Ring-opening polymerization of ɛ-caprolactone catalyzed by a novel thermophilic lipase from Fervidobacterium nodosum

Process Biochemistry 46 (2011) 253–257

Contents lists available at ScienceDirect

Process Biochemistry journal homepage: www.elsevier.com/locate/procbio

Ring-opening polymerization of ␧-caprolactone catalyzed by a novel thermophilic lipase from Fervidobacterium nodosum Quanshun Li a , Guangquan Li a , Shanshan Yu a , Zuoming Zhang a , Fuqiang Ma a , Yan Feng a,b,∗ a b

Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, 2519 Jiefang Road, Changchun 130023, China Key Laboratory of MOE for Microbial Metabolism and School of Life Science & Technology, Shanghai Jiao Tong University, Shanghai 200240, China

a r t i c l e

i n f o

Article history: Received 14 April 2010 Received in revised form 12 July 2010 Accepted 24 August 2010 Keywords: Thermophilic lipase ␧-Caprolactone Ring-opening polymerization Kinetic evaluation Molecular docking

a b s t r a c t The paper explored the catalytic activity of a novel thermophilic lipase from Fervidobacterium nodosum for polyester synthesis, using the ring-opening polymerization of ␧-caprolactone as the model. Effects of enzyme concentration, reaction medium, temperature and reaction time on monomer conversion, product molecular weight and distribution were systematically investigated. Remarkably, the enzyme could be effectively performed at high temperatures, and showed the highest activity towards the polymerization of ␧-caprolactone at 90 ◦ C. Through the optimization of reaction conditions, poly(␧-caprolactone) was obtained in almost 100% monomer conversion, with a number-average molecular weight of 2340 g/mol and a polydispersity index of 1.34 in toluene at 90 ◦ C for 72 h. Michaelis–Menten kinetic analysis indicated that compared with Candida antarctica lipase B, the enzyme had higher affinity for ␧-caprolactone with a Km value of 0.35 mol/L. Furthermore, the possible structural and energetic basis of the interaction of enzyme and the monomer ␧-caprolactone was elucidated using molecular docking. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Poly(␧-caprolactone) (PCL) has been considered as an important material in various biomedical applications because it has many fascinating characteristics, such as biodegradability, biocompatibility and permeability [1]. In the PCL synthesis, organometallic catalysts with emphasis on tin carboxylates and aluminium alkoxides were usually employed as catalysts [2–4]. However, residues of organometallic catalysts were not tolerated in biomedical applications due to their toxicity. Recently, non-metal catalysts from natural sources like lipases were increasingly employed as biocatalysts for the preparation of PCL [5–11]. Moreover, mild reaction conditions, high enantio- and regioselectivity, and recyclability of enzyme gave it an edge over conventional chemical polymerization. To date, various lipases or esterases have been employed in the ring-opening polymerization of ␧-caprolactone, such as porcine pancreatic lipase, Pseudomonas cepacia lipase, Candida antarctica lipase B (CALB) and Humicola insolens cutinase (HIC) [12–15]. Among them, CALB immobilized on a macroporous acrylic polymer resin Lewatit, showed an extraordinary catalytic activity towards the polymerization of ␧-caprolactone, and the number-

∗ Corresponding author at: Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, 2519 Jiefang Road, Changchun 130023, China. Tel.: +86 431 85155218; fax: +86 431 85155218. E-mail address: [email protected] (Y. Feng). 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.08.019

