Influence of monomer composition of poly(butylene succinate-co-hexamethylene succinate)s on their enzymatic hydrolysis by Fusarium solani cutinase

Abstract Poly(butylene succinate) (PBS), poly(hexamethylene succinate) (PHS) and three kinds of poly(butylene succinate-co-hexamethylene succinate)s (P(BS-co-HS)s) were enzymatically hydrolyzed by Fusarium solani cutinase. The results showed that the characteristics of the enzymatic hydrolysis of these polyesters were mainly affected by crystallinity, thermal properties and the BS/HS ratio. The enzymatic hydrolysis rates of the polyesters are as follows: P(BS-co-HS52) ≈ P(BS-co-HS71) > PHS > P(BS-co-HS32) > PBS. Furthermore, with increasing HS content, both the degree of crystallinity (Xc) and melting temperature (Tm) of the polyesters first decreased and then increased. P(BS-co-HS52) and P(BS-co-HS71) had the lowest Xc and the lowest Tm, thus had the highest hydrolysis rate; this shows that the hydrolysis rate is affected by Xc and Tm. The results also showed that BS/HS ratio could affect the physical properties and degradability of polyesters. Thus, it is possible to prepare polyesters with various physical properties and degradability for different applications by adjusting BS/HS ratio. The crystalline and amorphous regions of the polyesters were both hydrolyzed, during which parts of the crystalline regions were converted into the amorphous regions. Finally, we found that the crystal structure and thermal stability of the polyesters were not affected by the enzymatic hydrolysis.


Introduction
Bio-based and biodegradable polymers have increasingly gained widespread attention due to not only shortages of traditional, non-biodegradable polymers, but also environmental pollution problems that they have caused. [1,2] Poly(butylene succinate) (PBS) is a readily available biodegradable polyester that has excellent heat, mechanical and thermoplastic processing properties; it is also fully biodegradable. [3][4][5] All of these properties make PBS a suitable material that can be used in disposable packaging, agricultural film and biomedical industries. [6][7][8] However, due to poor biodegradation ability and low availability of PBS-degrading microorganisms in the environment, the applications of PBS in different fields remain limited. Thus, it is necessary to change or ameliorate the properties of PBS in order to expand its application range. [9] An effective way to achieve this is to blend PBS with another polymer to obtain a copolymer that has improved crystalline properties, thermal stability and degradability. Debuissy et al. have reported that the the degree of crystallinity (X c ) and melting temperature (T m ) of poly(3-hydroxybutyrate-co-butylene succinate) copolyesters decrease with increasing amount of hydroxybutyrate added to the copolymer and the randomness of the copolymer structure. [10] Papageorgiou et al. have demonstrated that poly(butylene-co-propylene succinate) copolymers have lower melting points and higher enzymatic degradation rates than those of neat PBS. [1] Shi et al. have described that increasing the amount of cellulose triacetate that is added to PBS may affect the crystal behavior of PBS. [11] Raw materials including 1,6-hexanediol (HD) and succinic acid (SA) required for the synthesis of poly(hexamethylene succinate) (PHS), which is fully biodegradable, are available from renewable sources. [12,13] Compared to PBS, PHS contains two additional methylene moieties (-CH 2 CH 2 -) on its diol unit; and these moieties cause the thermal, crystalline, and mechanical properties of the two materials to be significantly different. Bai et al. have compared the biodegradability of three kinds of polyesters and discussed the impact of hydroxyl monomers on their biodegradability; they found that the degradation rate of PHS is highest. [14] However, PHS has a poor tensile strength and a low melting point, thus can only have a few applications.
Considering their properties, the physical and chemical properties of poly(butylene succinate-co-hexamethlene succinate) P(BS-co-HS) are complementary to the performance and properties of neat PBS or PHS. Bi et al. have reported that the melting point and crystallinity of P(BS-co-HS) can control the rate of biodegradation. However, the changes of P(BS-co-HS) after degradation have not been assessed in detail. [15] Wang et al. have described that PBS, PHS, and P(BS-co-HS) have the crystallization mechanism. [16] Tan et al. have reported that the crystallinity of P(BS-co-HS) copolyesters predominantly depends on the content of the major component in the copolyesters. [17] Cutinases (EC 3.1.1.74) are serine esterases and are members of the a/b hydrolase family. Cutinases are multifunctional lyases that can catalyze the hydrolysis of various synthetic polyester. [18] Ping et al. reported that both Fusarium solani cutinase and Aspergillus fumigatus cutinase had degradation ability on poly(e-caprolactone) (PCL). [19] Sulaiman et al. cloned a LC-cutinase from a fosmid library of a leaf-branch compost metagenome LC-cutinase had an ability to degrade PCL and polyethylene terephthalate (PET). [20] In previous reports in our laboratory, we found that F. solani cutinase has the ability to degrade PBS and its copolyesters. [14] Therefore, In this study, we prepared PBS, PHS, P(BS-co-HS32), P(BS-co-HS52) and P(BS-co-HS71). We then compared their properties, including film morphology, crystallinity, and thermal stability, before and after enzymatic hydrolysis by F. solani cutinase. The present research aims to examine the monomer composition of P(BS-co-HS) on the degradation process and to determine the difference in the polyesters before and after degradation.

