Synthesis Of Aliphatic Polyesters Catalyzed By Novel Thermophilic Esterases

Aliphatic polyesters have been widely used as the packaging and biomedical materials, due to their favorable biocompatibility, biodegradability and mechanical properties. In the filed of biomedical materials, they are usually used as controlled release drug carrier and biomedical engineering materials. To date, they are mainly synthesized in the chemical route. In the conventional chemical route, reaction conditions must be strictly controlled, such as high temperature, reduced pressure, anhydrous and no oxygen. Moreover, concern has been raised about the harmful effects for medical applications because of trace metallic residues and potential toxicity. Recently, enzymatic polymerization has become a hot-spot of research in the biotechnology, as it possessed the advantages of mild conditions, eco-friendly, high enantio- and regioselectivity. However, due to the lack of stability in high temperatures and organic solvents, enzymatic polymerization was not well suited for the harsh reaction conditions, and hard to combine with chemical polymerization. Therefore, it is of great significance to explore novel biocatalysts and get deeper insight into the catalytic mechanism. In the paper, thermophilic esterases of high thermostability and resistance against organic solvents were employed as catalysts in polyester synthesis to systematically investigate the catalytic kinetics, mechanism and process.Herein, two thermophilic esterases from Archaeoglobus fulgidus and Fervidobacterium nodosum were employed as catalysts in the aliphatic polyesters synthesis. First, we performed the condensation polymerization of diols and diacids and ring-opening polymerization of lactones in organic solvents. It was proved that these two enzymes had the promising polyester synthesis activity. The obtained products were of low molecular weight (Mn<2500 g/mol) and narrow molecular weight distribution. Meanwhile, in the condensation polymerization, both of these two enzymes exhibited the preference of longer substrates. Compared with condensation polymerization, there were no small molecules as water produced in the ring-opening polymerization, and thus polymers of higher yield and molecular weight were more easily obtained. To the best of our knowledge, we for the first time reported that esterase had the promising polyester synthesis activity, and thus reliazed the highly efficient polyester synthesis at high temperatures.In order to get deeper insight into the polyester synthesis in organic solvents, ring-opening polymerization ofε-caprolactone was performed as the model. First, we optimized the reaction conditions, such as enzyme concentration, temperature, reaction time, reaction medium and water activity. In the optimal reaction conditions, monomer conversion values were almost 100% in AFEST and FNE-catalyzed poly(ε-caprolactone) synthesis. The polymers obtained were of lower molecular weight (1400 and 2340 g/mol) and polydispersity index (1.21 and 1.35), and expected to be widely used as the soft segment of thermoplastic elastomers and controlled release drug carrier. Structural characterization indicated the presence of linear and cyclic polymers, and the polymers were of low melting temperature and favorable thermostability through thermal analysis. Notably, compared with Humicola insolens cutinase recently developed by Novozymes, these two thermophilic esterases were highly stable and active in high temperatures and organic solvents, and thus it was beneficial for the one-pot integration of chemical and enzymatic routes to prepare the polymers of novel architecture and performance. Second, Michaelis-Menten kinetic analysis revealed that AFEST and FNE showed higher affinity forε-caprolactone (Km=0.093 and 0.35 mol/L) than commercially available mesophilic enzyme, CALB (Km=0.72 mol/L). Molecular modeling and docking indicated that there was strong hydrogen bonding interaction in the theoretically modeled complex, and the interaction energy was also lower than that of CALB. Through the above analysis, we obtained the deeper understanding of catalytic mechanism in both the structural and energetic levels.To reduce the cost of enzymes in the process, hydrophobic Sepabeads EC-OD was selected as the support to immobilize the enzyme AFEST. In addition, FNE was expressed in an insoluble and active form, and thus the recombinant E. coli whole-cell biocatalyst harboring FNE gene was also employed as the catalyst. The immobilized AFEST and recombinant E. coli whole-cell biocatalyst were of high operational stability and catalytic activity, and could be efficiently reused more than 5 and 12 times, respectively. During the reactions, monomers were completely consumed, and the products obtained were homogeneous oligopolyesters. The immobilization technique and whole-cell biocatalyst provided outstanding advantages over conventional enzymatic process with regard to high catalytic efficiency, inexpensive biocatalyst production and excellent operation stability, and thus it was propitious to the establishment of green, low-cost polyester synthesis process.In all, we established a novel process for thermophilic esterase-catalyzed synthesis of aliphatic polyesters, and provided an important foundation for mild, metal-free polyester synthesis at an industrial scale. With the development of genetic engineering and protein engineering, and the emergence of numerous biocatalysts with high activity and stability, sustainable enzymatic synthesis of polyesters is expected to be a basic technology in future chemical industry.