average molecular weight (Mn ) of PCL obtained was more than 4 × 104 g/mol [14]. However, due to the lack of stability, these mesophilic enzymes were not well suited for the harsh reaction conditions required in the industrial preparation of PCL, such as high temperature and exposure to organic solvents. Furthermore, their instability in harsh reaction conditions would obstruct the integration of enzymatic and chemical polymerization in a onepot reaction to synthesize novel polymers [16]. Enzymes from extremophiles, especially thermophiles, have been recognized as the potential catalysts in various biotechnological applications due to their excellent stability against high temperature, organic solvents and chemical denaturants [17,18]. To the best of our knowledge, no previous investigations have been published on the thermophilic enzyme-catalyzed synthesis of PCL, except for thermophilic esterase AFEST from Archaeoglobus fulgidus [19]. The enzyme AFEST was classified as a member of hormone-sensitive lipase group of the lipase/esterase superfamily, and exhibited the highest activity towards p-nitrophenyl hexanoate among the pnitrophenyl esters tested, with a kcat /Km value of 92.2 s−1 ␮M−1 [20,21]. However, no activities towards longer p-nitrophenyl esters (≥C10 ) and triglycerides were detected [20]. Recently, our laboratory cloned, expressed and characterized a novel thermophilic lipase FNL from Fervidobacterium nodosum, an extremely thermophilic, glycolytic anaerobic bacterium [22]. Compared with AFEST, FNL could effectively catalyze the hydrolysis of a much broader range of esters, including longer p-nitrophenyl esters (C12 to C16 ), triglycerides of short to mid-acyl chain (C4 to C10 ) and

254

Q. Li et al. / Process Biochemistry 46 (2011) 253–257

olive oil. Among the p-nitrophenyl esters, it exhibited the highest activity towards p-nitrophenyl decanoate, with a kcat /Km value of 22.5 s−1 ␮M−1 . Therefore, it was of great significance to investigate the differences between these two enzymes in polyester synthesis, and the work would facilitate the exploration of new biocatalysts for polyester synthesis and the understanding of kinetics and mechanism of enzymatic polymerization. Herein we reported a detailed investigation on the ringopening polymerization of ␧-caprolactone using FNL as the catalyst. Effects of reaction conditions on monomer conversion and product molecular weight were systematically evaluated. Using Michaelis–Menten kinetic analysis and molecular docking, we gained a deeper insight into the kinetics and mechanism of enzymatic polymerization.

The 1 H and DEPTQ 13 C NMR spectra of the product PCL were as follows: 1 H NMR (CDCl3 ; ppm) 1.39 (m, –COCH2 CH2 CH2 –), 1.66 (m, –COCH2 CH2 CH2 CH2 CH2 O–), 2.31 (t, –COCH2 –), 4.06 (t, –CH2 O–), 3.65 (t, –CH2 OH end group), 2.36 ppm attributable to –COCH2 – of cyclic oligomers; DEPTQ 13 C NMR (CDCl3 ; ppm) 24.9 (–COCH2 CH2 CH2 –), 25.9 (–COCH2 CH2 –), 28.7 (–CH2 CH2 O–), 34.5 (–COCH2 –), 62.8 (–CH2 OH, end group), 64.5 (–CH2 O–), 173.8 (–CO–, negative resonance).

2. Materials and methods

2.6. Determination of Mn and polydispersity index (PDI)

2.1. Materials

The Mn and PDI values of products were determined by GPC. Analyses were carried out using a Shimadzu HPLC system equipped with a refractive index detector and Shim-pack GPC-804 and GPC-8025 ultrastyragel columns in series. Tetrahydrofuran was used as the eluent with a flow rate of 1.0 mL/min at 40 ◦ C. The sample concentration and injection volume were 0.3% (w/v) and 20 ␮L, respectively. The GPC system was calibrated with polystyrene standards of narrow molecular weight distribution.

