One-Pot Multienzyme Synthesis of Rare Ketoses from Glycerol

Abstract

A facile approach is introduced here for the synthesis of rare ketoses from glycerol and d-/l-glyceraldehyde (d-/l-GA). The reactions were carried out in a one-pot multienzyme fashion in which the only carbon source is glycerol. In the enzymatic cascade, glycerol is phosphorylated and then oxidized at C2 to afford dihydroxyacetone phosphate (DHAP), the key donor for enzymatic aldol reaction. Meanwhile, the primary alcohol of glycerol is also oxidized to give the acceptor molecule GA in situ (d- or l-isomer could be formed stereospecifically with either alditol oxidase or horse liver alcohol dehydrogenase). Different DHAP-dependent aldolases were used to generate the aldol adducts (rare ketohexose phosphates) with various stereoconfigurations and diastereomeric ratios. It is worth noting that the enzyme that catalyzes the phosphorylation reaction in the first step could also help recycle the phosphate in the last step to provide free rare sugar molecules. This study provides a useful method for rare ketose synthesis on a 100 mg to g scale, starting from relatively inexpensive materials which solved the problem of supplying both glycerol 3-phosphate and GA in our previous work. It also demonstrates an example of green synthesis due to highly efficient carbon usage and recycling of cofactors.

Getting the Most Out of Enzyme Cascades: Strategies to Optimize In Vitro Multi-Enzymatic Reactions

“…Titrating one substance at a time and investigating the cascades performance under these conditions is the most common approach. Lit et al investigated the effect of the starting materials glycerol, pyrophosphate and D-glyceraldehyde sequentially on the product titer or the relative activity of their multi-enzymatic system [85]. With the substrates and four enzymes, the synthesis of rare ketoses such as D-sorbose and D-allulose is possible.…”

“…An increase in product titer or relative activity for each of these starting materials was observed with increasing concentrations, but the effect slowed down at certain concentrations. With the optimized reaction conditions and enzyme ratios, a conversion of 90% after h and full conversion after 36 h was reached in a 10 mL scale [85]. This titrating approach was also chosen for optimizing the production of (R)-acetoin from D-xylose [38].…”

Recent Advances in Enzymatic and Chemoenzymatic Cascade Processes

“…DHAP may be produced from glycerol 3-phosphate through oxidation by glycerol phosphate oxidase, and the by-product hydrogen peroxide must be degraded into water and oxygen by a catalase [28,29]. Considering that glycerol 3-phosphate and L/D-glyceraldehyde are still either expensive and/or relatively unstable, their production from glycerol has been investigated [29,30].…”

“…Recently, Li et al [30] introduced the one-pot synthesis of a series of rare ketohexoses, using glycerol as the sole carbon source, by a four-enzyme cascade reaction (Figure 2). Glycerol was initially phosphorylated by the acid phosphatase from Shigella flexneri (PhoN-Sf), and further oxidized by a recombinant glycerol phosphate oxidase (GPO).…”

“…According to the enzymes utilized, D-sorbose/D-allulose, L-fructose, L-tagatose/L-fructose, and L-sorbose were obtained. This study provides a useful method for rare ketose synthesis on a multi-milligram to gram scale, starting from relatively inexpensive materials [30]. Multi-enzyme synthesis of D-sorbose/D-allulose from glycerol.…”