Synthesis of polyesters and preparation of polyester films
PBS, PHS and three P(BS-co-HS)s containing three different BS/HS ratios (68/32, 48/53, 29/71) were synthesized using a two-stage reaction consisting of esterification and polycondensation reactions. [22] The molar ratio of SA and total diol(s) was 1:1.1. Initially, BA, BD and HD were used as primary materials to add into decahydronaphthalene (60 mL) containing TTIP (1/600 of the total molar mass of reactants). The esterification reaction was carried out at 140 C for 2 h under nitrogen atmosphere, and the polycondensation reaction was carried out at 230 C for 4 h under low pressure. The obtained products were dissolved in 100 mL chloroform and then precipitated in an excess amount of methanol. After washing several times until the solution became clear, the precipitates were collected and then dried at 37 C under vacuum before use. All polyesters were synthesized using the same process. The molecular weights of the polyesters were shown in Table 1. The BS/HS ratio of the polyesters was confirmed by 1 H-NMR ( Figure S1).
Polyester films were prepared by melt pressing at 160 C and cold pressing at room temperature, from which films, each with a thickness of 0.5 mm, were obtained. The polyester films were cut into rectangles, each with a length of 30 mm and a width of 10 mm. To ensure that the film crystalline remained intact, the film saples were stored in a desiccator for several weeks. [23] Enzymatic hydrolysis The polyester films (30 Â 10 Â 50 mm) were hydrolyzed in 10 mL of Na 2 HPO 4 -NaH 2 PO 4 buffer (0.1 M, pH 7.4) containing 1.2 mg/mL cutinase at 37 C. The films were removed from the solution and then rinsed with distilled water; after that, they were dried to a constant weight in vacuum. The weight loss of the polyester films was calculated by subtracting the weight after hydrolysis from the weight before hydrolysis. [14] The cutinase concentration was determined by Bradford method [24] and the cutinase activity was measured based on the liberation of p-nitrophenol from p-nitrophenyl butyrate according to Xu et al. [25] Characterization of P(BS-co-HS)

Gel permeation chromatography (GPC)
GPC was used for the analysis of the average molecular weights and molecular weight distribution of synthesized polyesters. GPC was conducted with a Waters 1515 Isocratic HPLC Pump (Milford, MA, USA). A Waters 1515 refractive index detector was used with a temperature controller. The polyesters were dissolved in chloroform to prepare a 1.5 mg/mL solution. After being fully dissolved, the solution was filtered by 0.22 lm membrane, and then 10 lL filtrate was directly injected for testing. Waters Styragel HT 3(7.8 Â 300 mm) and HT4 column (7.8 Â 300 mm) were used in tandem, and different molecular masses of polystyrene were utilized as standard. The pore size of the column was 10 lm. Other specific test conditions were detailed as follows: column temperature is 35 C, column pressure is 1600 psi, mobile phase used was chloroform and flow velocity is 0.8 mL/min.

Scanning electron microscopy (SEM)
Surface morphology of the polyester films before and after hydrolysis were observed by an SEM (SV810, Hitachi, Tokyo, Japan) operated at an acceleration voltage of 20 kV. The films were sputtered with gold before being placed on the sample stage.

Differential scanning calorimetry (DSC) analysis
Thermal properties of the polyester films were determined by DSC (TA Instruments, Q20, USA) under N 2 atmosphere. The polyester films were heated to 150 C at a rate of 10 C/min; and after holding at this temperature for 3 min, the temperature was reduced to 0 C at the same rate.