The recombinant Escherichia coli BL21 harboring the thermophilic lipase gene FN1333 from F. nodosum was constructed using expression vector pET-28a, and stored in our laboratory [22]. ␧-Caprolactone was purchased from Fluka Chemical Co. in the highest available purity and used as received. Chloroform-d was obtained from Aldrich Chemical Co. 4 A˚ molecular sieves were purchased from Tianjin Chemical Co. (Tianjin, China), and roasted at 500 ◦ C for 3 h. Dioxane, acetone, tetrahydrofuran, dichloromethane, chloroform, toluene, cyclohexane and n-hexane were purchased from Beijing Chemical Co. (Beijing, China), and dried over 4 A˚ molecular sieve before use. Yeast extract and tryptone were purchased from Oxoid Ltd. Kanamycin, p-nitrophenyl caprylate and isopropyl ␤-d-thiogalactopyranoside (IPTG) were purchased from Sigma. All other chemicals were of analytic grade, and used without further purification. 2.2. Purification of the recombinant enzyme FNL The recombinant E. coli BL21 harboring FN1333 from F. nodosum was cultured in 2YT medium (1% yeast extract, 1.6% tryptone and 0.5% NaCl) containing kanamycin (50 ␮g/mL) at 37 ◦ C. When the optical density at 600 nm of culture reached 1.0, the induction was carried out by adding IPTG at a final concentration of 1 mM and shaking for an additional 6 h at 37 ◦ C. The cells were harvested by centrifugation at 8000 rpm for 15 min at 4 ◦ C, and washed with 50 mM phosphate buffer (pH 8.0). The harvested cells were frozen and thawed three times, and then suspended in 50 mM phosphate buffer (pH 8.0). After ultrasonic cell disintegration and centrifugation at 5000 rpm for 20 min, the pellet was collected, suspended in 50 mM phosphate buffer (pH 8.0) and then incubated at 60 ◦ C for 20 min [22]. The suspension was centrifuged at 12,000 rpm for 20 min, and the supernatant was collected, analyzed by 12% SDS-PAGE, and then subjected to lyophilization. The lipase activity was determined by measuring the amount of p-nitrophenyl from the hydrolysis of p-nitrophenyl caprylate at 60 ◦ C [23], except that the enzyme was assayed in 50 mM phosphate buffer (pH 8.0) instead of the Tris–HCl buffer. One unit of enzymatic activity was defined as the amount of protein releasing 1 ␮mol p-nitrophenyl from p-nitrophenyl caprylate per minute. 2.3. FNL-catalyzed ring-opening polymerization of ␧-caprolactone The lyophilized enzyme FNL was dried in a desiccator overnight and transferred into a dried screwed vial containing 200 ␮L ␧-caprolactone and 600 ␮L organic solvent (no organic solvent addition for solvent-free system). Ethylbenzene (50 ␮L) was added as an internal standard to quantify the monomer conversion with gas chromatography (GC). The vial was sealed and then placed into a thermostatic reactor with stirring (180 rpm) at predetermined temperatures. An aliquot of reaction mixture (10 ␮L) was taken via a syringe at regular intervals, diluted with dichloromethane (100 ␮L) and then analyzed by GC to determine the monomer conversion. Reaction was terminated by adding dichloromethane and filtrating to remove the enzyme. The filtrate was collected and roto-evaporated to remove dichloromethane, and the remaining viscous sample was precipitated in methanol at −20 ◦ C. The cloudy solution was centrifuged (8000 rpm, 15 min), and the white precipitate was dried in a vacuum oven. The products were then characterized by nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). 2.4. Structural characterization of the product PCL The structure of polymer was characterized by 1 H and 13 C NMR. The spectra were recorded on an AVANCE DMX 500 spectrometer in chloroform-d. The chemical shifts for 1 H NMR spectrum were referenced relative to tetramethylsilane (0.00 ppm). The 13 C NMR spectrum was recorded using the distortionless enhancement by polarization transfer including the detection of quaternary nuclei (DEPTQ) program.

2.5. Determination of monomer conversion The monomer conversion was determined with a Shimadzu 2014 gas chromatograph equipped with an Rtx-1 capillary column (30 m × 0.25 mm × 0.25 ␮m) and a hydrogen flame ionization detector. Nitrogen was used as the carrier gas at an average velocity of 31 cm/s. The temperatures of injection pool and detector were set at 200 ◦ C and 240 ◦ C, respectively. Following injection, the column temperature was held at 70 ◦ C for 2 min, and then programmed to rise at 10 ◦ C/min to a final temperature of 140 ◦ C, which was maintained for 2 min. The injection volume was 1.0 ␮L. The monomer conversion values were the average of duplicate measurements.

