Eclipse sb c18 4.6 250 mm

Example 7

In this Example, a UDP-glucose regeneration system composed of sucrose, sucrose synthetase (AtSUS1 for short hereafter) from Arabidopsis thaliana, and UDP was used as a glucosyl donor.

150 mL of a 0.05 mol/L phosphate buffer (pH 7.0), 0.182 g of UDP, 51.3 g of sucrose, 0.17 g of Reb D, 1.5 g of the lyophilized UGT-A powder and 0.5 g of the lyophilized AtSUS1 powder were added successively into the reaction system, mixed uniformly, then placed in a water bath at 30° C., and stirred at 160 rpm to carry out reaction for 2 hours. After completion of the reaction, 500 μl of the reaction solution was taken and added into an equal volume of anhydrous methanol and mixed uniformly. The mixture was centrifuged for 10 min at 8,000 rpm. The supernatant was taken and passed through a filter membrane, followed by detection using high performance liquid chromatography (chromatographic condition: chromatographic column: Agilent eclipse sb-C18 4.6×250 mm; detection wavelength: 210 nm; mobile phase: 1% formic acid aqueous solution:methanol=20%:80%; flow rate: 1.0 mL/min; column temperature: 25° C.). A conversion rate of Reb D was more than 80%. 0.11 g of Reb M with a purity greater than 95% was obtained after purification by aftertreatments, such as, separation by silica gel resin, crystallization, etc.

Example 7

In this Example, a UDP-glucose regeneration system composed of sucrose, sucrose synthetase (AtSUS1 for short hereafter) from Arabidopsis thaliana, and UDP was used as a glucosyl donor.

150 mL of a 0.05 mol/L phosphate buffer (pH 7.0), 0.182 g of UDP, 51.3 g of sucrose, 0.17 g of Reb D, 1.5 g of the lyophilized UGT-A powder and 0.5 g of the lyophilized AtSUS1 powder were added successively into the reaction system, mixed uniformly, then placed in a water bath at 30° C., and stirred at 160 rpm to carry out reaction for 2 hours. After completion of the reaction, 500 μl of the reaction solution was taken and added into an equal volume of anhydrous methanol and mixed uniformly. The mixture was centrifuged for 10 min at 8,000 rpm. The supernatant was taken and passed through a filter membrane, followed by detection using high performance liquid chromatography (chromatographic condition: chromatographic column: Agilent eclipse sb-C18 4.6×250 mm; detection wavelength: 210 nm; mobile phase: 1% formic acid aqueous solution:methanol=20%:80%; flow rate: 1.0 mL/min; column temperature: 25° C.). A conversion rate of Reb D was more than 80%. 0.11 g of Reb M with a purity greater than 95% was obtained after purification by aftertreatments, such as, separation by silica gel resin, crystallization, etc.

Example 8

In this Example, a UDP-glucose regeneration system composed of sucrose, sucrose synthetase (referred to as AtSUS1 hereafter) from Arabidopsis thaliana, and UDP was used as a glucosyl donor.

150 mL of a 0.05 mol/L phosphate buffer (pH 7.0), 0,364 g of UDP, 51.3 g of sucrose, 0.145 g of Reb A, 1.5 g each of UGT-A and UGT-B, and 0.5 g of the lyophilized AtSUS1 powder were added successively into the reaction system, mixed uniformly, then placed in a water bath at 30° C., and stirred at 160 rpm to carry out reaction for 2 hours. After completion of the reaction, 500 μl of the reaction solution was taken and added into an equal volume of anhydrous methanol and mixed uniformly. The mixture was centrifuged for 10 min at 8,000 rpm. The supernatant was taken and passed through a filter membrane, followed by detection using high performance liquid chromatography (chromatographic condition: chromatographic column: Agilent eclipse sb-C18 4.6×250 mm; detection wavelength: 210 nm; mobile phase: 1% formic acid aqueous solution:methanol=20%:80%; flow rate: 1.0 mL/min; column temperature: 25° C.). A conversion rate of Reb A was more than 80%. 0.108 g of Reb M with a purity greater than 95% was obtained after purification by aftertreatments, such as, separation by silica gel resin, crystallization, etc.