2.2. Enzymatic Synthesis of Poly (ε-Caprolactone) Using Immobilized Y. lipolytica Lipase
As we reported previously, free Y. lipolytica
lipases are efficient catalysts for the polymerization of lactones in the presence of n-heptane, but the reaction times are long [10
Experiments were performed to determine the reaction order of monomer. The effect of incubation times in the polymerization of ε-CL at 70 °C was done and the results are shown in Figure 1
. It was found that lipases that are immobilized on amberlite beads are not efficient in the enzymatic polymerization of ε-CL. We can observe that 100% of monomer conversion was reached at 12 h when Y. lipolytica
lipase was immobilized in Lewatit K2629 (using decane as solvent, Mn 778 Da; isooctane, Mn 892 Da; Toluene, Mn 673 Da) and amberlyst 15 (isooctane, Mn 930 Da). Lowest conversion (0%) was seen when free Y. lipolytica
lipase was used in toluene and isooctane. The highest molecular weights were obtained when Y. lipolytica
lipase was immobilized in Lewatit 1026 and toluene was used as solvent at 60 h of reaction (Mn 7441 Da, 86% monomer conversion); the lowest molecular weight was reached at 120 h of reaction using free Y. lipolytica
lipase without solvent (Mn 683 Da, 42% of monomer conversion).
The time dependence of the polyesterification in four different solvents, showed the general trend of increasing the degree of polymerization with time.
Based on the kinetic curves, we conclude that immobilized lipases from Y. lipolytica are efficient catalysts for the polymerization of lactones in the presence of organic solvents.
In Figure 2
, the results for enzymatic polymerizations at 90 °C are shown. We can observe that 100% of monomer conversion was reached after 12 h of reaction time when Y. lipolytica
lipase was immobilized on Lewatit K2629 beads (bulk, Mn 899 Da; heptane, Mn 974 Da; toluene, Mn 863 Da; isooctane, Mn 1493 Da; decane, Mn 1441 Da); when free YLL was used, a conversion of 100% was reached at 72 h, and a Mn 1095 in bulk conditions. The highest molecular weight (Mn 10,685 Da) was obtained after 60 h (100% monomer conversion) using YLL that was immobilized on Lewatit 1026 and using decane as solvent, being the lowest molecular weight (Mn 708 Da) using free YLL in toluene after 48 h and a monomer conversion of 74%.
When YLL was immobilized on amberlyst 15, 100% of monomer conversions were reached after 12 h (bulk, heptane, toluene, isooctane, and decane). The highest molecular weight was obtained after 72 h in heptane (Mn 3369 Da) and the lowest after 12 h in toluene (Mn 862 Da).
The dependence of the catalytic activity of the enzymes on the polarity of solvents is attributed to the solvent’s ability to strip the enzyme from its essential water layer, or to penetrate into the water layer in order to distort the interaction between the water and the enzyme molecule. Polyesters of higher molecular weight were obtained in poor solvents, whereas in bulk and in good solvents only low-molecular weight oligomers were formed. This latter finding could be explained in terms of the good solubility and the consequent removal of the product oligomers from the enzyme surface, resulting in a lowering of the concentration of the oligomer-substrate near the enzyme. However, in a poor solvent, the growing substrate concentrates near the enzyme, and thus enables further reaction between the functional hydroxyl and ester groups of larger molecules, and consequently higher-molecular-weight polyesters were produced.
By raising the reaction temperature, a sharp increase in the degree of polymerization of the product polyesters could be observed. A sharp increase in the monomer conversion could be observed too. When comparing experiments carried out at prolonged times at 70 and 120 °C, showed that the molecular weights of the obtained polymers were about 683 and 3498 Da, respectively.
In Figure 3
, the results for enzymatic polymerizations at 120 °C are shown. A control was performed where no enzyme was used. In this control, no monomer conversion was observed after 24 h, and no polymer could be isolated. Monomer conversion of 100% was reached at 2 h using YLL immobilized on Lewatit K2629 (Mn 736 Da), and amberlyst 15 (Mn 788), and decane as solvent. When bulk conditions were used, free YLL reached the 100% of monomer conversion at 16 h and a molecular weight of 1155. The highest molecular weight (Mn 3498 Da) was obtained after 8 h using bulk conditions and YLL immobilized on Lewatit 1026 (100% monomer conversion); the lowest molecular weight (Mn 1059 Da) was obtained using free YLL after 24 h and using decane as solvent (100% monomer conversion). Lip2 is expressed as a 301 amino acid glycosylated protein of 38 kDa [25
], this glycosylation protects the enzyme from denaturation at higher reaction temperatures.