2.7. Michaelis–Menten kinetic evaluation The Michaelis–Menten kinetic evaluation was performed in toluene at 70 ◦ C, according to the methods previously reported [24,25]. In all cases, the amount of FNL was kept constant at 100 mg, while the concentrations of ␧-caprolactone were varied between 0.125 M, 0.25 M, 0.50 M, 0.75 M, 1.00 M and 2.00 M in toluene. Ethylbenzene (200 ␮L) was added as the internal standard, and the total volume was 3 mL. Samples (10 ␮L) were taken at appropriate time intervals and analyzed by GC to determine the monomer conversion values. The initial rate of reaction V was calculated from the initial rate constant for each ␧-caprolactone concentration, which was derived from the linear fitting of ln(1-conversion) vs time plot assuming firstorder kinetics. The Hanes–Woolf plot [S]/V = (Km /Vmax ) + ([S]/Vmax ) was selected to calculate the Michaelis–Menten constant Km and the maximal rate of reaction Vmax , where [S] represented the concentration of ␧-caprolactone. 2.8. Molecular docking studies Due to the lack of crystallographic structure, the theoretical three-dimensional (3D) model of FNL [22] was employed to elucidate the detailed catalytic mechanism. The 3D model of FNL was constructed by the combination of structure prediction on the Phyre server and molecular dynamics simulation. A serine hydrolase involved in the carbazole degradation (PDB code 1J1I) [26], was used as the template. The predicted structure of FNL was in accordance with the ‘canonical’ ␣/␤ hydrolase fold [27]. Based on the 3D model, Ser119, Asp260 and His282 were predicted as the catalytic triad, and Ile53 and Met120 were probably the residues of oxygen hole. The theoretical enzyme–monomer complex was constructed with Insight II package, version 2000 (Accelrys, San Diego, CA, USA) to obtain the structural and energetic information. The 3D structure of the monomer ␧-caprolactone was built with Builder program and optimized using AM1 method. Docking experiment was performed with Affinity program, using a combination of Monte Carlo-type and simulated annealing procedures. The potential of enzyme–monomer complex was assigned by using the consistent-valence force field (CVFF). To consider the solvent effects, the centered enzyme–monomer complex was solvated in a sphere of TIP3P ˚ This provided 10 structures from SA dockwater molecules with a radius of 10 A. ing, and the generated conformations were clustered according to root mean square deviation (RMSD) value. The global structure with the lowest energy was chosen for calculating the interaction energy between the monomer and enzyme.

3. Results and discussion The recombinant thermophilic lipase FNL was purified as described in Section 2.2. SDS-PAGE analysis showed a major band at 30 kDa, which corresponded to the enzyme FNL (Fig. 1). The gel was analyzed with an ARTHUR 1442 multi-wavelength fluoroimager, and the purity calculated was about 95%. The FNL solution in 50 mM phosphate buffer (pH 8.0) was then subjected to lyophilization. The lyophilized FNL, with a specific activity of approximately 6 U/mg for p-nitrophenyl caprylate at 60 ◦ C, was used as catalyst in the ring-opening polymerization of ␧-caprolactone.

Q. Li et al. / Process Biochemistry 46 (2011) 253–257

255

Table 1 Monomer conversion and product molecular weight Mn in various organic solvents at 60 ◦ C for 72 h. Solvent

Log P

Monomer conversion (%)

Mn (g/mol)

PDI

Dioxane Acetone Tetrahydrofuran Dichloromethane Chloroform Toluene Cyclohexane n-Hexane Solvent-free

−1.10 −0.23 0.49 0.93 2.00 2.50 3.09 3.50 –

45 49 53 57 46 76 81 94 61

710 730 980 760 760 1330 1690 1850 1690

1.54 1.52 1.22 1.57 1.50 1.35 1.17 1.26 1.50

increase of monomer conversion. Similar to monomer conversion, Mn increased slowly from 1330 g/mol (30 mg FNL) to 1600 g/mol (100 mg FNL). Therefore, in the viewpoint of cost-effectiveness, 30 mg FNL was the optimal amount of enzyme in the reaction system, and employed in the subsequent research.

Fig. 1. SDS-PAGE analysis of the recombinant thermophilic lipase FNL. Lane 1: molecular mass marker; Lane 2: the supernatant before lyophilization.

