Lipase-Mediated Mechanoenzymatic Synthesis of Sugar Esters in Dissolved Unconventional and Neat Reaction Systems

Abstract

Mechanochemical and biocatalytic approaches in modern research are two major assets to develop greener processes. In the present study, these modular tools of sustainability are pointed toward the production of versatile and daily employed compounds such as surfactants. Toward this aim, glycolipids, a class of nonionic surfactants composed of ubiquitous and primary metabolites such as sugar and fatty acid moieties, represent a promising alternative to petroleum-derived surface-active agents. Therefore, the combination of biocatalysis with mechanochemistry aiming at glycolipid synthesis seemed a logical step that was taken in this study for the first time. The monoacylated model compound glucose-6-O-decanoate was synthesized with the help of a bead mill apparatus using two different unconventional dissolved reaction systems, namely, menthol-based hydrophobic deep eutectic solvents and 2-methyl-2-butanol, thus reaching up to 12% yield in the latter based on the conversion of vinyl decanoate, after only 90 min of reaction. In addition, a neat reaction system using an excess of vinylated fatty ester as an adjuvant allowed a 27 mM/h space-time yield. The overall significant increase in productivities, up to 6 times, compared to standard heating and shaking methods, shows the tremendous potential of mechanoenzymatic synthesis.

Scheme 1. Reaction Scheme to Produce Glucose Monodecanoate

Figure 1

Bead mill apparatus for processing the samples.

Figure 2

Influence of different solvents on the mechanoenzymatic synthesis of glucose monodecanoate. Highest product titers were achieved in 2M2B. Reaction conditions: substrate ratio glucose: VD is 1:1 (0.5/0.5 M); 33 mg/mL Novozym 435; 0.5 g of glass beads (710–1180 μm); 600 μL of solvent; bead milling frequency: 25 Hz. 2M2B: 2-methyl-2-butanol; DES: hydrophobic (−)-menthol: decanoic acid DES; DES-VD: hydrophobic (−)-menthol: decanoic acid DES without addition of VD. n = 3, p-value < 0.05.

Figure 3

Influence of the milling frequency and a pre-grinding step on mechanoenzymatic glycolipid production yields. Reaction conditions: substrate ratio glucose/VD is 1:1 (0.5/0.5 M) at a reaction time of 45 min; 33 mg/mL Novozym 435; 0.5 g glass beads (710–1180 μm); 600 μL of solvent. (a–c) Statistically significant differences (p < 0.05).

Figure 4

Influence of substrate ratio on product titers and yields. (A) In 2M2B; (B) in (−)-menthol:decanoic acid DES. Tested ratios: 1:1 (0.5 M glucose: 0.5 M VD), 1:2 (0.25 M glucose: 0.5 M VD), and 2:1 (0.5 M glucose: 0.25 M VD). For equimolar ratio and 2:1, the curves of yield and concentration are overlapping. Reaction conditions: 33 mg/mL of Novozym 435; 0.5 glass beads (710–1180 μm); 600 μL of solvent; 25 Hz milling frequency. Data are presented as mean values ± standard deviations (n = 3).

Figure 5

Influence of enzyme loading on mechanoenzymatic production of glucose monodecanoate. (A) In 2M2B; (B) in DES. Reaction conditions: substrate ratio glucose: VD is 1:1 (0.5/0.5 M); 0.5 g glass beads (710–1180 μm); 600 μL solvent; 25 Hz milling frequency; 90 min. (a–c) Statistically significant differences (p < 0.05).

Figure 6

Solvent-free glycolipid synthesis. (A) Reaction time course of glucose monodecanoate production in a solvent-free mechanoenzymatic synthesis (substrate ratio glucose:VD is 1:1). (B) Influence of VD ratio on glucose monodecanoate yields in solvent-free mechanoenzymatic synthesis (substrate ratio glucose:VD is 2:1; 1:1; 1:2; 1:4; 1:8). Reaction conditions: 20 mg Novozym 435; 50 mg glucose; 0.5 g glass beads (710–1180 μm); 90 min, 25 Hz milling frequency. (a–c) Statistically significant differences (p < 0.05).