Fig 1.
Enzymatic system for the synthesis of D-fagomine.
Fig 2.
Tridimensional structure of fructose-6-phosphate aldolase (FSA).
The entrances to the two active sites are located between the Lys85 from five subunits, in yellow. Acid residues are colored in red; basic residues are in blue. His-tag is not shown; not included in the file used from the Protein Data Bank (PDB) [7]. View: (A) front, (B) back, (C) side. Represented using UCSF Chimera software.
Table 1.
Immobilization yield and retained activity of FSA immobilized by different methods on mesoporous supports.
The immobilizations were performed at room temperature under mild agitation.
Fig 3.
Aldol addition reaction of DHA and β-CHO (●) producing pre-D-fagomine (■) catalyzed by soluble FSA and FSA immobilized in mNC-NH2 (A), in Co-IDA (B), in MANA-agarose (C) and in glyoxal-agarose (D).
The reaction catalyzed by soluble enzyme is represented with the dashed line in all the reaction plots. The reaction medium contained: 45 mM DHA, 30 mM β-CHO and 1 U mL-1 of reaction. The aldol addition was performed at 25°C and pH 8.0 and 10 mL total reaction volume (1 mL reaction volume for the mNC-NH2 derivative).
Table 2.
Reaction yield, conversion and initial reaction rate of pre-D-fagomine synthesis catalyzed by FSA.
Reactions conditions: 1 U mL-1, 10 mL reaction volume*, 30 mM β-CHO, 45 mM DHA and 50 mM HEPES buffer, pH 8.0, 25°C, orbital stirring.
Fig 4.
Pre-D-fagomine reaction yields catalyzed by FSA immobilized in mNC-NH2, Co-IDA, MANA-agarose and Glyoxal-agarose in subsequent reaction cycles.
The reaction medium contained: 45 mM DHA, 30 mM β-CHO and 1 U mL-1 of reaction. The aldol addition was performed at 25°C and pH 8.0 for 5 hours.
Table 3.
Total product quantity, total reaction yield and biocatalyst yield for the synthesis of pre-D-fagomine using immobilized FSA in 6 reaction cycles and soluble FSA in a single cycle.
Reactions conditions: 1 U mL-1, 10 mL reaction volume, 30 mM β-CHO, 45 mM DHA and 50 mM HEPES buffer, pH 8.0, 25°C, orbital stirring.