3.1. Effects of reaction conditions on FNL-catalyzed synthesis of PCL 3.1.1. Effect of enzyme concentration In the enzymatic polymerization of ␧-caprolactone, enzyme concentration had a profound effect on both the monomer conversion and product molecular weight [28]. Effect of enzyme concentration was investigated using different amounts of FNL, 200 ␮L ␧-caprolactone and 600 ␮L toluene at 60 ◦ C for 72 h. Monomer conversion and Mn as a function of amount of FNL were shown in Fig. 2. It was obvious that increasing the enzyme concentration would result in the increased monomer conversion and product molecular weight. When the amount of enzyme used in polymerization exceeded 30 mg, the monomer conversion values were more than 75%, and increased smoothly from 75% (30 mg FNL) to 80% (100 mg FNL). This phenomenon was probably caused by the fact that large amounts of insoluble enzyme in the reaction system would lead to mass transfer limitation, and thus the slight

Fig. 2. Monomer conversion () and Mn () as a function of amount of enzyme for FNL-catalyzed ring-opening polymerizaton of ␧-caprolactone. The reactions were carried out using 200 ␮L ␧-caprolactone and 600 ␮L toluene at 60 ◦ C for 72 h.

3.1.2. Effect of reaction medium The reaction medium played an important role in determining enzyme stability and regulating the partition of substrates and products between the solvent and enzyme [29]. Effects of organic solvents on monomer conversion and Mn at 60 ◦ C for 72 h were summarized in Table 1. It was apparent that lower monomer conversion and Mn values were observed in the relative hydrophilic solvents (log P < 2.0). Highly efficient production of PCL was achieved in hydrophobic hydrocarbon solvents (toluene, cyclohexane and n-hexane), with monomer conversion more than 75%. These results could be ascribed to the deactivation of enzyme by the relative hydrophilic solvents, since these solvents might disrupt the functional structure of enzyme or strip off the essential water from enzyme [29,30]. Though higher monomer conversion and Mn values were obtained in cyclohexane and n-hexane, the synthesized PCL was of lower solubility in these two solvents and hard to be separated from the reaction system, which would definitely limit the future application of enzymatic polymerization at an industrial scale. Therefore, toluene was selected as the reaction medium for synthesizing PCL and further kinetic studies. In toluene, PCL was obtained in 76% monomer conversion, with Mn value of 1330 g/mol. 3.1.3. Effect of reaction time and temperature Time-course studies of FNL-catalyzed ring-opening polymerization of ␧-caprolactone were performed at different temperatures in the range of 60–90 ◦ C. Fig. 3 shows the corresponding plots of monomer conversion vs reaction time. A large increase in FNL activity for ␧-caprolactone polymerization was found when the reaction temperature increased from 60 ◦ C to 90 ◦ C. After 72 h, the monomer conversion increased from 76% at 60 ◦ C to almost 100% at 90 ◦ C. The increased monomer conversion values at higher temperatures were probably caused by the decreased diffusion constraints in the reaction mixture and increased water molecules accessible in the chain initiation reaction. Compared with the complete loss of enzymatic activity of HIC at temperatures more than 70 ◦ C [31], FNL was highly thermostable, and could efficiently catalyze the ring-opening polymerization of ␧-caprolactone at much higher temperatures. This would be of great significance for the simultaneous, single-step chemoenzymatic synthesis of novel polymers. To get a further understanding of advantages of enzymatic polymerization at high temperatures, effect of temperature on monomer conversion and product molecular weight was investigated at different temperatures in toluene for 72 h. As shown in Fig. 4, both the monomer conversion and product molecular weight at 90 ◦ C were higher than other reaction temperatures.

256

Q. Li et al. / Process Biochemistry 46 (2011) 253–257

Fig. 3. Monomer conversion as a function of reaction time for FNL-catalyzed ringopening polymerization at 60 ◦ C (), 70 ◦ C (䊉), 80 ◦ C () and 90 ◦ C (). The reactions were carried out using 30 mg FNL, 200 ␮L ␧-caprolactone and 600 ␮L toluene.

