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Sericin: Structure and Properties

1.1 Type of Silk Sericin

Sericin is a natural product from the silkworm. Sericin is one of the major protein components in the cocoons of Lepidopteron insects. Sericin is a glue-like coating protein surrounded with filament protein, fibroin (Figure 1.1). In manufacturing silk, sericin is a waste product from the degumming process. The silk sericin is classified into two types based on the feeding source of the silkworms: mulberry and non-mulberry sericin. The mulberry silkworm, Bombyx mori, is a well-known source of commercial silk production. This worm is a completely domesticated species that feeds on mulberry leaves. B. mori had long been developed for an indoor cultivation for the silk industry, whereas non-mulberry silkworm or wild silkworm is the group that feeds on other leaves such as oak leaves and castor oil leaves. Most of the non-mulberry silkworms cannot be reared indoors for their whole life cycles. The well-known non-mulberry silkworms are Antheraea, Samia ricini (or Philosamia ricini), and Cricula trifenestrata. Antheraea is a genus of silkworm that feeds on oak leaves and produces "tasar" silk, such as Antheraea assamensis (producing muka silk), Antheraea mylitta, Antheraea pernyi, and Antheraea yamamai. S. ricini produces the famous "eri" silk. In the wild environment, S. ricini feeds on castor oil plant leaves. C. trifenestrata is a wild silkworm producing "cricula" silk. The diversity of silkworm sources (genus, species, and diet) may produce distinct sericin characteristics.

1.2 Localization of Silk Sericin

Sericin is located at several sites of silkworms and cocoons. In the mulberry silkworm, B. mori, it has been reported that sericin is present in three components including silk gland, cocoon, and floss (Gamo et al. 1977; Kikkawa 1953; Yamada 1978). For non-mulberry silkworms, sericin is also secreted in the cocoon peduncle (Dash et al. 2006). The silk gland is the site that produces sericin. In a histological study, sericin was found to be mainly synthesized in the middle and posterior of the silk gland. Sericin protein is then sent to anterior silk glands via the lumen for secretion and cocoon construction (Consortium 2008; Kikkawa 1953; Yamanouchi 1922).

Figure 1.1 Scanning electron microscope (SEM) of a silk filament that contains fibroin and sericin.

1.3 Molecular Mass of Sericin

The molecular mass of sericin has been observed using sodium dodecyl sulfate and polyacrylamide gel electrophoresis (SDS-PAGE). The diversity pattern of sericin was investigated from various extraction sites, extraction methods, species, and strains. Figure 1.2 shows the molecular mass of sericin from different extraction methods using SDS-PAGE.

1.3.1 Middle Silk Gland Sericin

The middle silk gland (MSG, Figure 1.3) is a synthesis site for sericin in silkworm. Sericin obtained from MSG is known as native sericin. The MSG sericin measured by gel electrophoresis was found in intact bands and various protein sizes. Sericin extracted from the silk gland of mulberry silkworm was identified with three sizes of sericin including 130, 210, and 220?kDa as shown in Figure 1.4 (Sprague 1975). The study of sericin extracted from four MSG sections, including the anterior, middle to anterior, middle, and posterior sections, found five different sizes of sericin polypeptides. Two polypeptides, 177 and 134?kDa, were isolated from the anterior MSG. The middle section of the MSG had two polypeptides (309 and 145?kDa). One polypeptide (80?kDa) was found in the posterior section of the MSG (Gamo et al. 1977). Therefore, various sericin polypeptides were observed in mulberry sericin extracted from the MSG in the range between 80 and 309?kDa.

