The recombinant E. coli BL21 strain was first cultured and the cells were harvested through centrifugation. Afterwards, through ultrasonic cell disintegration and unique thermal precipitation strategy [22], the recombinant enzyme AFEST was considerably purified. Finally, ultrafiltration and lyophilization were employed to remove the fractions of low molecular weight and obtain the enzyme preparation, respectively. The SDS-PAGE analysis showed a major band at 35.5 kDa, which corresponded to the enzyme AFEST, and the purity was more than 92% (Figure 1). The lyophilized AFEST, with a specific activity of 168 U/mg towards p-nitrophenyl caprylate at 80 °C [20], was then used as catalyst in the ring-opening polymerization of δ-valerolactone.

The ring-opening polymerization of δ-valerolactone was first performed using 30 mg AFEST in toluene at 70 °C for 72 h, and the product obtained was characterized by ¹H NMR. As shown in Figure 2, the spectrum confirmed the structure of PVL as follows: 1.68 (m, –COCH₂CH₂CH₂CH₂O–), 2.35 (t, –COCH₂–), 3.64 (t, –CH₂OH end group), 4.09 (t, –CH₂O–). Through the calculation of peak areas at 3.64 and 4.09 ppm ((Area_4.09/Area_3.64 + 1) × 100), the M_n value of the product was ca. 1540 g/mol. Compared with the conventional chemical route (e.g., PVL with molecular weight of 21,200 g/mol was obtained using phosphido-diphosphine Group 3 complexes [3]), the synthesized polymers in the present research are of low molecular weight, and might be used as the soft segments in thermoplastic elastomers, or carriers for controlled drug delivery and release.

In the lipase-catalyzed polyester synthesis, enzyme concentration played an important role in both monomer conversion and product molecular weight [23]. The effect of enzyme concentration was investigated using different amounts of enzyme AFEST, 200 μL δ-valerolactone and 600 μL toluene at 80 °C for 72 h. Monomer conversion and M_n as a function of the amount of enzyme are shown in Figure 3. Obviously, increasing the enzyme concentration should be favorable for increasing monomer conversion, and when the amount of enzyme exceeded 30 mg, the monomers were completely converted (>97% monomer conversion). Similar to monomer conversion, M_n values also exhibited a tendency to increase at relatively low amounts of enzyme (<30 mg), and at large amounts of enzyme (>30 mg), M_n values remained constant (1490–1650 g/mol). However, in the AFEST-catalyzed ring-opening polymerization of ɛ-caprolatone, M_n values were almost independent of the monomer conversions, and remained constant in the range of 30–100 mg amount of enzyme [20]. This phenomenon was probably caused by the fact that large amounts of insoluble enzyme in the reaction system could lead to mass transfer limitation, and thus in the subsequent research, 30 mg AFEST was selected as the optimal amount of enzyme.

Effect of temperature on monomer conversion and product molecular weight was investigated at different temperatures in toluene for 72 h. As shown in Figure 4, high monomer conversions were achieved at high temperatures, and in the range of 70–90 °C, the monomers were almost completely consumed, with monomer conversion values of higher than 97%. For the product M_n values, reaction at 70 °C exhibited the highest value of 2225 g/mol, which was higher than that calculated through ¹H NMR (1540 g/mol) as described above. In the AFEST-catalyzed poly(ɛ-caprolatone) synthesis [20], high temperature was favorable for the product molecular weight; and thus the results in the present study are different, but it is a common phenomenon, mainly due to the different binding affinity of AFEST towards different monomers. With regard to monomer conversion and M_n values, 70 °C was employed as the optimal reaction temperature.

Time-course studies of AFEST-catalyzed ring-opening polymerization of δ-valerolactone were performed in toluene at 70 °C for different reaction times. Figure 5 shows the corresponding plots of monomer conversion and M_n vs. reaction time. During the polymerization before 72 h, the monomer conversion and M_n increased with reaction time. After 72 h, monomer conversion values could still increase from 97% (72 h) to 100% (96 and 120 h). However, the M_n values exhibited an obvious tendency to decrease after 72 h. In enzymatic ring-opening polymerization, enzymatic hydrolysis and transesterification are also involved in the reaction system [24], and thus we can conclude that before 72 h, rapid chain initiation and propagation could lead to the increasing monomer conversion and M_n values, and after 72 h, hydrolysis and transesterification of the formed polyester chains play important roles in the reaction system, which is probably the main reason for the decreasing M_n values. Interestingly, higher PDI values were also obtained in hydrophobic solvents. In addition to catalyzing polymerization, lipases and esterases can also cleave the ester bonds of polyesters, thus catalyzing the degradation, inter-transesterification and intra-transesterification of the polymer chain [24]. The use of hydrophobic solvents results in an improved degree of polymerization, but might also lead to increased rates of hydrolysis and transesterification; this in turn results in a polymer with a broader molecular weight distribution.