Compared with the reaction at 50 ◦ C, the monomer was completely consumed, and Mn increased almost 2-fold to 2340 g/mol at 90 ◦ C. Meanwhile, the product obtained was of narrow molecular weight distribution (PDI = 1.34). Although the hydrolytic activity of thermophilic lipase FNL preparation (6 U/mg) was much lower than AFEST (168 U/mg) using p-nitrophenyl caprylate as the substrate [19], after optimizing the reaction conditions, monomer conversion could still reach 100%, and the product Mn value was 2340 g/mol, even higher than PCL obtained via AFEST-catalyzed ring-opening polymerization (1400 g/mol) [19]. The improved monomer conversion and product molecular weight might be attributed to the relatively lower hydrolytic activity of FNL towards the generated polyester chain in organic solvents. 3.2. Michaelis–Menten kinetic evaluation To get insight into the kinetics of FNL-catalyzed ring-opening polymerization of ␧-caprolactone, Michaelis–Menten kinetic evaluation was performed. The Km and Vmax values calculated were 0.35 mol/L and 2.54 × 10−2 mol/(L h), respectively. To calculate the turnover number kcat , we determined an active protein content of 95% (w/w) of the enzyme preparation and a molecular

Fig. 5. The hydrogen bonding interactions of the theoretically modeled complex FNL-␧-caprolactone. The monomer ␧-caprolactone was in yellow, and the hydrogen bonds were represented in dashed lines (distances were shown in Å). The conformation was developed by Affinity program. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

mass of 30 kDa of FNL using SDS-PAGE analysis. The kcat value calculated from Vmax was 6.71 × 10−3 s−1 . Compared with CALBcatalyzed polymerization of ␧-caprolactone at 45 ◦ C in toluene (Km = 0.72 mol/L; Vmax = 1.97 mol/(L h); and kcat = 72.9 s−1 ) [24], FNL showed higher affinity for ␧-caprolactone. However, its catalytic efficiency (kcat /Km = 1.92 × 10−2 s−1 M−1 ) was much lower than that observed for CALB-catalyzed polymerization of ␧-caprolactone (kcat /Km = 101.25 s−1 M−1 ). These results were similar to AFESTcatalyzed ring-opening polymerization of ␧-caprolactone [19]. In enzymatic ring-opening polymerization, it was generally accepted that the lactone was activated via the formation of an acylenzyme intermediate by the nucleophilic attack of serine residue towards the carbonyl of lactone [32]. The chain initiation and propagation occurred by the deacylation of the nucleophiles, such as water molecules and terminal hydroxyl group of the growing polymer chain, which produced the ␻-hydroxycarboxyl acid and a one unit elongated polymer chain, respectively [32]. According to this mechanism, our data suggested that, compared with CALB, FNL could incorporate the monomer into its active site more easily, but the formation of acyl-enzyme intermediate, and the chain initiation and propagation were much slower. 3.3. Molecular docking studies

Fig. 4. Effects of temperature on monomer conversion and product molecular weight Mn . The reactions were carried out using 30 mg FNL, 200 ␮L ␧-caprolactone and 600 ␮L toluene at different temperatures for 72 h.

Based on the modeled structure, molecular docking of the monomer ␧-caprolactone to FNL was undertaken to investigate the structural and energetic basis of high affinity between FNL and ␧caprolactone. Generally, hydrogen bond played an important role for the stability of enzyme–substrate complex, and was chosen to assess the binding ability. As shown in Fig. 5, hydrogen bonding interactions formed between the carbonyl group of ␧-caprolactone and the active site residues of FNL. Three strong hydrogen bonds were formed between carbonyl O of ␧-caprolactone and OH of active residue Ser119, and NH of residues of oxyanion hole (Ile53 and Met120), respectively. Furthermore, the interaction energy, including the total, van der Waals and electrostatic ones were calculated from Affinity program (Table 2). Compared with CALB (Etotal = −30.17 kcal/mol) [19], Etotal value of the theoretically modeled complex FNL-␧-caprolactone (−32.39 kcal/mol) was lower, which indicated that FNL had a stronger interaction with ␧-caprolactone than CALB, and thus a higher affinity for the

