Ring-opening Polymerization Of ε-caprolactone Catalyzed By Thermophilic Esterase

Aliphatic polyesters have been widely used in the field of agriculture, dope, biomedical material, adhesive and packing due to their good biocompatibility, biodegradability and mechanical properties. In the field of biomedical material, they are mainly used as sutures, bone fixing materials and drug delivery systems. The biodegradable aliphatic polyesters are synthesized mainly in the chemical route. However, there are many disadvantages in the chemical route, such as strict reaction conditions (high temperature, reduced pressure, et al.), multi-byproducts and complex process. In addition, concern has been raised about the harmful effects for medical applications because of trace metallic residues and potential toxicity. Mild conditions, eco-friendly, high enantio- and regioselectivity of enzymatic synthesis give it an edge over the traditional route. Therefore, enzymatic synthesis route has become a hot-point of research in the biotechnology. However, enzymatic synthesis also has its disadvantages: (1) enzymes can be easily denatured in the presence of organic solvents, strong acid and base, or under high temperature; and (2) the cost of enzymes is too high. Recently, enzymes from extremophiles, especially thermophiles, have been recognized as making up the deficiencies in the industrial applications and having the potential for polyester synthesis due to their high stability against organic solvents.In the paper, the ring-opening polymerization ofε-caprolactone in organic solvents was investigated using the thermophilic esterase AFEST from the archaeon Archaeoglobus fulgidus as the catalyst. In the previous research, the enzyme has high stability and catalytic activity, especially for the hydrolysis of acyl esters of long chain. First, the enzyme to be used in non-aqueous medium was prepared by the fermentation of recombinant E. coli harboring gene AF1716 of thermophilic esterase AFEST, ultrasonication cell disintegration, thermal denaturation, ultrafiltration and lyophilization. Then the reaction conditions were optimized, such as temperature, enzyme concentration, reaction time, reaction medium and water activity. In the optimized conditions (25 mg/mL enzyme concentration, in toluene, water activity 0.75, 80 oC for 72 h), poly(ε-caprolactone) was obtained in almost 100% monomer conversion with a number-average molecular weight of 1400 g/mol. The product was oligomer and expected to be widely used as the soft segment of polyurethanes and drug carrier. Meanwhile, the properties of poly(ε-caprolactone) obtained were characterized by IR, DSC, 1H and 13C NMR. IR analysis showed the presence of ester bond, carbonyl, hydroxyl group and C-H bond. The structure of poly(ε-caprolactone) was also confirmed by 1H and 13C NMR. From DSC profile, we calculated that the melting and crystallization temperature was at 58 oC and 25 oC, respectively. The Michaelis-Menten kinetic analysis revealed that compared with CALB-catalyzed ring-opening of polymerization ofε-caprolactone in toluene at 45 oC (K_m=0.72 mol/L and k_cat/K_m=72.9 s^-1), AFEST showed much higher affinity forε-caprolactone (K_m=0.093 mol/L) and lower catalytic efficiency (k_cat/K_m=0.064 s^-1). Molecular docking studies showed that AFEST had stronger hydrogen bonding interactions with the monomerε-caprolactone, and therefore the higher affinity. In the viewpoint of energy, the interaction energies of AFEST to the monomerε-caprolactone (-33.81 kcal mol^-1) was lower than that of CALB (-30.17 kcal mol^-1); and the free energy of binding (-5.15 kcal mol^-1) was also lower than that of CALB (-3.27 kcal mol^-1). Therefore, AFEST had higher affinity forε-caprolactone than CALB, which was consistent with the Michaelis-Menten kinetic analysis. In subsequent research, the immobilization of thermophilic esterase AFEST was preliminarily performed. We employed hydrophobic poly(methyl methacrylate) Sepabead EC-OD as the immolization support, and physical absorption was successfully achieved in room temperature. The reaction conditions of ring-opening polymerization ofε-caprolactone were optimized as follows: enzyme concentration of 75 mg/mL, in toluene, with the ratio of monomer to toluene of 1: 3 at 80 oC for 72 h. The selection of immobilization supports and optimization of the immobilization conditions are now underway in our laboratory.In this study, we employed the thermophilic esterase AFEST as the catalyst in the synthesis of poly(ε-caprolactone) . The process of poly(ε-caprolactone) was established by optimizing the reaction conditions and charactering the properties of the polymer. In addition, the Michaelis-Menten kinetic analysis and molecular docking studies were also undertaken to gain the deeper insight of catalytic mechanism. Furthermore, we performed the immobilization of thermophilic esterase AFEST and employed it in the ring-opening polymerization ofε-caprolactone, which provided a base to the industrial application of thermophilic enzymes. With the development of genetic engineering, sustainable enzymatic synthesis of polyesters is expected to be a basic technology in future chemical industry.