and SacI restriction sites, as illustrated in Fig. . A domain rearrangement was achieved by substituting the DNA regions encoding for the GeoT domains A1, B, A2, and CDE with the corresponding G825‐6 fragments.…”

Engineered cyclodextrin glucanotransferases from Bacillus sp. G‐825‐6 produce large‐ring cyclodextrins with high specificity

“…Plasmid isolation and DNA sequencing of the CGTase encoding gene were performed to confirm the correct incorporation of mutations. Recombinant protein expression in Escherichia coli BL21(DE3) and purification based on starch adsorption were performed as described elsewhere (Sonnendecker et al, ).…”

“…A pET20b(+) expression vector encoding the DacD signal peptide fused to the mature CGTase G825-6 sequence from Bacillus sp. G825-6 (GenBank: AB201304) was used as template (Sonnendecker et al, 2017). Mutagenic primers were designed and mutagenesis was performed as described elsewhere (Liu & Naismith, 2008).…”

“…Recombinant protein expression in Escherichia coli BL21(DE3) and purification based on starch adsorption were performed as described elsewhere (Sonnendecker et al, 2017).…”