Q. Li et al. / Process Biochemistry 46 (2011) 253–257 Table 2 The total energy Etotal , van der Waal energy Evdw , and electrostatic energy Eele of the monomer ␧-caprolactone tested for FNL and CALB binding. Enzymes

Evdw a (kcal/mol)

Eele a (kcal/mol)

Etotal a (kcal/mol)

FNL CALBb

−14.93 −16.62

−17.46 −13.55

−32.39 −30.17

a b

Calculated from Affinity program. The data were quoted from Ref. [19].

monomer. These results were consistent with the experimental results obtained using Michaelis–Menten kinetic analysis. 4. Conclusion In this paper, PCL was efficiently synthesized using a novel thermophilic lipase FNL from F. nodosum as the catalyst. The synthesized PCL was of low molecular weight and narrow molecular weight distribution, and expected to be widely used as the soft block of thermoplastic elastomers, or controlled release drug carrier. Moreover, compared with commercially available enzymes, such as CALB and HIC, FNL could be effectively performed at much higher temperatures, and thus it was of great potential in combining with the conventional chemical route to synthesize the polymers of new architecture and performance. This work would provide a new route for sustainable polyester synthesis and increase our understanding of how thermophilic lipases work in polyester synthesis. Acknowledgment The research was supported by National High Technology Program of China (863 Program, 2006AA020203 and 2007AA021306) and 985 Graduate Innovation Program of Jilin University (20080220). References [1] Srivastava RK, Albertsson AC. Porous scaffolds from high molecular weight polyesters synthesized via enzyme-catalyzed ring-opening polymerization. Biomacromolecules 2006;7:2531–8. [2] Duda A. Polymerization of ␧-caprolactone initiated by aluminum isopropoxide carried out in the presence of alcohols and dols: kinetics and mechanism. Macromolecules 1996;29:1399–406. [3] Penczek S, Duda A, Kowaiski A, Libiszowski J, Majerska K, Biela T. On the mechanism of polymerization of cyclic esters induced by tin(II) octoate. Macromol Symp 2000;157:61–70. [4] Vaida C, Takwa M, Martinelle M, Hult K, Keul H, Moller M. ␥-Acyloxy-␧caprolactones: synthesis, ring-opening polymerization vs. rearrangement by means of chemical and enzymatic catalysis. Macromol Symp 2008;272:28–38. [5] Gross RA, Kumar A, Kalra B. Polymer synthesis by in vitro enzyme catalysis. Chem Rev 2001;101:2079–124. [6] Kobayashi S, Uyama H, Kimura S. Enzymatic polymerization. Chem Rev 2001;101:3793–818. [7] Uyama H, Kobayashi S. Enzyme-catalyzed polymerization to functional polymers. J Mol Catal B: Enzym 2002;19–20:117–27.