X-ray diffraction (XRD) analysis
Crystal structures of the polyester films were determined by XRD (S8 Tiger, Bruker, Germany) equipped with a Cu Ka radiation source. Scan angles ranged from 5 to 50 at a scan rate of 5 / min. All samples were measured at 40 kV, 200 mA and 25 C.

Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) analysis
FTIR analysis of the polyester samples was carried out in ATR mode using an FTIR spectrometer (Agilent Cary 660, USA) equipped with a slide-on ATR accessory (Agilent, USA). The samples were scanned at a frequency range of 4000 to 400 cm À1 .

Thermogravimetric (TG) analysis
Thermal behaviors of polyester films were examined by TG analysis (TA Instruments, Q600, USA) under a nitrogen gas stream flew at a flow rate of 50 mL/min. During the analysis, the films were heated from 20 to 500 C at a rate of 10 C/min.

Results and discussion
Degradability Figure 1 shows the weight loss of the polyester films after being hydrolyzed by cutinase at pH 7.4 and 37 C. As blank controls, polyester films were hydrolyzed without the presence of enzyme, from which the enzymatic hydrolysis rate was undoubtedly zero (data not shown). The weight loss of the polyester films markedly increased with increasing hydrolysis time. The enzymatic hydrolysis rate of the five kinds of polyester films were could be ranked in the following order: P(BS-co-HS52) % P(BS-co-HS71) > PHS > P(BS-co-HS32) > PBS. The biodegradation curves of PBS, PHS and P(BS-co-HS32) films showed that the enzymatic hydrolysis could be divided into two stages: a fast stage (PBS: 0-4 h, P(BS-co-HS32): 0-6 h, PHS: 0-4 h); and a slow stage (PBS: 4-18 h, P(BS-co-HS32): 6-12 h, PHS: 4-10 h). In the fast stage (the first stage), the ester bonds in the polymers are cleaved to produce shorter polymer fragments, which causes a significant weight loss. In the slow stage (the second stage), the hydrolysis occurs at the end fragments of the polymers, from which water-soluble oligomers are produced and then removed from the film surfaces. [23,26] Within the experimental range, in the hydrolysis of P(BS-co-HS52) and P(BS-co-HS71), only the fast stage was observed. This clearly shows that the copolyesters that consist primarily of HS have faster enzymatic hydrolysis rates than those that consist primarily of BS; a similar observation has also been reported. [15] Morphology Figure 2 shows the SEM micrographs of films of neat PBS, PHS and their copolyesters. Before enzymatic hydrolysis, the polyester films had a smooth surface (Figure 2(a,g,m,p,s)). After 2 h of enzymatic hydrolysis, the surface became rough and ununiform, as a result of chain scission caused by the enzymatic hydrolysis. [27] Pits appeared and became deeper as the hydrolysis time was increased, and increasing numbers holes were observed as the hydrolysis progressed toward completion. Thandengco and Tokiwa have revealed the mechanism of this enzyme, describing that it first attacks the center of the crystal. [28] Cracks and holes that were observed in the crystal are due to the penetration of water into the amorphous region, which in turn causes the enhancement of the enzymatic hydrolysis. [26] The polyester films were initially degraded layer-by-layer, and each layer was then turned into pits and holes. [7] Melting temperature One of the factors that can influence the enzymatic hydrolysis rate of polyesters is T m ; [29,30] and for this reason, a polyester with a lower T m has a faster enzymatic hydrolysis rate. [29,31] Table 2 shows the T m and enthalpy of fusion (᭝H m ) of polyesters before and after enzymatic hydrolysis. The T m before and after hydrolysis were not significantly different. P(BS-co-HS52) and poly(BSco-HS71) had a similar T m that was also lower that the T m of other polyesters; thus, their enzymatic hydrolysis rates were higher. A similar trend has also been observed in Bi and Marten's studies, in which they described that a polyester with lower T m has higher weight loss. [15,29] The DSC curves of the polyester films degraded by the enzyme were shown in Figure S2.

