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The effect of [alpha]-chymotrypsin on some properties of silk fibroin: comparison between Thai Sumrong (B. mori) and Eri (Samia ricini) silks.

Silk fibroin (SF) has become a promising natural material and found gradually increasing applications [1,2]. It showed various advantages excellent properties include good oxygen and water vapor permeability, good cell adhesion, biodegradability, high tensile strength, minimal inflammatory response and susceptible to biological degradation by proteolytic enzymes [3-5]. Degradation is an important character of material for its designs and applications. About SF, the degradation rate might be highly variable depending on their structural and morphological features as well as characteristics of the biological environment [6]. The degradation of SF by protease enzyme has been reported. Many enzymes such as protease XIV [7,8], [alpha]-chymotrypsin [9], protease XXI [10] were studied for their effect on SF degradation. However, proteolytic degradation study on Thai B. mori and Eri (Samia ricini) silks were rarely available occurred. Therefore, the aims of this work were to study and compare the effect of [alpha]-chymotrypsin, a protease enzyme, on some properties of Thai B. mori and Eri SF.

Materials and Methods

Materials

Thai B. mori silk variety called Sumrong and Eri silk called SIC varieties were kindly supplied by Silk Innovation Center (SIC), Mahasarakham University, Maha Sarakham, Thailand. [alpha]-chymotrypsin type II was purchased from Sigma-Aldrich, USA. All of chemical used were analytical grade.

Preparation of silk fibroin

Silk fibers of B. mori and Eri were firstly boiled in 0.5% (w/v) [Na.sub.2]C[O.sub.3] solution at 90[degrees]C to help for reeling fiber. The raw silk fibers were then degummed to exclude sericin by boiling twice in 0.5% (w/v) [Na.sub.2]C[O.sub.3] solution to obtain silk fibroin (SF). After degumming in each step, the silk fibers were washed with distilled water.

Incubation of SF with [alpha]-chymotrypsin type II

Firstly, the enzyme was prepared by dissolving in phosphate buffer saline (PBS) to give 1 unit of enzyme activity. The SF was submersed in the enzyme solution at 37 [degrees]C for 49 days. The enzyme solution was changed every 7 days and replaced at the same volume of fresh enzyme solution. The incubated SF was rinsed twice with distilled water before further submersing.

Morphological observation

All of SF were dehydrated and cut (~1cm in length) before observing their morphology under the scanning electron microscope (SEM) (JEOL, JSM-6460LV, Tokyo, Japan). The samples were sputter coated with gold by double side of carbon for enhancing surface conductivity. Current and voltage were adjusted to give power of 2W (3mA, 15 kV) for 3 min.

Secondary structure analysis

SF samples were investigated their secondary structures using attenuated total reflection infrared (ATR-IR) spectroscopy (Perkin Elmer-Spectrum Gx, USA) in the spectral region of 4000-400 [cm.sup.-1] at 4 [cm.sup.-1] spectral resolution and 32 scans.

Thermal properties measurement

SF samples with 8-10 mg were loaded in a platinum crucible. The thermogravimetric analysis (TGA) was then performed using TA instruments, SDT Q600 (Luken's drive,

New Castle, DE). The samples were non-isothermal heated from 50 [degrees]C to 800 [degrees]C at a heating rate of 20 [degrees]C/min. The TGA was carried out in nitrogen with the flow rate of 100 mL/min.

Results

Fig. 1 showed SEM micrographs of Thai B. mori (Sumrong) silk fibers after incubation with [alpha]-chymotrypsin type II in different days. The results found that SF with the homogeneous surface without fractions was obtained when incubated the SF in the enzyme solution for 1, 7 and 14 days. The degradation of SF was appeared after 28 days of incubation. SF was destroyed by [alpha]-chymotrypsin that indicated by the pore on the fiber surfaces. The fraction of SF was gradually increased when the incubation timed increased. The wide range of pore as well as length of destroyed areas was significantly occurred especially at 49 days after incubation with enzyme. Fig. 2 showed SEM micrographs of the Eri SF after submersing in the [alpha]-chymotrypsin solution at various days. Similar pattern of SF destroyed by [alpha]-chymotrypsin was observed when compared with domesticated B. mori.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

The degraded SF was also found after incubation the Eri fiber in [alpha]-chymotrypsin solution for 21 days. The highest areas destroyed by the [alpha]-chymotrypsin was also observed at the end of period (49 days). However, the degradation profile of Eri SF was differed from Sumrong since the pores of destroy area in Eri were more wide and long sizes than Sumrong silk.

