Enzymatic Hydrolysis of Triacetin and L-Lactide in Emulsified Microparticles Within a Cellulose Hydrogel Dispersion

Abstract

Triacetin (TA) is a solvent commonly used in pharmaceutical and food applications, and as a plasticizer in bioplastics such as poly(lactic acid) (PLA) and cellulose acetate (CA). L-lactide is the monomer used in the ring-opening polymerization of PLA. The structure of TA emulsions stabilized by a cellulose hydrogel (CH) was imaged in this study. The emulsions were prepared by mechanical homogenization or a two-step process with subsequent high-pressure homogenization (HPH). The two-step process yielded smaller TA droplets and a more homogeneous CH dispersion. The images demonstrate that emulsion stabilization is due to CH particles adsorbed at the TA-water interface. The ester hydrolysis of TA and a lactide/TA solution by two industrially important lipases, from Candida rugosa (CRL) and Burkholderia cepacia (BCL), was investigated, assessing the effect of CH as an emulsion stabilizer. Mechanically homogenized TA emulsions were effectively hydrolyzed. Lactide was found to inhibit the enzymatic hydrolysis of TA. This inhibition was mitigated by CH for CRL-catalyzed hydrolysis but not for BCL catalysis. These results indicate a synergistic effect of CH stabilization on the interfacial activation of CRL. Thise effect may also be relevant for the biodegradation of bio-derived plastics and their fibrous cellulose composites.

Keywords: biodegradation; cellulose hydrogel; lactide; lipase; polylactic acid (PLA); triacetin.

Figure 1

Confocal 3D rendering micrographs of TA droplets (red) in CH (green) dispersion. Each trio of images contains (from left to right) the merged red and green channels, the red channel alone and the green channel alone. (a) One-step homogenization; (b) two-step homogenization. Volume: 714 (X) × 714 (Y) × 100 (Z) µm.

Figure 2

Confocal cross-sectional XY (white lines), YZ (right) and XZ (bottom) views showing integration of TA droplets (red) in CH (green). The rectangles indicate identical fields of view in the images beside (from left to right, XY slices captured at 5 µm intervals). (a) One-step homogenization; (b) two-step homogenization.

Figure 3

LM images of TA droplets in CH: (a) one-step homogenization; (b) two-step homogenization. Images on the right are enlargements of regions from those at left, as indicated. Arrows point to particular structures: small-size TA droplets; mid-size TA droplets; TA droplets with water inclusions.

Figure 4

Cryo-SEM images of the fracture surface of rapidly frozen TA/CH dispersions made by the two-step process: (a) a large TA droplet; (b) a small droplet. White arrows point to water inclusions. Black arrows indicate possible CH particles at the droplet edge. (c) A combined secondary electron image and EDS elemental mapping of the large droplet shown in (a). Red color: carbon-rich regions; blue color: oxygen-rich regions. (d) An image of a dispersed cellulose gel particle, exposed by the specimen fracture. Images on the right are enlargements of the areas indicated in (b,d).

Figure 5

¹H NMR spectra (295 K, 400 MHz) of samples: (N 1) TA in CDCl₃; (N 2) L-lactide in CDCl₃; (N 3) L-lactide/TA solution in CDCl₃; (N 4) the aqueous phase of L-lactide/TA mixture in D₂O; (N 5) TA in D₂O. (N 6) 1 wt.% of commercial 90 wt.% of L-LA solution in D₂O. The solution concentrations are given in Table 1.

Figure 6

Sequential hydrolysis of TA showing progressive formation of diacetin, monoacetin, glycerol and acetic acid. Annotated proton C positions (green) identify corresponding peaks in the ¹H NMR spectra.

Figure 7

¹H NMR spectra of products of TA’s enzymatic hydrolysis by lipases CRL and BCL in aqueous TA emulsions and emulsions prepared with CH dispersion, after incubation at 45 °C for 120 h. The emulsions were prepared by mechanical homogenization.

Figure 8

Enzymatic conversion of TA after incubation at 45 °C for 120 h with 5 mg mL⁻¹ of lipases BCL and CRL versus control samples, with or without L-lactide, in both aqueous and CH dispersion: (a) total conversion to diacetin, monoacetin and glycerol; (b) yield of TA complete hydrolysis to glycerol. Emulsions were prepared by the one-step mechanical homogenization process.

Figure 9

¹H NMR spectra of the products of TA’s and L-lactide’s enzymatic hydrolysis by lipases CRL and BCL in aqueous TA emulsions and emulsions prepared with CH dispersion, after incubation at 45 °C for 120 h. The emulsions were prepared by mechanical homogenization.

Figure 10

Enzymatic hydrolysis of L-lactide by CRL and BCL lipases (5 mg mL⁻¹), in emulsions prepared by a one-step mechanical homogenization process, after 120 h at 45 °C, compared to lipase-free controls, in both aqueous solution and CH dispersion.

Figure 11

A schematic presentation of an emulsion of TA droplets containing dissolved lactide, with adsorbed lipases, stabilized by CH microparticles, demonstrating CH-assisted activation of the lipase active site at the triacetin–water interface. The purple circles represent enzymes with an open or closed lid. The enlargement on the right indicates (schematically) the role of CH in promoting the open-lid conformation that enables the access of triacetin and lactide to the inner cavity and the catalytic site. The solid purple contour lines represent the lipase’s shape, with large orange dashes marking the inner cavity and small red dashes the open lid (the sketch is not to scale and does not reflect accurate molecular structures).