| Products/Services Used | Details | Operation |
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| Gene Synthesis | The OleD (ASP) glycosyltransferase gene was generated by Genscript Biotechnology (Nanjing, China) and was cloned into DH5α (Takara, Japan) and pET28a (Novagen, USA) expression vector. Single colony of Escherichia coli BL21 (DE3) pLysS (Tiangen, China) transformed with pET28a/OleD vector was inoculated in Luria Bertani (LB) medium (3 mL) supplemented with 50 μg mL−1 kanamycin. The medium was cultured overnight at 37 °C with shaking (200 rpm). The entire starter culture was then transferred to 1 L LB medium supplemented with 50 μg mL−1 kanamycin and grown at 37 °C with shaking (200 rpm) until the OD600 reached 0.6. Isopropyl β-D-thiogalactoside (IPTG) was subsequently added (final concentration to 0.4 mM) and the culture was incubated at 18 °C for 18 h. Then the cell pellets were collected by centrifugation at 10000g at 4 °C for 20 min and the supernatant was discarded.24 Pellets were resuspended in 10 mL PBS buffer (20 mM phosphate buffer, 0.2 M NaCl, 2 mM KCl, pH 7.4) and then lysed by sonication. Cell debris was removed by centrifugation at 10000g at 4 °C for 20 min and the clear supernatant was immediately submitted to His60 Ni super flow resin (Clontech, USA). The resin was balanced by the equilibration buffer (50 mM sodium phosphate buffer, 0.3 M NaCl, 20 mM imidazole, pH 7.4). The enzyme was allowed to bind for 1 h at 4 °C with gentle agitation, and the resin was washed with 10 mL mild buffer (20 mM phosphate buffer, 0.5 M NaCl, 50 mM imidazole, pH 7.4). Finally, the protein was eluted by with 20 mL strong buffer (50 mM sodium phosphate buffer, 0.3 M NaCl, 300 mM imidazole, pH 7.4). The enzyme aliquots were immediately frozen in liquid nitrogen and stored at −80 °C. Protein purity was confirmed by SDS-PAGE to be >95% and protein concentration for all studies was determined using the Bradford Protein Assay Kit from Bio-Rad (TransGen Biotech, China). | Get A Quote |
The glycosyltransferase OleD variant as a catalyst for the glycosylation of four pairs of epimers of cardiotonic steroids (CTS) are assessed. The results of this study demonstrated that the OleD-catalyze glycosylation of CTS is significantly influenced by the configuration at C-3 and the A/B fusion mode. 3β-OH and A/B ring cis fusion are favoured by OleD (ASP). An epoxide ring at C-14 and C-15 further increases the bioconversion rate; while an acetyl group at C-16 and lactone ring type at C-17 did not influence the biotransformation. A high conversion rate corresponded to a low Km value. A molecular docking simulation showed that filling of hydrophobic pocket II and interaction with residue Tyr115 may play... More