Secondary structures of SF of B. mori Sumrong was shown in Fig. 3. It was found that the important peaks were appeared at ~1615, 1514 and 1226 [cm.sup.-1] for amide I, II and III, respectively. In comparison among the incubation periods, amide III region did not change of IR spectra. In contrast, amide I and amide II peaks were slightly shifted into higher absorption peaks after incubation the SF in [alpha]-chymotrypsin solution for 14 and 28 days, respectively. The Eri SF showed different characteristics of secondary structures compared to B. mori. This was due to it has no change of IR spectra even at 49 days of incubation with enzyme solution. The amide I, II and III peaks of the Eri SF indicated at 1623, 1507 and 1221 [cm.sup.-1], respectively [Fig. 4].

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Thermal properties of SF were observed from heat flow curves. The SF of Sumrong started to degrade at about 100[degrees]C and then gradually decomposed with the highest about 330[degrees]C. Considering from endo-thermic profiles, the SF incubated with [alpha]-chymotrypsin at 1 day has temperature maximum of decomposition ([T.sub.d,max]) at 330 [degrees]C and it was shifted into lower at 320[degrees]C after incubation for over 21 days [Fig. 5]. The Eri SF has 2 steps of [T.sub.d,max] at about 320[degrees]C and 380[degrees]C. The clearly details was shown by heat flow curve [Fig. 6]. Finally, it was also found that the [T.sub.d,max] was changed into lower about 10[degrees]C after incubation the Eri SF with [alpha]-chymotrypsin comparison to the first day of study.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

Discussion

Silk fibroin (SF) composed of various excellent properties which are suitable for application. Publications about SF applications have been reported [11,12]. SF is composed of sequentially small hydrophobic amino acids [13,14]. This was reflected to the mechanical properties of the fibers. However, SF was varied by food, environmental or genetics. Degradation of SF was considered to be an important for designing and application. Previous study showed that biodegradation of SF vary greatly and mechanisms are complex. However, SF has been classified as enzymmatical degradable polymer. Many proteolytic enzymes were used for degradation SF study including [alpha]-chymotrypsin [9]. The SEM micrographs indicated that both B. mori "Sumrong" and Eri "SIC" SF were destroyed after incubation with [alpha]-chymotrypsin. The fractures of silk fiber should be started from the surfaces on some area along the fibers and then gradually moved inside the texture. The obtained results were related to previous study. Generally, enzymatic degradation comprised of 2 steps. The first step is adsorption of the enzyme on the surface and the second step is hydrolysis of the bonding [15]. In addition, factors influenced the degradation of materials were records. These are including original preparation method and structural characteristics [16,17]. Comparison between B. mori and Eri silks, the degradation profiles slightly varied since the degradation of Eri SF showed pore sizes more wide and long than B. mori. This different point may from the amino acid component in each silk which affected on the secondary structure. The conformational structure is significantly affected on protein properties [18]. ATR-IR spectra indicated typical absorption bands sensitive to the molecular conformation of SF. The structures of the SF protein are indicated in amide regions called amide I (1700-1600 [cm.sup.-1]), amide II (1600-1500 [cm.sup.-1]), and amide III (1300-1200 [cm.sup.-1]) [19,20]. The present work found that secondary structures of Sumrong and Eri SF were differed. This was due to the variation of amino acid types and ratios. The absorption peaks of SF of Sumrong after incubation with [alpha]-chymotrypsin were changed into lower wave number, while Eri was not. This also indicated the different conformational structures between Sumrong and Eri SF. In addition, thermal study was also confirmed that different varieties have directly affected on the silk characteristics. The result found in this study was similar to another report that wild silk take various decomposition steps than domesticated silk.

Conclusion

The effect of [alpha]-chymotrypsin enzyme on degradation of Thai B. mori and Eri SF was reported in this study. SEM micrographs indicated this protease enzyme can destroy SF observing by all fractures on SF fibers. The action of enzyme to degrade the SF slightly differ comparison between Sumrong and Eri. The differences might be influenced by both amino acid compositions and conformational structures. ATR-IR spectra and thermal analysis were also confirmed the different action of enzyme on the silks. The results should be concluded that Eri SF has higher strength and thermal stability than that of Sumrong.

Acknowledgements

The authors would like to thank Division of Research Facilitation and Dissemination, Department of Chemistry, Faculty of Science, Mahasarakham University and Center of Excellence for Innovation in Chemistry, Commission on Higher Education, Ministry of Education, Thailand for financial support of this work.

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Chuleerat Wongnarat (1), Darunee Puangpronpitag (2) and Prasong Srihanam (1) *

(1) Department of Chemistry, Faculty of Science, Department of Pre-clinic, Faculty of Medicine and Center of Excellence for Innovation in Chemistry, Mahasarakham University, Maha Sarakham 44150, Thailand

E-mail: prasong.s@msu.ac.th; psrihanam@gmail.com

* Corresponding Author
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Author:Wongnarat, Chuleerat; Puangpronpitag, Darunee; Srihanam, Prasong
Publication:International Journal of Applied Chemistry
Date:Jan 1, 2012
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