| Aliphatic polyesters have been widely used in the field of agriculture, dope, adhesive, biomedical material 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 by a 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, trace residua and potential toxicity are harmful for biomedical materials. Mild conditions, environmentally biodegradability, high enantio- and regioselectivity of enzymatic synthesis give it an edge over the traditional route. Therefore, enzymatic synthesis route has become a point of research in the biotechnology. In the study, poly(ε-caprolactone) was the goal through ring-opening polymerization. By screening the mesophilic lipases, Candida antarctica lipase B, CALB, was found to be of high efficiency. Then effects of amount of enzyme, reaction time, medium, initiators on monomer conversion and product molecular weight were further investigated. The structure was characterized by IR and 1H NMR. In the optimized conditions (45 oC, 10 h, in toluene), the polymer was obtained in 99.7% monomer conversion with a molecular weight of 41540 g/mol. In addition, alcohols as initiator in the reaction system would increase the monomer conversion and improve the product molecular weight. The chain-initiation rate increased as the adding of initiators, while the chain-propagation rate was inhibited as the competition ofω-hydroxyl fatty acid. The results were significant for investigating the mechanism and kinetics of enzymatic polymerization. By analyzing the structures, terminal-functionalized polyesters were synthesized, and the method provided a basis for further modification to obtain the polymer of new structure and function.Poly(ε-caprolactone) synthesized by CALB catalysis was of high molecular weight and expected to have good mechanical properties. The product showed excellent thermal stability, with the main degradation at 400 oC in the TGA curve. Its melting point and melting enthalpy were 60 oC and 108.70 J/g calculated from DSC profile, respectively. Its crystallization point was 30 oC. Compared with a commercial PCL, CAPA 6501, poly(ε-caprolactone) synthesized by enzymatic catalysis was of higher degree of crystallinity observed by DSC, WAXD and POM. Therefore, enzymatic polymerization was an oriented method and polymer chain was more regular. Tensile and machining testing revealed that the polymer was of good mechanical properties (tensile strength and elongation at break were 12.28 MPa and 5.88 %, respectively).Though great achievement was attained in the lipase-catalyzed polymerization ofε-caprolactone, there were still many problems to be solved, such as low stability in organic solvents and at high temperature. Therefore, hyperthermophilic esterases from archaeon were employed as the catalyst in the ring-opening polymerization ofε-caprolactone.The hyperthermophilic esterase gene (APE1547) from the archaeon Aeropyrum pernix K1 has been over-expressed in Escherchia coli, and the recombinant protein has been characterized in our previous work. R526V was obtained by directed evolution and showed a 6-fold increase in activity higher than the wild-type enzyme. The enzyme to be used in non-aqueous medium was prepared by the fermentation of recombinant E. coli harboring APE1547, ultrasonication cell disintegration, thermal denaturation, ultrafiltration and lyophilization. The activity of enzyme preparation was 664 U/g when p-nitrophenyl caprylate was employed as the substrate. The monomer and molecular weight of polymer were 70% and 5280 g/mol when the thermophilic esterase was employed in the polymerization in the hydrophobic organic solvents. Based on the problem, hyperthermophilic esterase AFEST from archaeon Archaeoglobus fulgidus was employed as the catalyst. In previous research, the enzyme was of high thermal stability and catalytic activity, and could catalyze the hydrolysis of long chain acyl ester. The hydrolytic activity of AFEST prepared in the same method was 250-fold than R526V. Then the reaction conditions were optimized, such as temperature, amount of enzyme, reaction time, reaction medium and water activity. In the optimized reaction conditions (100 mg/ml enzyme concentration, 80 oC for 48 h), poly(ε-caprolactone) was obtained in almost 100% monomer conversion with a number-average molecular weight of 1399 g/mol. The product was oligomer and expected to be widely used as the soft segment of polyurethanes and drug carrier. The Michaelis-Menten kinetic analysis revealed that AFEST showed much higher affinity forε-caprolactone (Km=0.093 mol/L) and lower catalytic efficiency (kcat/Km=0.064 s-1).From the above results, the ability to catalyze the polymerization ofε-caprolactone was different according to the source of lipase. Therefore, it was of great significance to investigate the structure-function relationship and construct recombinant enzyme of high activity to catalyze polymerization through rational design. This work is underway in our laboratory.In all, poly(ε-caprolactone) of high molecular weight (Mn>40000 g/mol) was synthesized by CALB catalysis, and had good thermal and mechanical properties. Low molecular weight poly(ε-caprolactone) (Mn=2000 g/mol) was synthesized using thermophilic esterase APE1547, R526V and AFEST as catalyst. The reaction conditions was optimized and kinetic analysis was performed.In this study, we established a method of enzymatic synthesis of aliphatic polyesters. By screening the enzymes of different resources, polyesters were synthesized in a series of molecular weight and could be used as different functional materials. It provided a base to the industrial production of aliphatic polyesters. With the development of genetic engineering, sustainable enzymatic synthesis of polyesters is expected to be a basic technology in future chemical industry.
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