The nature of solvents employed plays a crucial role in determining the stability of the biocatalyst and in the partitioning of substrates and products between the solvent and the biocatalyst in non-aqueous biocatalytic systems [25]. The effect of organic solvents with different Log P values on monomer conversion and M_n at 70 °C for 72 h is summarized in Table 1 (Log P is the octanol/water partition coefficient value [26]). Similar to AFEST-catalyzed ring-opening polymerization of ɛ-caprolactone [20], high monomer conversion (>97%) and M_n values (1860–2225 g/mol) were achieved in hydrophobic solvents (toluene, cyclohexane and n-hexane), which could be ascribed to the deactivation of enzyme by the relatively hydrophilic solvents, since these solvents might disrupt the functional structure or strip off the essential water layer from the enzyme [27,28]. In addition, enzymatic ring-opening polymerization of δ-valerolatone could also proceed in a solvent-free system, with monomer conversion and M_n values of 94% and 1620 g/mol, respectively. In all, toluene was selected as the optimal reaction medium in the present research.

Recombinant E. coli BL21 harboring the thermophilic esterase gene AF1716 from A. fulgidus was kindly provided by Dr. Giuseppe Manco (Istituto di Biochimica delle Proteine, Italy). Yeast extract and tryptone was purchased from Oxoid Ltd (Basingstoke, UK). Isopropyl β-d-thiogalactopyranoside (IPTG), ampicillin and p-nitrophenyl caprylate were purchased from Sigma (Shanghai, China). δ-Valerolactone was purchased from Sigma (Shanghai, China) in the highest available purity and used as received. Molecular sieves (4 Å) were purchased from Tianjin Chemical Co. (Tianjin, China), and heated at 500 °C for 3 h. Organic solvents of analytical grade were purchased from Beijing Chemical Co. (Beijing, China), and dried over 4 Å molecular sieves before use. All other reagents were of the highest reagent grade commercially available, and used without further purification.

The recombinant E. coli BL21 strain was cultured in 2YT medium (1% yeast extract, 1.6% tryptone, and 0.5% NaCl) containing ampicillin (100 μg/mL) at 37 °C. When the optical density at 600 nm of the culture reached a value of 1.8, the induction was performed by adding IPTG at a final concentration of 1 mM and shaking for an additional 3 h at 37 °C. The cells were harvested by centrifugation at 8000 rpm for 15 min, 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) at a ratio of 1:6 (w/v). After ultrasonic cell disintegration and incubation at 80 °C for 30 min, the suspension was then centrifuged at 8000 rpm for 15 min. The solution was collected, ultrafiltrated under a 10 kDa membrane and lyophilized to obtain the enzyme preparation.

The esterase activity was monitored by measuring the amount of p-nitrophenyl from the hydrolysis of p-nitrophenyl caprylate at 80 °C [29]. One unit of enzymatic activity was defined as the amount of protein releasing 1 μmol p-nitrophenyl from p-nitrophenyl caprylate in one minute.

The thermophilic esterase AFEST was dried in a desiccator overnight, and then transferred to a dried screw-capped vial containing 200 μL δ-valerolactone and 600 μL organic solvent (no solvent addition for solvent-free system). Ethylbenzene (50 μL) was added as an internal standard for the quantification of monomer conversion by gas chromatography (GC). The vial was sealed and then placed into a thermostatic reactor with stirring (180 rpm) at the appropriate temperature. After the reaction, 5 mL dichloromethane was added, and the enzyme was removed by filtration. The enzyme was washed three times with dichloromethane, and the filtrate was collected and evaporated under reduced pressure to remove dichloromethane. Afterwards, the viscous sample was precipitated in methanol at −20 °C, and the cloudy solution was centrifuged (8000 rpm, 15 min, 4 °C). The white precipitate was then collected, dried in a vacuum oven, and then characterized by ¹H NMR.

The structure of the polymer was characterized by ¹H NMR. The ¹H NMR spectrum was recorded in chloroform-d (CDCl₃) on an AVANCE DMX 500 spectrometer at 500 MHz. The chemical shifts for ¹H NMR spectra were referenced relative to tetramethylsilane (TMS, 0.00 ppm).

Monomer conversion values were determined by GC using a Shimadzu 2014 gas chromatograph (Shanghai, China) equipped with an Rtx-1 capillary column (30 m × 0.25 mm × 0.25 μm) and a hydrogen flame ionization detector. Nitrogen was used as the carrier gas. The temperatures of injection pool and detector were set as 200 °C and 240 °C, respectively. 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 then maintained for 2 min. The injection volume was 1.0 μL.

The M_n, weight-average molecular weight (M_w) and PDI (M_w/M_n) values of products were determined by gel permeation chromatography (GPC). Analyses were carried out using a Shimadzu HPLC system (Shanghai, China) 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 μL, respectively. The GPC system was calibrated with polystyrene standards of narrow molecular weight distribution.