Crystal structure and crystallinity
The changes of crystal structure and crystallinity of the polyesters before and after enzymatic hydrolysis are displayed in Figure 3 and Table 2. In Figure 3, the diffraction peaks at 19.6 , 21.9 and 22.6 are the (020), (021) and (110) planes of PBS, respectively, and the peaks at 21.4 and 24.4 belong to the (220) and (040) planes of PHS, respectively. The crystal structure of P(BS-co-HS) that contained a higher BS content resembled that of PBS, and the crystal structure of P(BS-co-HS) that contained a higher HS content was similar to that of PHS. The polyesters after hydrolysis also exhibited similar diffraction peaks. We also observed that as the hydrolysis time increased, the area of the diffraction peak of the polyesters decreased, which is an indication that the crystallinity decreased. This might be due to that the hydrolysis converts the crystalline region into the amorphous region, in turn causing the crystallinity to decrease. In addition, the low molecular weight segments, and the presence of oligomers and water absorption can also cause the crystallinity to decrease. [32] The same conclusion has also been previously reported. [14,33] However, the diffraction pattern and peak position of the polyesters before and after hydrolysis remained unchanged. These observations reveal that the crystal structures of the polyesters were unaffected by the hydrolysis. The crystallinity of the materials can also effect the enzymatic hydrolysis rate: the higher the crystallinity, the slower the enzymatic hydrolysis rate. [34] X c of the five kinds of polyesters are shown in Table 2. Among all the polyesters, P(BS-co-HS52) and P(BS-co-HS71) had the lowest X c ; therefore, they could be more quickly hydrolyzed.
PBS had the highest T m and crystallinity, followed by P(BS-co-HS32); thus, they have lower enzymatic hydrolysis rates than other polyesters. P(BS-co-HS52) and P(BS-co-HS71) had the lowest T m and crystallinity, thus had the highest enzymatic hydrolysis rate. Figure 4 shows the FTIR spectra of the polyesters. According to the spectra, the stretching vibration of C¼O appeared at around 1711-1730 cm À1 . These absorption peaks are shifted compared to the C¼O group of free ester, which is most likely due to the crystal stacking arrangement of the polyester. These peaks are associated with C¼O in the crystalline region of the polyesters . [35][36][37][38] The bands at around 1154 cm À1 are due to C-O-C in the crystalline regions and the amorphous regions, respectively. [35,36] Furthermore, the FTIR spectra of the polyesters before the hydrolysis were not significantly different from the spectra of those after the hydrolysis. However, the intensities of C¼O and C-O-C peaked could be observed to decrease as the hydrolysis time increased. This is related to the thinning of polyesters films thickness under the influence of enzyme. Based on the FTIR and XRD results, it can be inferred that the crystal structure of the polyester did not change during the enzymatic hydrolysis, but the structure was disrupted, leading to a decrease in crystallinity.

Thermal decomposition behavior
The thermal decomposition behavior of the polyesters was investigated under nitrogen atmosphere by TG, and the results are shown in Figure 5. The decomposition of polyesters follows two different mechanisms. [38] The initial weight loss, which was not prominent, may be attributed to the volatilization of some small molecules, including catalysts, SA, BD, or HD. [16] With prolonged hydrolysis time, the molecular chains of carboxyl and vinyl groups are cleaved, causing the decomposition of the polyesters to increase. [39,40] The cleavage of the polymer chains also leads to the decrease of the thermal decomposition temperature. [41,42] After that, ester bonds can undergo random cracking to further generate carboxyl and vinyl groups. [29] As illustrated in Figure 5, the thermal decomposition of the polyesters before and after hydrolysis were not significantly different. The optimal decomposition temperatures of PBS, P(BS-co-HS32), P(BS-co-HS52), P(BS-co-HS72), and PHS were 396, 394, 398, 402, and 397 C, respectively. The main factor affecting the thermal stability of polyesters is the number of methylene group, the higher the number of the methylene group, the lower the thermal stability. [22,38,43] The crystallinity and PB/HS ratio of the polyesters also affect their thermal stability. [10,44]

Conclusions
The work aims to demonstrate that adjusting the monomer composition of copolyesters can change their melting temperature, crystallinity, thermal stability and enzymatic hydrolysis rate, which in turn can expand their application scope. The results showed that among the five kinds  of polyesters that were synthesized and compared, P(BS-co-HS52) and P(BS-co-HS71) had the lowest X c and T m , and could be most rapidly hydrolyzed by cutinase. Additionally, the two polyesters could be completely hydrolyzes within about 5 h. The result also showed that the enzymatic hydrolysis rate was affected by crystallinity, melting temperature and PB/HS ratio of the polyesters. The crystalline structures of the polyesters were not affected by the hydrolysis; however, the hydrolysis could cause their crystallinity to decrease by transform their crystalline regions into non-crystalline regions.

Disclosure statement
No potential conflict of interest was reported by the author(s).