257

[8] Varma IK, Albertsson AC, Rajkhowa R, Srivastava RK. Enzyme catalyzed synthesis of polyesters. Prog Polym Sci 2005;30:949–81. [9] Albertsson AC, Srivastava RK. Recent developments in enzyme-catalyzed ringopening polymerization. Adv Drug Deliver Rev 2008;60:1077–93. [10] Kobayashi S. Recent developments in lipase-catalyzed synthesis of polyesters. Macromol Rapid Commun 2009;30:237–66. [11] Kobayashi S, Makino A. Enzymatic polymer synthesis: an opportunity for green polymer chemistry. Chem Rev 2009;109:5288–353. [12] Henderson LA, Svirkin YY, Gross RA, Kaplan DL, Swift G. Enzyme-catalyzed polymerizations of ␧-caprolactone: effects of initiators on product structure, propagation kinetics, and mechanism. Macromolecules 1996;29:7759–66. [13] Namekawa S, Suda S, Uyama H, Kobayashi S. Lipase-catalyzed ring-opening polymerization of lactones to polyesters and its mechanistic aspects. Int J Biol Macromol 1999;25:145–51. [14] Kumar A, Gross RA. Candida antarctica lipase B catalyzed polycaprolactone synthesis: effects of organic media and temperature. Biomacromolecules 2000;1:133–8. [15] Hunsen M, Azim A, Mang H, Wallner SR, Ronkvist A, Xie W, et al. A cutinase with polyester synthesis activity. Macromolecules 2007;40:148–50. [16] Duxbury CJ, Wang W, de Geus M, Heise A, Howdle SM. Can block copolymers be synthesized by a single-step chemoenzymatic route in supercritical carbon dioxide? J Am Chem Soc 2005;127:2384–5. [17] Hasan F, Shah AA, Hameed A. Industrial applications of microbial lipases. Enzyme Microb Technol 2006;39:235–51. [18] Levisson M, van der Oost J, Kengen SWM. Carboxylic ester hydrolases from hyperthermophiles. Extremophiles 2009;13:567–81. [19] Ma J, Li Q, Song B, Liu D, Zheng B, Zhang Z, et al. Ring-opening polymerization of ␧-caprolactone catalyzed by a novel thermophilic esterase from the archaeon Archaeoglobus fulgidus. J Mol Catal B: Enzym 2009;56:151–7. [20] Manco G, Giosue E, D’Auria S, Herman P, Carrea G, Rossi M. Cloning, overexpression, and properties of a new thermophilic and thermostable esterase with sequence similarity to hormone-sensitive lipase subfamily from the archaeon Archaeoglobus fulgidus. Arch Biochem Biophys 2000;373:182–92. [21] Simone GD, Menchise V, Manco G, Mandrich L, Sorrentino N, Lang D, et al. The crystal structure of a hyper-thermophilic carboxylesterase from the archaeon Archaeoglobus fulgidus. J Mol Biol 2001;314:507–18. [22] Yu S, Yu S, Han W, Wang H, Zheng B, Feng Y. A novel thermophilic lipase from Fervidobacterium nodosum Rt17-B1 representing a new subfamily of bacterial lipases. J Mol Catal B: Enzym 2010;66:81–9. [23] Gao R, Feng Y, Ishikawa K, Ishida H, Ando S, Kosugi Y, et al. Cloning, purification and properties of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1. J Mol Catal B: Enzym 2003;24–25:1–8. [24] Van der Mee L, Helmich F, de Bruijn R, Vekemans JAJM, Palmans ARA, Meijer EW. Investigation of lipase-catalyzed ring-opening polymerizations of lactones with various ring sizes: kinetic evaluation. Macromolecules 2006;39:5021–7. [25] Veld MAJ, Fransson L, Palmans ARA, Meijer EW, Hult K. Lactone size dependent reactivity in Candida antarctica lipase B: a molecular dynamics and docking study. ChemBioChem 2009;10:1030–4. [26] Habe H, Morii K, Fushinobu S, Nam JW, Ayabe Y, Yoshida T, et al. Crystal structure of a histidine-tagged serine hydrolase involved in the carbazole degradation (CarC enzyme). Biochem Biophys Res Commun 2003;303:631–9. [27] Nardini M, Dijkstra BW. ␣/␤ Hydrolase fold enzymes: the family keeps growing. Curr Opin Struct Biol 1999;9:732–7. [28] Deng F, Gross RA. Ring-opening bulk polymerization of ␧-caprolactone and trimethylene carbonate catalyzed by lipase Novozym 435. Int J Biol Macromol 1999;25:153–9. [29] Dong H, Cao S, Li Z, Han S, You D, Shen J. Study on the enzymatic polymerization mechanism of lactone and the strategy for improving the degree of polymerization. J Polym Sci A: Polym Chem 1999;37:1265–75. [30] Chua LS, Sarmidi MR. Effect of solvent and initial water content on (R, S)-1phenylethanol resolution. Enzyme Microb Technol 2006;38:551–6. [31] Hunsen M, Abul A, Xie W, Gross R. Humicola insolens cutinase-catalyzed lactone ring-opening polymerizations: kinetic and mechanistic studies. Biomacromolecules 2008;9:518–22. [32] Peeters JW, Leeuwen O, Palmans ARA, Meijer EW. Lipase-catalyzed ringopening polymerizations of 4-substituted ␧-caprolactones: mechanistic considerations. Macromolecules 2005;38:5587–92.