The degradation behaviors of Thai Bombyx mori called Samrong and Nanglai silk fibroins exposure to protease enzymes; protease XIV, protease XXIII and α-chymotrypsin type II were studied in this work. The degradation behaviors were expressed by their weight loss, morphological and secondary structure changes as well as thermal properties. Samrong showed higher percentage of weight loss than Nanglai. SEM micrographs indicated that silk fibroin were de- stroyed and showed many holes on their fiber surfaces. All of silk samples were increasable destroyed when exposure to the protease enzyme for long incubation period. With thermal analysis, both silk fibroin presented the thermal stability in the same profile. The result suggested that the selected silk fibroin should be composed of similar pattern of amino acids and their ratios. However, the protease susceptibility of each silk fibroin slightly varied in case of morphology observation. This might be affected by their genetic variety.
Recently, various biomaterials have been explored for applications [1,2]. Various ideal characteristics including non-immunogenic, biocompatible, biodegradable and high mechanical strength are main criterions for decision use [
Silk is a fibrous protein produced by a variety of insects [
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Previous literatures have been investigated the degradation behaviors of silk fibroins exposed to different proteolytic enzymes such as protease XIV [
Thai Bombyx mori silk cocoon of 2 varieties (Nanglai and Samrong) were kindly supplied from Silk Innovation Center (SIC), Mahasarakham University, Thailand.
Domesticated silk; B. mori (locally called “Nang lai” and “Samrong”) silk cocoons were used for preparation of silk fiber and subjected as materials in this study. The cocoons were firstly boiled in 0.5% (w/v) Na2CO3 solution at 90˚C for excluding sericin to obtain silk fibroin (SF). After degumming in each step, silk fiber was washed with distilled water.
Degradation of SF by action of the protease enzymes: α-chymotrypsin II, protease XIV and protease XXIII were investigated as previously reported [
where Wi is initial weight of sample while Wf is the weight of sample after immersing in enzyme solution.
Samples were dehydrated and segmented (~1 cm in length), then mounted on the stub with double-sided carbon tapes. Yarns were sputter-coated with gold for enhanced surface conductivity. Current and voltage were adjusted to give power of 2 W (3 mA, 15 kV) for 3 min. The samples were examined using scanning electron microscope (SEM) (JEOL-JSM 6460LV).
A TA-Instruments TG SDT Q600 (Leken’s drive, New Castle, DE) thermogravimetric analyzer was be used to determine the thermal decomposition patterns of the SF. Samples were loaded in platinum crucible. The analysis condition would be 50˚C - 800˚C at heating rate of 20˚C /min under nitrogen atmosphere. The TGA data were recorded online in TA instrument’s Q series explorer software. The analyses of the TG data would be done using TA Instrument’s Universal Analysis 2000 software (version 3.3B).
The weight losses of SF after incubation with protease enzymes are shown in Figures 1-3. The SF of Samrong showed dominantly higher weight loss than Nanglai after exposure to protease XIV. The weight loss of the Samrong SF gradually increased from the initial day up to the end of incubation for 49 days at about 27.5%. Nanglai SF was stable of its weight in protease XIV until 14 days,
and then slightly increased at about 7.5% of weight loss after exposure to enzyme for 35 days. The degradation of Nanglai SF was dramatically increased when the incubation time over the 35 days until the end at about 25% of weight loss.
Exposure to protease XXIII, Samrong SF degraded at first day of incubation and showed exponential pattern up to 49 days. The weight loss of Samrong SF by protease XXIII was about 17.5%. Nanglai SF started to degrade from initial day until 35 days of investigation. Incubation SF with α-chymotrysin type II, both of SF was found after incubation for 14 days. The effect of this enzyme on SF showed different profiles compared to protease XIV and protease XXIII. Nanglai SF degraded in the highest at 28 days and prolonged until 42 days of incubation time. Samrong showed the highest of weight loss at 35 days. At the end of degradation, Samrong has the highest weight loss at about 13.6% whereas Nanglai about 12.3%.
The results showed that the extent weight loss depending on the type of enzyme, silk variety and on the treatment time. However, the actions of each enzyme on different types of SF were varied. It can be concluded that all of enzyme can be degraded SF of B. mori, but with different characteristics. The amount and accessibility of the sites of proteolysis have probably acted as important factor in examining of SF degradation [
In general, all of SF can be destroyed by protease XIV, especially in their surfaces. SF of Samrong showed higher susceptibility to the protease XIV than Nanglai silk. The results indicated that Samrong SF started degrade at the 14 days of incubation (
Samrong SF showed higher surface erosion than that of Nanglai SF at the same incubation time. The action of α-chymotrysin type II on SF degradation was similar profile with protease XXIII. It can be degraded SF both of domesticated silks. Samrong SF was destroyed by this enzyme more than Nanglai SF. The erosion of Nanglai SF surfaces was observed after exposure to enzyme for 21 days (
Since protease XIV showed the highest degradation effect on SF. Therefore it is chosen enzyme model to study thermal stability of SF after immersing in enzyme solution. With protease XIV, Samrong SF (
The poly(Ala)n sequences are expected to be location
for β-sheet and α-helix structures, whereas all Gly residues were reported to take random coil conformation; nearly all tyrosine residues [
The effects of some proteases; protease XIV, protease XXIII and α-chymotrypsin enzyme on degradation of Thai B. mori SF were reported in this study. The obtained results from percentage of weight loss, SEM micrographs, and thermal study found that all protease enzymes can be destroyed the surfaces of SF. The ability of each enzyme on SF degradation was varied. However, thermal property of SF was affected by proteases since they destroyed SF surface and resulted to decrease silk stability.
The authors would like to thank Division of Research Facilitation and Dissemination, Graduate School, Department of Chemistry, Faculty of Science, Mahasarakham University and the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education, Thailand for financial support of this work.