Kozo Tsubouchi

Preparation of undegraded native molecular fibroin solution from silkworm cocoons

Abstract The molecular mass of solubilized fibroin prepared from silk was analyzed by SDS-PAGE. It was found that the fibroin molecule was degraded during reeling, degumming (removal of sericin), and dissolution of silk threads. A protocol for the preparation of solubilized fibroin conserving its native molecular size is offered, that is, (1) to use only fresh cocoons (not dry cocoons or reeled silk threads), (2) to degum by heating in 8 M aqueous urea, and (3) to dissolve by saturated (∼9 M) aqueous lithium thiocyanate at room temperature.

Isolation of Three Main Sericin Components from the Cocoon of the Silkworm, Bombyx mori

To characterize the sericin components of the cocoon of silkworm Bombyx mori, fresh cocoon shells were dissolved in saturated aqueous lithium thiocyanate containing 2-mercaptoethanol, and fractionated by ethanol precipitation. Cocoon sericin was found to mainly consist of three polypeptides having molecular masses of the 400, 250, and 150 kDa estimated by SDS-PAGE, which corresponds to the sericin present in the middle, anterior, and posterior part of the middle silk gland. The amino acid compositions of the 400 and 150 kDa components were similar to each other, but that of the 250 kDa component was different. This suggests differences in the coding gene and properties of the 250 kDa sericin from the other two.

Sericin Enhances Attachment of Cultured Human Skin Fibroblasts

Human skin fibroblasts were cultured on sericin prepared from cocoon shells. The living cell number after 72 h was enhanced to 250% of the no-sericin control. The increase was due to the acceleration of the initial attachment of the cells. It was found that sericin M, the main component of about 400 kDa, and its serine-rich repetitive domain were the active principles.

Identification of fibroin-derived peptides enhancing the proliferation of cultured human skin fibroblasts

We previously reported that the fibroin of the silkworm Bombyx mori enhanced the proliferation of cultured human skin fibroblasts. In this work, the fibroin was digested by chymotrypsin, and the resulting peptide fragments were fractionated and assayed for their biological activity. Two peptides that promoted fibroblast growth were isolated and identified to be VITTDSDGNE and NINDFDED. Both sequences are found in the N-terminal region of the fibroin polypeptide and are thought to be the active principle of fibroblast growth-promoting activity.

Preparation of silk fibroin-supported Pd(0) catalyst for chemoselective hydrogenation: reduction of palladium(II) acetate by methanol on the protein

The Pd/fibroin (Fib) was easily prepared by the auto-reduction of the silk-fibroin conjugated Pd(OAc)2 using MeOH as a solvent and a reductant and exhibited good chemoselectivity in the hydrogenation of olefins and azides in the presence of aromatic carbonyls and/or halogens or an O-benzyl protective group.

Thickness of the Still Air Layer Adhering to Perforated Plastic Plates and Fabric

The thickness of the still air layer adhering to perforated plastic plates and a fabric was investigated in terms of the water vapor pressure gradient in the zone of diffusion above the samples. The water vapor pressures above the samples were determined from measurements using a temperature and relative humidity sensor. To analyze the thickness of the still air layer for a wide range of evaporation rates, the hole diameter and the number of holes per unit area were changed and the measurements were under different temperature and humidity/gradient conditions. The thickness of the still air layer corresponded to a distance of 10 mm from the sample surface and was not related to the surface construction and materials.

Water Vapor Pressure in the Still Air Layer Adhering to Perforated Plastic Plates

Although relative humidity and temperature sensors have become smaller and more compact, still some bulk size persists. It is conceivable that the sensor may be im permeable to the water vapor in the zone close to the sample. The water vapor pressure in the zone was investigated using two different kinds of sensors (fiber and flat). The water vapor pressure gradients were straight, and water vapor diffusion occurred ev erywhere in the still air layer. If a flat humidity sensor is used to measure the water vapor pressure close to a sample through which vapor is diffusing, an error will result when the sensor is too close, as described in our previous paper. For a sensor 3 mm wide, a 4 mm distance is too close, but a thinner sensor such as a hair can be used down to about 1 mm.