1.3.2 Mulberry Cocoon sericin

Cocoon sericin has been isolated, and the molecular mass of the protein was studied. Multiple sericin polypeptides have been extracted by several approaches including temperature, pressure, urea, acid, and alkali solution. In 1980, Tokutake reported that sericin and fibroin protein could be separated by the precipitation of sericin in acidic conditions (pH 5.5). Later, the gel filtration approach was used to separate the precipitated sericin into five fractions (Tokutake 1980). This result suggests that sericin comprises a variety of forms. In 1982, Gamo and colleagues reported the molecular size of sericin proteins obtained by boiling with an alkali solution for extracting the protein from the cocoon. SDS-PAGE analysis revealed the molecular size of sericin in three different bands including the sizes >226.5, 226.5, and 218.8?kDa (Gamo 1982). In 2002, Takasu et al. compared the molecular mass between cocoon sericin and sections of the MSG. Cocoon proteins were extracted by saturated aqueous lithium thiocyanate solution. The four cocoon proteins were identified from SDS-PAGE analysis and named after the similar sizes found in subparts of the MSG. The two close bands around 250?kDa were named sericin A, as a reference to the specific size found in the anterior of the MSG. The fraction of 400?kDa was named sericin M, which was abundantly found in the middle of the MSG. The molecular size 150?kDa was named sericin P; this protein was the lowest expressed and found only in the posterior subpart of the MSG (Takasu et al. 2002). Moreover, the observation of sericin patterns was different depending on the extraction methods. In 2010, Aramwit et al. compared the extracted sericin protein pattern from four different extraction methods, including urea, autoclave (high heat and high pressure), acidic solution, and alkaline solution. Clear bands were observed with urea extraction, which found sericin in various sizes from 10 to >225?kDa. Sericin extracted by autoclave showed smear patterns ranging from 20 to 150?kDa. Acid and alkaline extraction solutions revealed band patterns mixing between clear bands and smears between 50-150?kDa and 15-75?kDa, respectively. In addition, both acid and alkaline extractions shared a clear band pattern at 50-70?kDa (Aramwit et al. 2010b). From all this information, it is evident that the extraction method affected the size and pattern of cocoon sericin proteins and is related to its biological properties.

Figure 1.2 Sodium dodecyl sulfate and polyacrylamide gel electrophoresis (SDS-PAGE) of sericin extracted from white, green, and yellow cocoons using the following methods: urea solution (U), high temperature-high pressure (H), citric acid solution (A), and sodium carbonate solution (B). Different silk strains with various extraction methods show different molecular weights ranging from 10 to >225?kDa.

Source: Reprinted with permission from Aramwit et al. (2010a).

Figure 1.3 Diagrammatic representation of the silk gland in the mature larvae of silkworms. Shadowed parts in the figure indicate the section used for the extraction of silk proteins, fibroin, and sericin. A, anterior gland; AM, anterior section in the middle gland; MM, middle section in the middle gland; PM, posterior section in the middle gland; P, posterior gland.

Source: Reprinted with permission from Gamo et al. (1977). © 1977 Elsevier.

Figure 1.4 Separation of the protein components of sericin by sodium dodecyl sulfate and polyacrylamide gel electrophoresis on slabs of (a) 4% acrylamide and 0.1% bisacrylamide; (b) 8% acrylamide and 0.2% bisacrylamide.

Source: Reprinted with permission from Aramwit et al. (2010a).

Additionally, the different silkworm strains gave different patterns of sericin proteins. The study of B. mori from three strains based on the pigment color (different concentrations of flavonoids and carotenoids in cocoons), white shell, greenish shell, and yellow shell (Figure 1.5), had variations in sericin molecular mass. Urea-extracted sericin revealed the clear bands in all strains, but different size ranges for the white shell and yellow shell strains with proteins ranging from 10 to >225?kDa, while the greenish shell strain had mass ranging from 10 to 150?kDa. The autoclave method showed smear bands ranging from 50-150, 35-100, and 35-75?kDa for the white shell, greenish shell, and yellow shell strains, respectively. For acid and alkali extraction methods, all strains also displayed different bands within the range of 35-150 and 15-75?kDa, respectively (Aramwit et al. 2010b). This suggests that not only the sericin extraction method but also the strain of silkworms affected the molecular mass structure of extracted-sericin proteins.

1.3.3 Non-mulberry Cocoon and Peduncle Sericin

Non-mulberry cocoon sericin has been studied in S. ricini, A. assamensis, and A. mylitta. In 2004, Ahmad et al. observed non-mulberry sericin at 66?kDa from S. ricini and A. assamensis (Ahmad et al. 2004). In 2007, Dash et al. reported a 70?kDa sericin extracted from A. mylitta (Dash et al....